Literature Supporting The Use Of Dietary Nucleotides

1
Literature Supporting The Use Of Dietary
Nucleotides

• Dr. Garry Gordon
• MD, DO, MD(H)

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DIETARY NUCLEOTIDES

• Dietary nucleotides have been shown to have
  important effects on several components of the
  immune system they may enhance intestinal
  absorption of iron they affect lipoprotein and
  long-chain polyunsaturated fatty acid metabolism
  they may alter intestinal flora and they have
  been demonstrated to have trophic effects on the
  intestinal mucosa and liver in several
  experimental situations. Clinical studies have
  shown nucleotide supplementation of infant
  formula reduces the incidence of diarrheal
  episodes
• Cosgrove M. Nucleotides. Nutrition
  19981474851.

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RNA CONTAINING FOODS CURRENTLY ON THE MARKET

• IMPACT (novartis/sandoz)
• Primary indications
• Trauma
• Major Surgery
• Infections of pneumonia
• Cancer
• Ventilator dependent
• Burn Injury

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RNA CONTAINING FOODS CURRENTLY ON THE MARKET

• There have been numerous studies of the
  clinical, immunological, nutritional and
  biochemical effects of IMPACT.
• benefits were clinically confirmed by a
  significant reduction in infection rates and a
  reduction in wound healing complications, while
  average length of admission fell (Senkal et al.
  1997)
• Clinically, this group had a significantly
  shorter length of hospital stay, a lower sepsis
  score and a trend to decreased infection. (Braga
  et al. 1996)
• Complication rates and length of hospital stay
  were reduced (Daly et al. 1995)

5
RNA CONTAINING FOODS CURRENTLY ON THE MARKET

• SMA Baby Food
• contains added nucleotides which have been
  shown to be involved in the development of an
  infants immune system and may also reduce the
  incidence of diarrhoeal disease in babies.
• Advanced Formula ENFAMIL
• (Mead Johnson)
• Nucleotide levels in ENFAMIL are patterned after
  the actual average free nucleotide levels found
  in breast milk.

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RNA CONTAINING FOODS CURRENTLY ON THE MARKET

• Breast milk has been reported to contain 1-12
  mg/dL of DNA and 10-60 mg/dL of RNA.
• Sanguansermsri J, 1974 Am. J. Clin. Nutr. 2 859

7

• Humans typically eat several grams of nucleotides
  in their diet each day.

8
(No Transcript)
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LONGEVITY

• research indicates that nucleotides such as
  RNA may help to reverse the effects of aging.
• One Swedish study found that supplemental RNA
  enhances the effects of antioxidants
• many patients followed a RNA supplement
  regime for many years without exhibiting harmful
  side effects. Instead, they demonstrate many
  benefits, including diminished skin wrinkling,
  improved circulation and peripheral nerve
  function.
• rats injected weekly with RNA and DNA lived
  far longer than rats that did not receive the
  supplements.
• LE Magazine August 1997

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LONGEVITY

• efficacy of RNA was its ability to increase
  cellular energy levels so as to facilitate the
  movement of young cells to the surface where they
  could replace unsightly senescent cells.
• The nucleic acid RNA seemed to restore this
  repair mechanism.
• LE Magazine January 2003

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MEMORY

• Other studies indicate that RNA may play a
  role in helping elderly people to avoid illness
  and remember things more clearly
• LE Magazine August 1997

12
ENERGY

• exogenous nucleosides increased the
  intercellular concentrations of UTP, UDP-glucose,
  CDP-choline and NAD
• Arnaud A, Exogenous nucleosides alter the
  intracellular nucleotide pool in hepatic cell
  cultures. Implications in cell proliferation and
  function. Clin Nutr, 2003 Aug Vol. 22 (4), pp.
  391-9

13
ENERGY

• Nucleotide food deprivation significantly
  decreased the ATP pool
• Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A,
  (1994) Dietary nucleotides accelerate intestinal
  recovery after food deprivation in old
  rats.Symposium Nucleotides and Nutrition
  Supplement in J. Nutr. (WA Walker, ed.), vol.
  124, pp1413-1418

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BLOOD FLOW

• the difference in blood flow velocities
  between baseline and 90 min was significantly
  greater with the formula with added nucleotides
  versus the formula with no added nucleotides
  These data suggest that orally administered
  nucleotides are associated with affects on the
  intestinal vasculature.
• The higher blood flow velocity measured after
  nucleotide-supplementation may reflect dilation
  of the intestinal vascular bed.
• Carver JD et al The Effects of Dietary
  Nucleotides on Intestinal Blood Flow Pediatric
  Research 52 (3) (2002)

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BLOOD FLOW

• Nucleotides may decrease the inflammatory
  response to ischemia-reperfusion. the number
  of leukocytes accumulated was reduced in the
  presence of nucleotides. Furthermore,
  nucleotides may have decreased protein leak and
  the production of nitric oxide during ischemia.
• Bastamante, S. A., N. Sanches, J. Crosier, D.
  Miranda, G. Colombo, and M. J. S. Miller. 1994.
  Dietary nucleotideseffects on the
  gastrointestinal system in swine. J. Nutr.
  124149S-156S.

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BLOOD CELLS

• Studies on the rat everted gut have found that
  the nucleotides inosine, hypoxanthine and uric
  acid all significantly increased iron
  absorption.
• Faelli A, Esposito G. Effect of inosine and its
  metabolites on intestinal iron absorption in the
  rat. Biochem Pharmacol 1970 19 2551-4.

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DETOXIFICATION

• Mycotoxin levels in the liver of pigs and
  poultry fed with nucleotide supplemented feed
  were significantly lower than of animals fed a
  standard diet, whereas the mycotoxin levels in
  the faeces of nucleotide fed animals were higher
  than of standard fed animals.
• Koppel P, Physiological and nutritional
  functions of nucleotides. Chemoforma LTD
  Switzerland. Glasgow Veterinary School February
  (2003.)

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INFLAMMATION

• peri-operative IMPACT administration reduced
  C-reactive protein release in the IMPACT group.
• enteral formula containing nucleotides
• O'Flaherty L, Immunonutrition and surgical
  practice. Proc Nutr Soc, 1999 Nov Vol. 58 (4),
  pp. 831-7

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INFLAMMATION

• enhancement of IL-12 production is responsible
  for the augmentation of the Th1 immune response
  by dietary nucleotides feeding of dietary
  nucleotides was found to decrease the
  antigen-specific IgE response significantly.
• Nagafuchi et al, Dietary nucleotides can
  up-regulate antigen specific TH1 immune responses
  in mice International Archives of Allergy and
  Immunology 122, 33-41 (2000)

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ALLERGIC INFLAMMATION

• report that mice fed nucleotide-supplemeted
  diets had lower serum IgE levels, serum
  IgGIIgG2a ratios and IL-4
• dietary nucleotides may upregulate the Th1
  immune response in systemic immunity.
• Nagafuchi S, Katayanag T, Nakagawa E, Takahashi
  T, Yajima T, Yonekubo A, et al. Effects of
  dietary nucleotides on serum antibody and splenic
  cytokine production in mice. Nutr Res 1997 17
  1163-74

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IMMUNE SURVELLIENCE SYSTEM

• To date no side effects of nucleotides have
  been shown in clinical trials.
• Lerner A and R. Shamir. Nucleotides in Infant
  Nutrition A Must or an Option
• IMA. Vol 2, October 2000

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IMMUNE SURVELLIENCE SYSTEM

• Mice maintained on a nucleotide-free diet have
  been found to exhibit deficient macrophage
  activity.
• Kulkarni AD, Fanslow WH, Drath DB, Rudolph FB,
  Van Buren Ct Influence of dietary nucleotide
  restriction on bacterial sepsis and phagocytic
  cell function in mice. Arch Surg 1986 121
  169-172.

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HUMORAL IMMUNE SYSTEM

• Infants fed the nucleotide-supplemented formula
  had significantly higher serum antibody
  concentrations
• the nucleotide-supplemented formula was
  associated with higher antibody responses to two
  vaccines, Hib conjugate vaccine and DPT
• Pickering L, Granoff D, Erickson JR, Masor M,
  Cordle CT, Scheller JP, Winship TR, Paule CL,
  Hilty MD. Modulation of the immune system by
  human milk and infant formula containing
  nucleotides. Pediatrics 19981012429.
• Carver, JD. Dietary nucleotides effects on the
  immune and gastrointestinal systems. Acta
  Paediatrica, 09/21/99, Vol. 88 Issue 9

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HUMORAL IMMUNE SYSTEM

• using murine spleen cells showed that
  polynucleotides significantly increase in vitro
  antibody production in response to
  T-cell-dependent antigen. They seem to exert
  actions on T-helper cells at antigen
  presentation, perhaps during cognitive cell-cell
  interactions. Polynucleotides increase in vitro
  human immunoglobulin production in response to
  T-cell-dependent stimuli and antigen. Humoral
  immune responses to T-cell-dependent antigen were
  depressed in mice fed a nucleotide-free diet, but
  were restored by a mononucleotide-nucleoside
  mixture.
• Jyonouchi H, (1994) Nucleotide Actions on the
  Humoral Immune Response
• J. Nutr. 124, Suppl. January 1994 138S -143S

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HUMORAL IMMUNE SYSTEM

• higher plasma immunoglobulin M and A
  concentrations were found in preterm infants fed
  nucleotide-supplemented formula.
• Martinez-Augustin O, Boza J, Navarro J,
  Martinez-Valverde A, Araya M, Gil A. Dietary
  nucleotides may influence the humoral immunity in
  immunocompromised children Nutrition 1997 13
  465-9.

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HUMORAL IMMUNE SYSTEM

• In mice and in humans, a nucleotide free diet
  significantly depressed IgM and IgG antibody
  production from spleen cells.
• Jyonouchi, H. (1994). Nucleotide actions on
  humoral immune responses. The journal of
  Nutrition. Nucleotides and nutrition. Vol 124. No
  1S. 138S-143S.

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HUMORAL IMMUNE SYSTEM

• the specific immunoglobulin A (IgA) level
  significantly increased in mice fed a
  nucleotide-supplemented diet compared with those
  fed a nucleotide-free control diet.
• Shinya N et al Dietary nucleotides increase
  mucosal IgA response and secretion of TGF
  Cytotechnology 4049-58(2002 ).

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CELLULAR IMMUNE SYSTEM

• oral nucleotides enhanced the response to
  some T dependent protein antigens.
• Pickering L, Granoff D, Erickson JR, Masor M,
  Cordle CT, Scheller JP, Winship TR, Paule CL,
  Hilty MS. Modulation of the immune system by
  human milk and infant formula containing
  nucleotides. Pediatrics 1998 101 242-9.

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CELLULAR IMMUNE SYSTEM

• In animal models, nucleotides have been shown
  to improve delayed cutaneous hypersensitivity
  and alloantigen-induced lymphoproliferation
• Pickering L, Granoff D, Erickson JR, Masor M,
  Cordle CT, Scheller JP, Winship TR, Paule CL,
  Hilty MD. Modulation of the immune system by
  human milk and infant formula containing
  nucleotides. Pediatrics 19981012429.

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CELLULAR IMMUNE SYSTEM

• the effect of a nucleotide-free diet on the
  immune function of mouse syngeneic bone marrow
  radiation chimeras. These results suggest that
  dietary nucleotides are important for the normal
  function of mouse T-lymphocytes.
• Kulkarni, S. S., D. C. Bhately, A. R. Zander, C.
  T. Van Buren, F. B. Rudolph, K. A. Dicke, and A.
  D. Kulkarni. 1984. Functional impairment of
  T-lymphocytes in mouse radiation chimeras by a
  nucleotide-free diet. Exp. Hematol. 12694-699.

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IMMUNE SYSTEM

• Feeding nucleotide-supplemented rodents has
  been associated with
• increases in the following immune responses
• Graft-versus-host disease mortality
• Rejection of allogenic grafts
• Delayed cutaneous hypersensitivity
• Alloantigen- and mitogen-induced
  lymphoproliferation
• Reversal of malnutrition and starvation-induced
  immunosuppression
• Natural killer cell activity and macrophage
  activation
• Resistance to microbial challenge
• Macrophage phagocytic capacity
• Spleen cell production of interleukin-2 (IL-2)
  and expression of IL-2 receptors and lyt-1
  surface markers
• The number of antibody-secreting spleen cells
  produced in cluture
• Popliteal lymp-node cytokine secretion
• Peripheral blood total leukocyte counts and
  neutrophil numbers following infection.
• Carver, JD. Dietary nucleotides effects on the
  immune and gastrointestinal systems. Acta
  Paediatrica, 09/21/99, Vol. 88 Issue 9

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IMMUNE SYSTEM

• Dietary nucleotides are reported to play a role
  in the maintenance of the immune response.
• Carver, JD. Dietary nucleotides effects on the
  immune and gastrointestinal systems. Acta
  Paediatrica, 09/21/99, Vol. 88 Issue 9

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IMMUNE SYSTEM

• Spleen cells of mice maintained on a
  nucleotide-free diet produce less interleukin-2
  (IL-2) and have lower natural killer (NK) cell
  cytotoxiicity and macrophage activiation compared
  with animals fed nucleotide-supplemented diet.
  In vivo lymphoproliferative response, macrophage
  phagocytic activity, and expression of IL-2
  receptor and Lyt1 surface marker are also lower
  in animals fed nucleotide-free diet.
• Carver JD, Pimentel B, Cox WI, Barness LA.
  Dietary nucleotide effects upon immune function
  in infants. Pediatrics 19918835963.

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RNA therapy shows signs of enhancing the immune
system, improving transplanted organ viability
and even fighting off cancers.
supplemental ribonucleic acid (RNA) may help
boost our immune systems, increase energy
levels, neutralize toxins, repair cell
damage and improve skin elasticity.LE
Magazine August 1997
IMMUNE SYSTEM
35
studies suggesting potential benefits to
intestinal flora, immunity, iron absorption,
lipid metabolism and gut development.
Nucleotides in Infant Nutrition A Must or an
Option, Aaron Lerner and Raanan Shamir
IMMUNE SYSTEM
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IMMUNE HEALTH

• In human, parenteral solutions with nucleotides
  given to postoperative cancer patients improved
  immune function and infectious complications and
  length of hospital stay was reduced compared to a
  control group.
• Van Buren, C.T., A.D. Kulkarni, F.B. Rudolph.
  (1994). The role of nucleotides in adult
  nutrition. The journal of Nutrition. Nucleotides
  and nutrition. Vol 124. No 1S. 160S-164S.

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CANCER

• Because dietary nucleotides are particularly
  essential for individuals under conditions of
  stress, oral administration of dietary
  nucleotides may result in tumor regression
  through enhancement of the production of IFN-?
  and IL-12.
• Nagafuchi et al, Dietary nucleotides can
  up-regulate antigen specific TH1 immune responses
  in mice International Archives of Allergy and
  Immunology 122, 33-41 (2000)

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CANCER

• increased toxicity and tumor promotion
  observed in animals may in part be due to
  resulting imbalances in dNTP pools and
  interference with DNA replication.
• Jackson C. et al Dietary NucleotidesEffects on
  Cell Proliferation in Rats Fed Folate/Methyl-Defic
  ient Diets. Journal of Nutrition 127(5)
  834S-837S.(1997)

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INFECTION

• Anil D. Kulkarni and his colleagues fed mice a
  diet supplemented with RNA and compared them with
  a control group of mice fed a nucleotide-free
  diet. Then they injected the mice with a strain
  of powerful bacteria. Half of the mice on the
  nucleotide-free diet died. The mice that had
  taken RNA, however, were able to fight off the
  infection.
• LE Magazine August 1997

40
INFECTION

• There was an inhibition of gram-negative enteric
  bacteria in mice fed a diet supplemented with
  a nucleoside-nucleotide mixture indicating that
  the nuclotide-nucleoside mixture blocked
  bacterial translocation by preventing
  endotoxin-induced mucosal or epithelial damage.
  These results suggest that the nucleoside-nucleoti
  de mixture could be used ot inhibit or reduce the
  incidence of bacterial translocation, and
  improve survival in a lethal model of
  endotoxemia.
• Adjei AA, Yamamoto S. A dietary
  nucleoside-nucleotide mixture inhibits
  endotoxin-induced bacterial translocation in mice
  fed protein-free diet. J Nutr 1995125428.

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INFECTION

• Mice on the nucleotide-free diet suffered 100
  mortality following intravenous challenge with
  Staphylococcus aureus while RNA- or
  uracil-repleted mice demonstrated significantly
  greater resistance to this bacterial challenge.
  Macrophages from mice on the nucleotide-free diet
  had decreased phagocytic activity compared with
  mice maintained on the nucleotide-free diet
  supplemented with adenine, uracil, or RNA.
  provision of nucleotides to defined diets appears
  vital to maintain host resistance to bacterial
  challenge.
• Kulkarni AD, Fanslow WC, Drath DB, Rudolph FB and
  Van Buren CT, (1986a)
• Influence of dietary nucleotide restriction on
  bacterial sepsis and phagocytic cell function in
  mice Arch. Surg. 121 169-172

42
INFECTION

• dietary nucleotide restriction increases the
  mortality from staphylococcal sepsis. Addition
  of certain dietary nucleotides appears to
  decrease this susceptibility to bacterial
  challenge.
• Kulkarni AD et al Effect of Dietary nucleotides
  on response to bacterial infections. J Parenter
  Enteral Nutr. March 10(2)169-171 (1986)

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INFECTION

• mice maintained on a nucleotide-free diet
  exhibit a significantly decreased mean survival
  time and a significantly increased viable
  organism recovery in the spleen following
  intravenous injection of graded inocula of C.
  albicans compared to mice fed diets containing
  RNA or uracil as a nucleotide source.
• Fanslow WC, Kulkarni AD, Van Buren CT and Rudolph
  FB (1988) Effect of nucleotide restriction and
  supplementation on resistance to experimental
  murine candidiasis
• J. Parenter. Enteral Nutr. 12 49-52

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HEART

• An immune-boosting nutritional supplement can
  lower the risk of infection and death in patients
  undergoing heart surgery, researchers report.
  high-risk patients who took a supplement
  containing yeast RNA had lower rates of
  infection and organ dysfunction following
  coronary artery bypass than those who took an
  inactive placebo pill.
• Tepaske R, et al. Effect of preoperative oral
  immune-enhancing nutritional supplements on
  patients at high risk of infection after cardiac
  surgery a randomised placebo-controlled trial.
  Lancet 358 696-701(2001)

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ICU PATIENTS

• Early enteral feeding of the formula containing
  dietary nucleotides was safe and well tolerated
  in ICU patients. In patients who received the
  nucleotide formula, particularly if they were
  septic on admission to the study, a substantial
  reduction in hospital length of stay was
  observed, along with a significant reduction in
  the frequency of acquired infections.
• Bower RH, Cerra FB, Bershadsky B, Licari JJ, Hoyt
  DB, Jensen GL, Van Buren CT, Rothkopf MM, Daly
  JM, Adelsberg BR. Early enteral administration of
  a formula (Impact) supplemented with arginine,
  nucleotides, and fish oil in intensive care unit
  patients results of a multicenter, prospective,
  randomized, clinical trial.
• Crit Care Med. 1995 Mar23(3)436-49.

46
KIDNEY

• beneficial effects support use of an oral
  immune-enhancing nutritional supplement in
  patients who are undergoing surgery and are at
  high risk of infection. The study included 50
  patients who took the supplement or a placebo for
  at least 5 days before surgery. About 17 of
  patients who took the supplement developed an
  infection, compared with 55 of those who took
  the placebo. Patients who took the supplement
  also had better kidney function and remained in
  the hospital for fewer days than patients who
  were given the placebo.
• Tepaske R, et al. Effect of preoperative oral
  immune-enhancing nutritional supplements on
  patients at high risk of infection after cardiac
  surgery a randomised placebo-controlled trial.
  Lancet 358 696-701(2001)

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KIDNEY

• Oral administration of nucleotides to fish daily
  resulted in enhanced activities in kidney
  phagocytic cells. This activation of kidney
  cells was observed for at least 10 days
  post-treatment.
• Sakai, M Taniguchi, K Mamoto, K Ogawa, H
  Tabata, M. Immunostimulant effects of nucleotide
  isolated from yeast RNA on carp, Cyprinus carpio
  L. Journal of Fish Diseases, Oct2001, Vol. 24
  Issue 8, p433, 6p

48
LIPID BALANCE

• It has been reported that early childhood levels
  of serum lipids and lipoproteins are good
  predictors of lipid levels in adulthood.
• neonates fed formula supplemented with
  nucleotides from birth have significantly higher
  HDL-C and lower LDL-C serum levels than do
  neonates fed an unsupplemented formula.
• the only known difference between groups was
  the supplementation of formula with nucleotides.
  This factor was identified as one of the
  independent variables influencing the HDL-C and
  LDL-C levels in the multiple regression analysis
  models. Therefore, it can be assumes that
  dietary nucleotides have an effect on
  lipoprotein-cholesterol serum concentrations
• Siahanidou. Serum Lipids in Preterm Infants Fed
  a Formula Supplemented With Nucleotides. Journal
  of Pediatric Gastroenterology Nutrition,
  Jan2004, Vol. 38 Issue 1, p56, 5p
• Webber LS, Srinivasan SR, Wattigney WA, et al.
  Tracking of serum lipids and lipoproteins from
  childhood to adulthood. Am J Epidemiol 1991 133
  884-9

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LIPID BALANCE

• previous studies all conclude that formula
  supplementation with nucleotides influences
  circulating lipid levels
• Sanchez et al. found higher HDL and lower VLDL
  plasma levels in supplemented neonates.
• The mechanism(s) by which dietary nucleotides
  affect HDL-C and LDL-C serum concentrations
  is/are not known.
• It has been suggested that dietary nucleotides
  increase the synthesis of long-chain
  polyunsaturated fatty acids
• Siahanidou. Serum Lipids in Preterm Infants Fed
  a Formula Supplemented With Nucleotides. Journal
  of Pediatric Gastroenterology Nutrition,
  Jan2004, Vol. 38 Issue 1, p56, 5p
• Sanchez-Pozo A, Pita ML, Martinez A, et al.
  Effects of dietary nucleotides upon lipoprotein
  pattern of newborn infants. Nutr Res 1986 6
  763-71

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LIPID BALANCE

• It is well known that polyunsaturated fatty
  acids have a significant lowering effect on LDL-C
  circulating levels by increasing LDL receptor
  activity or reducing apoB secretion, and they
  also may increase the HDL lipoprotein levels by
  enhancing the apoA-1 secretion.
• The combination of high HDL-C and low LDL-C
  circulating levels is considered to be a
  protective factor against the development of
  vascular disease.
• Siahanidou. Serum Lipids in Preterm Infants Fed
  a Formula Supplemented With Nucleotides. Journal
  of Pediatric Gastroenterology Nutrition,
  Jan2004, Vol. 38 Issue 1, p56, 5p
• .
• American Academy of Pediatrics. Committee on
  Nutrition. Cholesterol in Childhood. Pediatrics
  1998 101 141-7.

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LIPID METABOLISM

• Dietary nucleotides supplementation had
  higher lecithin cholesterol acyl transferase
  (LCAT) activities and apoA-IV levels than those
  receiving the nucleotide-free formula. These
  findings indicate that nucleotide supplementation
  may improve dietary lipid tolerance by
  enhancing plasma LCAT activity, probably as a
  result of an increase in apoA-IV plasma
  concentrations
• Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J,
  van Biervliet JP, Rosseneu M. Dietary nucleotides
  enhance plasma lecithin cholesterol acyl
  transferase activity and apolipoprotein A-IV
  concentration in preterm newborn infants. Pediatr
  Res 19953732833.

52
LIPIDS

• nucleotides are involved in the synthesis of
  proteins and other macromolecules such as
  phospholipids, and thereby facilitate lipoprotein
  synthesis. . . . These findings suggest that
  dietary nucleotides may enhance the synthesis of
  lipoproteins during the early neonatal period,
  especially in preterm infants.
• Sanchez-Pozo, A., J. Morillas, I. Molto, R.
  Robles, A. Gils. (1994). Dietary nucleotides
  influence lipoprotein metabolism in newborn
  infants. Pediatr. Res. 35. 112-116.

53
STRESS

• nucleotide-supplemented groups shoed
  significantly lowered corticosterone levels,
  resulting in a decreased stress response such a
  decrease in corticosterone levels may be one of
  the mechanisms of immune restoration. Thus it
  is possible that RNA and uracil may have
  suppressed the production of this stress hormone
  and enhanced certain cytokines that resulted in
  the significant increase in immune response.
• Yamauchi K, Dietary nucleotides prevent decrease
  in cellular immunity in ground-based microgravity
  analog. J Appl Physiol, 2002 Jul Vol. 93 (1),
  pp. 161-6

54
STRESS

• under certain stress conditions and extremely
  unusual environments including space flight, the
  endogenous supply of these compounds (nucleotides
  and nucleosides) may not be adequate for optimal
  functions
• The results of our study have demonstrated that
  mice fed both CDR (RNA supplemented) and CDU
  (uracil supplemented) enhanced in vivo and ex
  vivo lymphocyte cell proliferation and IL-2 and
  INF-? production and decreased the stress level,
  indicated by corticosterone levels, compared with
  mice fed control CD (control diet).
• Yamauchi K, Dietary nucleotides prevent decrease
  in cellular immunity in ground-based microgravity
  analog. J Appl Physiol, 2002 Jul Vol. 93 (1),
  pp. 161-6

55
STRESS

• Roles for dietary sources of performed purines
  and pyrimidines such as RNA in various systems
  have been demonstrated in recent years. These
  compounds were previously thought to be
  non-essential but are now considered to be
  conditionally requires when various stresses
  are present.
• Rudolph FB. The metabolic effects of enterally
  administered ribonucleic acids. Curr Opin Clin
  Nutr Metab Care, 1998 Nov Vol. 1 (6), pp. 527-30

56
STRESS

• The effect of dietary nucleotide restriction was
  tested during stress These studies clearly
  indicate the nutritional role of nucleotides
• Pizzini R, Kumar S, Kulkarni Ad, Rudolph FB and
  Van Buren CT, (1990)
• Dietary nucleotides reverse malnutrition and
  starvation-induced immunosuppression
• Arch. Surg. 125 86-90

57
ULCERS

• We conclude that the yeast RNA accelerated
  ulcer healing, as indicated by decreased ulcer
  number and length. We postulate that the
  underlying mechanism(s) contributing to ulcer
  healing may be related, in part, to increased
  cell proliferation.
• Sukumar P, Loo A, Magur E, Nandi J, Oler A,
  Levine RA. Dietary supplementation of nucleotides
  and arginine promotes healing of small bowel
  ulcers in experimental ulcerative ileitis. Dig
  Dis Sci. 1997 Jul42(7)1530-6.

58
ULCERS

• enteral and parenteral supplementation of
  nucleotides accelerates healing of ulcers in
  rats
• Sukumar et al Dietary Nucleotides Augment Distal
  Colitis in Rats Journal of Nutrition
  1291377-1381 (1999).

59
STOMACH

• dietary nucleotides are reported to play a role
  in growth and differentation of the
  gastrointestinal tract.
• Uauy R. Dietary nucleotides and requirements in
  early life. In Lebenthal E, editor. Textbook of
  gastroenterology and nutrition. New York Raven
  Press, 1989 265-80

60
ENZYMES

• Maltase activity was significantly greater the
  largest increase, 87, was seen in the proximal
  gut mucosa. The maltase/lactase ratio was also
  higher in this segment.
• Uauy, R. G. Stringel, R. Thomas, and R. Quan.
  1990. Effect of dietary nucleotides on growth and
  maturation of the developing gut in the rat. J.
  Pediatr. Gastroenterol. Nutr. 10497-503.

61
ENZYMES

• Nucleotide food deprivation significantly
  decreased maltase, sucrase, lactase and
  aminopeptidase activities.
• Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A,
  (1994) Dietary nucleotides accelerate intestinal
  recovery after food deprivation in old
  rats.Symposium Nucleotides and Nutrition
  Supplement in J. Nutr. (WA Walker, ed.), vol.
  124, pp1413-1418

62
INTESTINAL TRACT

• investigators report decreased protein
  synthesis in the small intestine of rats fed
  nucleotide-free diets and accelerated intestinal
  recovery following food deprivation in rats fed
  nucleotide-supplemented diets. Dietary
  nucleotides may also be beneficial following
  intestinal injury.
• intestinal histology and ultrastructure were
  improved by feeding nucleotide-supplemented
  versus nucleotide-free diets to rats following
  chronic diarrhea.
• Dietary nucleotide supplementation promoted
  healing of small bowel ulcers in experimental
  ulcerative ileitis in rats
• Carver, JD. Dietary nucleotides effects on the
  immune and gastrointestinal systems. Acta
  Paediatrica, 09/21/99, Vol. 88 Issue 9

63
INTESTINAL TRACT

• Infants receiving the supplemented formula
  experienced fewer first episodes of diarrhea
• infants fed nucleotide-supplemented formula
  had a significantly lower incidence of diarrhea
  compared with infants fed unsupplemented
  formula.
• Carver, JD. Dietary nucleotides effects on the
  immune and gastrointestinal systems. Acta
  Paediatrica, 09/21/99, Vol. 88 Issue 9

64
INTESTINES

• studies have shown that rapidly dividing cells
  of human intestinal tract are dependent on
  dietary nucleotides for their replication.
• Sanderson, I.R., H. Youping. (1994). Nucleotide
  uptake and metabolism by intestinal epithelial
  cells. The journal of Nutrition. Nucleotides and
  nutrition. Vol 124. No 1S. 131S-137S.

65
DIARRHEA

• The effects of a nucleotide-supplemented formula
  on diarrhoeal disease was studied The
  supplemented group experienced less episodes of
  diarrhoea including less first episodes and for a
  lesser number of days and a larger proportion
  never developed episodes of diarrhoea. The
  mechanisms through which nucleotides decrease the
  incidence of diarrhoeal disease in infants remain
  unclear.
• Brunser O, Espinoza J, Araya M, Cruchet S, Gil A.
  Effect of dietary nucleotide supplementation on
  diarrhoeal disease in infants. Acta Paediatr
  19948318891.

66
DIARRHEA

• the incidence and duration of acute diarrhoea
  is lower in infants fed supplemented-nucleotide
  formulas.
• Gil A Modulation of the immune response mediated
  by dietary nucleotides. Eur J Clin Nutrition
  August 56(3)S1-4 (2002)

67
DIARRHEA

• results suggest that dietary nucleotides
  promote earlier restoration of the ileal
  mitochondrial function after chronic diarrhea.
• Arnaud, Journal of Pediatric Gastroenterology
  Nutrition Aug2003, Vol. 37 Issue 2, p124, 8p

68
LIVER

• aspartate aminotransferase (AST), alanine
  aminotransferase (ALT), glutamic acid- oxalacetic
  transaminase (GOT) and glutamic acid- pyruvic
  transminase (GPT) levels were significantly lower
  in animals given a nucleotide-supplemented diet
  than those given the standard nutrition,
  indicating faster recovery from liver injury.
• Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y,
  Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects
  of total parenteral nutrition with nucleotide and
  nucleoside mixture on D-galacosamine-induced
  liver injury in rats.
• J. Paranter. Enter. Nutr. 12 53-57

69
LIVER

• Parenterally administered nucleotides promote
  recovery of liver injury and improve liver
  function and nitrogen balance after liver injury
  or partial hepatectomy.
• Ogoshi, S., Iwasa, M., Kitagawa, S., Ohmori, Y.,
  Mizobuchi, S., Iwasa, Y. Tamiya, T. (1988)
  Effects of total parenteral nutrition with
  nucleosides and nucleotide mixture on
  D-galactosamine-induced live injury in rats. J.
  Parenter. Enteral Nutr. 18 62-66.

70
LIVER

• In pre-term infants nucleotide supplement may
  improve lipid tolerance by enhancing plasma
  lecithin cholesterol acyltransferase activity.
• Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J,
  van Biervliet JP, Rosseneu M. Dietary nucleotides
  enhance plasma lecithin cholesterol
  acyltransferase activity and apolipoprotein A-IV
  concentration in preterm newborn infants. Pediatr
  Res 1995 37 328-33.

71
LIVER

• The use of dietary nucleotides was found to
  decrease the percentage area of fibrous septae.
  In animals with liver cirrhosis fed the
  nucleotide-supplemented diet for two weeks, the
  total area of fibrosis was reduced. In
  conclusion, dietary nucleotides may be an
  important factor in the histological recovery of
  damaged liver in experimental cirrhosis.
• Torres M. I. Effect of Dietary Nucleotides on
  Degree of Fibrosis and Steatosis Digestive
  Diseases and Sciences , 42 (6) 1322-132.(1997) .

72
LIVER

• The serum GOT and GPT concentrations were
  significantly lower in the group supplemented
  with nucleoside-nucleotide mixture than those in
  other groups.
• the nucleoside/nucleotide mixture inhibited
  the liver injury. Thus infusion of a
  physiological and balanced mixture of nucleosides
  or nucleotides may improve liver function in rats
  with liver injury.
• Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y,
  Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects
  of total parenteral nutrition with nucleotide and
  nucleoside mixture on D-galacosamine-induced
  liver injury in rats.J. Paranter. Enter. Nutr.
  12 53-57

73
BRAIN

• The learning ability of rats fed the
  nucleotides-supplemented diet, which was
  evaluated by the water-filled multiple T-maze
  test and passive avoidance test, was superior to
  the rats fed the nucleotides-free diet. The
  results presented here suggest that dietary
  nucleotides may influence lipid metabolism of the
  cerebral cortex and contribute to the rise in
  learning ability of rats.
• Sato N, Effects of dietary nucleotides on lipid
  metabolism and learning ability of rats. Biosci
  Biotechnol Biochem, 1995 Jul Vol. 59 (7), pp.
  1267-71

74
FATTY ACIDS

• dietary nucleotides play a role in the in vivo
  desaturation and elongation of essential fatty
  acids to long chain PUFA during early life for
  the human newborn.
• DeLucchi C, Pita ML, Faus MJ, Molina JA, Uauy R,
  Gil A. Effects of dietary nucleotides on the
  fatty acid composition of erythrocyte membrane
  lipids in term infants. J Pediatr Gastroenterol
  Nutr 1987656874.

75
FATTY ACIDS

• Plasma polyunsaturated fatty acids with more
  than 18 carbons of the omega 6 family were
  significantly increased These results suggest
  that dietary nucleotides may be involved in the
  conversion of linoleic acid to longer chain
  polyunsaturated fatty acids during early life.
• Gil A, Pita M, Martinez J, Molina JA and Sanchez
  Medina F, (1985)
• Effect of dietary nucleotides on the plasma fatty
  acids in at-term neonates.
• Hum. Nutr. Clin. Nutr. 40 185-195

76
PROTEIN SYNTHESIS

• These findings suggest that the
  nucleoside-nucleotide mixture was used for the
  syntheses of RNA and DNA through the salvage
  pathway and that this resulted in enhanced
  protein turnover with simultaneous increase in
  protein synthesis.
• Ogoshi S, Iwasa M, Yonezawa T and Tamiya T,
  (1985) Effect of nucleotide and nucleoside
  mixture on rats given total parenteral nutrition
  after 70 hepatectomy.
• J. Paranter. Enter. Nutr. 9 339-342

77
APOPTOSIS

• in vitro modulation of cell proliferation and
  apoptosis has been described by ribonucleosides,
  in particular by modified components using human
  cell culture models.
• Schlimme, E., D. Martin, and H. Meisel. 2000.
  Nucleosides and nucleotides natural bioactive
  substances in milk and colostrums. Br. J. Nutr.
  84S59

78
CYTOKINES

• Dietary nucleotides have been shown to enhance
  the production and the genetic expression of IL-6
  and IL-8 Dietary nucleotides may affect the
  levels of intestinal cytokines.
• Gil A Modulation of the immune response mediated
  by dietary nucleotides. Eur J Clin Nutrition
  August 56(3)S1-4 (2002)

79
NERVES

• Guanine nucleotides regulate the affinity of
  melatonin receptors The effect of guanine
  nucleotides and related analogues on the binding
  of melatonin to membranes was studied. These
  results are consistent with a melatonin receptor
  existing in an equilibrium between high- and
  low-affinity states, with GTP and related
  analogues able to cause a shift in the
  equilibrium
• Morgan P.J.et al . Guanine nucleotides regulate
  the affinity of melatonin receptors on the ovine
  pars tuberalis. Neuroendocrinology, 50, 359362.
  (1989)

80
SKIN

• topical application of RNA improved cell
  energy metabolism and therefore the health and
  appearance of the skin.
• Dietary nucleotides affect tissue repair.
• LE Magazine January 2003
• Ramírez M, Effect of dietary nucleotides and
  orotate on the blood levels of prostacyclin
  (PGI2) and thromboxane (TXA2) in the weanling
  rat. Prostaglandins Leukot Essent Fatty Acids,
  1991 May Vol. 43 (1), pp. 49-54

81
MICROBES

• In animal models, nucleotides have been shown
  to enhance T-cell maturation and function.
• Pickering L, Granoff D, Erickson JR, Masor M,
  Cordle CT, Scheller JP, Winship TR, Paule CL,
  Hilty MD. Modulation of the immune system by
  human milk and infant formula containing
  nucleotides. Pediatrics 19981012429.

82
MICROBES

• One possible anti-infective mode of action of
  nucleotide may be that dietary nucleotide can
  inhibit endotoxin-induced bacterial translocation
  in protein-malnourished mice.
• Adjei AA, Yamamoto S. A dietary
  nucleoside-nucleotide mixture inhibits
  endotoxin-induced bacterial translocation in mice
  fed protein-free diet. J Nutr 1995 125 42-8.

83
WEIGHT LOSS

• Dietary RNA is required to restore lost immune
  function after protein deprivation. Adequate
  calories and protein alone do not return immune
  function to normal. Dietary nucleotides can
  restore lost immune function even during protein
  starvation and weight loss.
• Van Buren C.T., Kulkarni A.D.. Rudolph F.B., The
  Role of Dietary Nucleotides in Adult Nutrition,
  The Journal of Nutrition, January
  124124S-160S.(1994)

84
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86
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
RNAi What's All the Noise About Gene
Silencing? Posted 04/02/2004 Ernie
Hood Introduction It all began with petunias. In
the late 1980s, geneticist Richard Jorgensen,
then working at a California plant biotechnology
company, attempted to deepen the hue of purple
petunias by introducing more of the gene that
gives them their color, in the form of
double-stranded RNA (dsRNA). Instead, some of the
engineered flowers became variegated and others
turned white, indicating that expression of both
the introduced pigmentation gene and its
homologous endogenous gene had been knocked down
or knocked out altogether. Jorgensen had
serendipitously discovered an age-old natural
biologic process now recognized to be
evolutionarily conserved in most, if not all,
forms of life. Today, gene silencingor RNA
interference (RNAi), as it is now knownhas
revolutionized genetics and is on the verge of
spawning an entirelynew class of drugs to treat
human diseases with a genetic component. The
ability to selectively silence genes is one of
the hottest topics in biology today. Science
crowned RNAi as its "Breakthrough of the Year" in
2002. Nobel laureate and RNAi pioneer Phillip
Sharp, who is Salvador E. Luria Professor of
Biology and director of the McGovern Institute
for Brain Research at the Massachusetts Institute
of Technology (MIT), calls it "the most exciting
discovery in the last decade," adding that
"there's not an area of biological science this
will not touch." John Maraganore, who is
president, CEO, and director of Alnylam
Pharmaceuticals, touts RNAi as "presenting
perhaps the broadest new class of therapeutics
since recombinant proteins and monoclonal
antibodies." Can RNAi live up to the hype? That
remains to be seen, of course, but academic and
industrial researchers are optimistic that it can
and will, if the significant remaining barriers
to its progress can be overcome. Given the rapid
pace of discovery in the field, such optimism may
well be justified.
87
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNA Redefined It once seemed so simple, so
straightforward basically, DNA makes messenger
RNA (mRNA) mRNA makes proteins. But the
discoveries associated with RNAi have shown that
the real story is far more complex. RNA has been
unveiled as the "man behind the curtain" in the
cell, wielding previously unimagined control over
and influence upon cellular processes (including
gene expression and regulation) and organism
development. RNAi has been revealed to be an
ancient mechanism protecting cells from invading
viruses and from damage by transposable genetic
elements, performing a variety of cellular
housekeeping functions essential to survival,
health, and development. RNAi was first
described and so named by molecular biologists
Andrew Fire of the Carnegie Institute of
Washington and Craig Mello of the University of
Massachusetts, along with their colleagues, in a
landmark 19 February 1998 Nature paper that
electrified the biology community. The team found
that administering tiny amounts of dsRNA to
Caenorhabditis elegans resulted in potent
sequence-specific gene silencing. Tantalized by
the possibility of acquiring a powerful new tool
for genetic manipulation and analysis,
investigators around the world began
investigating RNAi. The flood of significant
discoveries that followed soon established the
basic outlines of the mechanisms involved in
RNAi. Researchers using Drosophila found in 2000
that long-strand dsRNA was processed in cells
into 21- to 23-nucleotide snippets of RNA, which
then cleaved to precisely matching homologous
mRNA sequences, degrading the mRNA and
effectively silencing the corresponding gene by
blocking its ability to encode for proteins. The
higher life forms, such as mammals, while
conserving this ability, use it in different
ways the response to dsRNA is more complicated,
triggering a cellular immune response involving
the release of interferon that ultimately kills
the cell.
88
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
89
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNA Redefined Then, in 2001, Thomas
Tuschl, then of the Max Planck Institute for
Biophysical Chemistry in Göttingen, Germany,
discovered with his colleagues that RNAi could be
prompted through the use of shorter pieces of RNA
known as small interfering RNAs (siRNAs). Soon
thereafter, they showed that duplexes of
21-nucleotide siRNAs mediated RNAi in cultured
mammalian cells and demonstrated that siRNAs
could be designed to silence specific genes
without activating the interferon response. In
other words, scientists could potentially silence
any gene of interest in a highly predictable,
reproducible, and accurate fashion. Research
scientist Gregory Hannon and his colleagues at
New York's Cold Spring Harbor Laboratory
contributed several key discoveries during the
same period. They identified, described, and
named the "Dicer" enzyme, which chops dsRNA into
siRNAs, as well as the RNA-induced silencing
complex (RISC), which mediates the silencing
process by degrading the homologous mRNA. In
2002, they described the use in mammalian cells
of so-called short hairpin RNAs (shRNAs), which
generate endogenous siRNAs within cells and thus
provide stable, heritable gene silencing (in
contrast, administered siRNAs are transient in
their silencing effect). They whimsically named
this effect "short hairpin-activated gene
silencing," or SHAGging. This discovery allowed
the development of cell lines and animal models
with permanently silenced genesa major step
forward for basic science in general, and
especially for functional genomics. Further
advances in the past few years have added the
ability to silence the expression of just the
mutant copy of a gene, leaving the normal copy
intact, as well as to modulate the level of
silencing in order to produce a range of
phenotypes. Plus, researchers can now induce
silencing in a controlled manner and target
multiple genes for silencing. These discoveries
alone are quite importantall of these
capabilities are crucial in a variety of critical
applications. But some proponents believe this is
only the beginning, and the best may be yet to
come.
90
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Knock Down Genes, Drag Out Knowledge With
the continual refinement and improvement of
techniques to silence genes with exquisite
specificity, RNAi has already had a major impact
on molecular biology. For example, the pace of
discovery in functional genomics has accelerated
as a consequence of researchers' enhanced ability
to practice reverse genetics, in which a gene's
function can be inferred by silencing its
expression. With complete sequences of several
genomes now on hand, including those of C.
elegans, Drosophila, the mouse, and the human,
investigators can now quickly, easily, reliably,
and relatively inexpensively use siRNAs to
silence genes of interest and determine their
functions. Several companies are already selling
made-to-order siRNAs for use in functional
genomics work, as well as for drug target
identification and validation. The ability to
knock down genes either stably (that is, creating
heritable phenotypes through germline
transmission of permanently silenced genes) or
transiently (as opposed to knocking them out
altogether) has some important advantages in the
production of animal models and in vitro cell
lines. When a gene is considered silenced by
RNAi, expression is typically reduced by 70 or
more. This allows the method to be used in
so-called essential genes, which cannot be
knocked out in animal models without killing the
animal. Also, "turning down" a gene by a certain
amount can sometimes more closely resemble a
disease state, allowing the fashioning of more
useful, refined models of some diseases. Russell
Thomas, director of the functional genomics
research program at the CIIT Centers for Health
Research in Research Triangle Park, North
Carolina, points out another advance that RNAi
has brought to animal studies "When you knock
out a gene in mice, you have to live with the
consequences," he says. "The targeted gene is
knocked out for the remainder of the animal's
life span. In contrast, shRNAs with inducible
promoters allow an investigator to control the
timing for the knockdown of the targeted gene.
With this technology, you don't have residual
developmental effects, and you can have more
sophisticated experimental designs since you can
look at wild-type expression and knockdown in the
same animal and at multiple times throughout the
animal's life span."
91
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Knock Down Genes, Drag Out Knowledge Sharp
observes that RNAi will in some cases obviate the
need for animal knockouts. "You can do gene
knockouts in mice, and then relate phenotype to
somatic cells in the human, but it's very
expensive, the gene has to be nonessential to get
an interesting phenotype, and you have to ask the
question in the context of the developing mouse,"
he says. By comparison, he explains, RNAi allows
researchers to inactivate the gene and observe in
real time the changes in the metabolic, cell
biologic, or other phenotype of the cell, and
characterize the role of the gene in that
particular situation. Although these applications
of RNAi are not yet perfected and are unlikely to
completely replace classic knockout studies, many
scientists are excited about adding these new
capabilities and efficiencies to their bag of
laboratory tricks. "It's fundamentally changing
how we do laboratory science," says Sharp. "It
will change how we do animal genetics, and we
have not even scratched the surface of all the
ways it will be used. RNAi and the Big
Picture The ability to reproducibly and robustly
silence every single gene in the genome is
expected to facilitate the acquisition of
profound new knowledge regarding function and
regulation at the cell and whole-organism levels.
Several organizations are in the process of
constructing large-scale RNAi libraries that
should be available for use very soon.
Genomewide screening using RNAi libraries will
help researchers learn more about global
questions in systems biology, elucidating the
nature and role of the complex, often
interrelated pathways and signaling networks at
work in organisms.
92
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. RNAi and the Big Picture Leona Samson,
director of the MIT Center for Environmental
Health Sciences, has already used such a library
for the 4,800 nonessential genes in Saccharomyces
cerevisiae. She says that higher-organism RNAi
construct libraries will aid toxicogenomics
research by allowing researchers to interrogate
each and every gene of an organismto examine a
specific gene to determine its function, or to
screen large amounts of genes in the context of a
specific function, to see which ones contribute
to the function, and possibly what roles they
might play and how they might interact. As a
result, Samson says, "you can start to identify
the important pathways for helping cells recover
from toxic insults. Samson says there are
important toxicogenomics treatment and prevention
end points to be achieved with this type of
large-scale screening. By interrogating every
gene, it's possible to compile a portrait of all
the pathways that are relevant to cellular
function and response, such that researchers
could look at which genes are being expressed in
members of a population and accurately predict
the effects of exposure to particular
environmental agents. "In the end," Samson says,
"we're going to get an integrated systems view,
and we need that to be able to make
predictions. There's More to the Machinery It
turns out that gene silencing through degradation
of mRNA by siRNAs is not the only cellular
mechanism regulated by small pieces of RNA.
Thanks to groundbreaking work in C. elegans,
researchers have discovered a class of natural
small RNA molecules called microRNAs that appear
to be crucial in regulating development. Although
they apparently use the same tools as siRNAs to
carry out their functionsDicer, RISC, and a
family of proteins known as ArgonautesmicroRNAs
differ in that their sequences do not precisely
match their mRNA targets. As such, they regulate
the expression of proteins by those targets,
rather than degrade them altogether, which leads
investigators to believe that they play an
important role in the timing and nature of
developmentperhaps to the point of controlling
differentiation in embryonic stem cells.
93
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. There's More to the Machinery Tuschl, who
is now group leader of the Laboratory of RNA
Molecular Biology at Rockefeller University,
says, "It's a very complicated regulatory
machinery that you have in your cells, with a
very complicated biology behind it. We know that
there are genes that express dsRNA and microRNAs,
and that this gene family in humans is about 250
genes, and many of these genes are conserved. . .
. The question is what all these genes are doing,
and how the RNAi machinery ties in to the gene
regulation mediated by the microRNA
genes." Recent studies further suggest there is
yet a third mechanism controlled by microRNAs, an
arm of the silencing machinery in the nucleus of
the cell that modifies heterochromatin. The
result is transcriptional repression of gene
expression. As Tuschl puts it, "This is one of
the real highlights of the discovery of RNAithat
it's a new cellular mechanism involved in
regulating gene expression, and it's as
complicated and as effective as transcriptional
gene regulation." More thorough understanding of
this mechanism could eventually lead to
beneficial insights into development and disease
processes, particularly carcinogenesis. Silencing
Disease Although biologists are excited about
the long-range additions to knowledge that could
emerge from studying the complexities of the RNAi
machinery, many are focusing major efforts on
exploiting what is already knownthat the
gene-silencing effect of RNAi holds tremendous
promise in treating human disease. RNAi
therapeutics will be judged on the same criteria
as any other prospective drug potency,
stability, and safety. Despite the great deal of
work yet to be done, many researchers believe
that RNAi-based agents will eventually pass
muster on those issues, and will actually have
inherent advantages over presently available
classes of drugs.
94
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. Silencing Disease For example, the fact
that RNAi is a natural cellular process may mean
that drugs based upon the phenomenon can be
expected to be quite efficacious. "The siRNA
process depends upon the endogenous pathway of
microRNA biology, a process that's present in all
cells," says Sharp. "Therefore, it's efficient,
it's a normal biological process, and we are
learning how to design siRNAs that are more
efficiently taken up in that process and more
efficiently used in silencing. Maraganore
agrees. "What's very unique about RNAi is that
it's the first natural mechanism that's ever been
discovered that would allow people to silence
genes," he says. "Because of that leveraging of
that natural mechanism, you have a high degree of
specificity and potency in the action of
siRNAs." According to Nassim Usman, chief
operating officer and senior vice president of
Sirna Therapeutics in Boulder, Colorado, there is
ample precedent for this opinion. "If you look at
the history of successful biotechnology drugs, a
lot of them that have been successful are the
ones that either use or replace a naturally
occurring mechanism," he says. "The two clearest
examples are recombinant proteins and
antibodies. The potential therapeutic value of
RNAi has been repeatedly demonstrated in a wide
variety of in vitro studies, and more and more in
vivo experiments are confirming that early
promise. Efficacy in gene silencing has been
shown in viral diseases (such as HIV/AIDS,
influenza, human papillomavirus infection,
various hepatitis strains, smallpox, and SARS),
neurodegenerative diseases (such as Parkinson
disease, amyotrophic lateral sclerosis, and
Alzheimer disease), cancer, inflammatory diseases
(such as rheumatoid arthritis), and autoimmune
diseases (such as type 1 diabetes mellitus). The
intense interest in RNAi therapeutics has come at
least in part because of its potential broad
applicability across such a wide spectrum of
disorders. Researchers also cite the specificity
of RNAi targeting siRNAs may be able to
effectively reach cellular targets that have
previously been inaccessible or highly resistant
to other forms of therapy. Success in reaching
such targets could lead to significant advances
in the treatment of several diseases with
currently unmet medical needs.
95
From Environmental Health Perspectives
112(4)A225-A229, 2004. National Institute of
Environmental Health Sciences
Cont. The Principle of the Thing Researchers in
academic and industrial labs around the world are
pursuing therapeutic applications of RNAi. One
good illustration is the work conducted by senior
investigator and associate pediatrics professor
Judy Lieberman and her group at the Institute for
Biomedical Research of the Harvard Medical School
Center for Blood Research. In 2003, the team
published two RNAi studies of landmark
importance. In one, an in vitro experiment
presented in the Journal of Virology in July 2003
(issue 13), the team achieved sustained
siRNA-mediated silencing of HIV-1 in primary
macrophages, in essence preventing infection from
taking hold. In previous studies conducted in
rapidly dividing cells, such as tumors, the RNAi
effect lasted only 3-5 days. In these
macrophages, however, as Lieberman explains, "the
silencing lasted very long, in fact for as long
as we could keep the cultures growing, for some
genes." The experiment co-targeted the viral p24
structural gene and CCR5, the major HIV-1
co-receptor in macrophages, cells that are known
to be reservoirs of HIV infection and that are
stubbornly resistant to current antiviral
therapies. The team found that they could
completely suppress HIV replication in
macrophages by using this co-targeting stratagem.
They also found that they could inhibit viral
replication in cells that were already infected,
in which HIV was integrated into the host cell.
As Lieberman summarized, those are "pretty
encouraging data for the possibility of using
siRNA against HIV." The experiment also sh