Probing Glycoproteins

1
Probing Glycoproteins
Strategy for glycoprotein labeling
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Most of eukaryotic proteins are glycoproteins,
and it is acknowledged that glycosylation process
plays a central role in mediating protein
function in living organisms. It is also known
that altered glycosylation is often associated
with inflammation and cancer metastasis.
Therefore, the detailed correlations between
glycosylation and biological or pathological
statuses are of great interest and may provide
information for disease diagnosis and treatment.
For this purpose, new chemical tools to probe
glycoproteins are developed by the research team
led by Dr. Chi-Huey Wong in GRC. Sugar analogs
are used to feed cells for incorporating into
cellular glycoproteins by glycosyltransferases.
With the chemical functionality on sugar analogs,
the labeled glycoproteins can thus be
specifically tagged on fluorogenic/fluorescent or
affinity probes for imaging or purification. The
studies were published in Proc. Natl. Acad. Sci.
USA in 2006 and 2007. This work was selected and
reported as a Research Highlight by Nature
Functional Glycomics Gateway-- this method adds a
powerful technique to the glycosylation and
glycoprotein analysis repertoire.
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Identification of pulmonary stem cells and their
susceptibility to SARS-CoV infection
----- Stem Cell Program
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The team had grown lung cells in vitro and
reported a serum-free culture system for primary
neonatal pulmonary cells that can support the
growth of Oct-4 epithelial colonies with a
surrounding mesenchymal stroma. In addition to
Oct-4, these cells also expressed other stem cell
markers such as SSEA-1, Sca-1 and Clara cell
secretion protein but not c-Kit, CD34 and p63,
indicating they represent a sub-population of
Clara cells which had been implicated as lung
stem/progenitor cells in lung injury models.
Lastly these Oct-4 epithelial colony cells were
shown to be the target cells for SARS-CoV
infection in primary cultures and also to support
active virus replication leading to their own
destruction. The study has implicated a possible
involvement of lung stem cells in SARS-CoV
infection, accounting for the continued
deterioration of lung tissues and apparent loss
of capacity for lung repair in the later stage of
infection. This discovery has opened a door for
future manipulation of lung stem cells in
cellular therapy of ARDS.
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Pioneering Mass Spectrometry Technology
One for All" Mass Spectrometer to detect masses
from the size of an atom to cancer cells
Dual-polarity MALDI-TOF Mass Spectrometer
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Dr. Yi-Sheng Wang and Dr. Chung-Hsuan Chen in the
Division of Key Technology Development in
Genomics Research Center (GRC) recently developed
a novel Dual-Polarity Matrix-assisted Laser
Desorption/Ionization Mass Spectrometer which can
be used to simultaneously measure both positive
and negative ions from a biomedical sample. Since
some biomolecules tend to form positive ions and
some to become negative ions, all present
commercial mass spectrometer cannot get a
complete biomolecular imaging. With this
invention, imaging of many more different
biomolecules can possibly be achieved. This work
has been published in a highly regarded journal.
(S. T. Tsai, C. W. Chen, L. C.L. Huang, M. C.
Huang, C. H. Chen and Y. S. Wang, Simultaneous
mass analysis of positive and negative ions using
a dual-polarity time-of-flight mass
spectrometer, Anal. Chem. 78, 7729-7734 (2006))
The patent application is in process. Dr.
Chung-Hsuan Chen at GRC, Dr. Huan-Chang Chang at
Institute of Atomic and Molecular Sciences (IAMS)
and their research team recently invented a
Charge-Monitoring Laser-induced Acoustic
Desorption Mass Spectrometer (LIAD-MS). This mass
spectrometer can be used to rapidly measure the
masses and mass distribution of cells and
microparticles. This instrument can measure
several thousand cells within one hour. It can be
further improved to measure mass range from atom
to cell in the future. This work has been
published in one of the top scientific journals.
(P. Peng, H. C. Lin, M. L. Chu, L.T. Yu, H. C.
Chang, and C. H. Chen Charge Monitoring
Laser-induced Acoustic Mass Spectrometry for
Microparticle Mass Distribution Measurement
Angew. Chem. Inter. 46, 3865-3869 (2007)) Patent
application is also in process.
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Human-specific (HS) insertions deletions
(indels)

• We found that HS indels located in CDSs are
  enriched in genes related to virus infection ?
  providing clues to studies of Homo-Pan difference
  in disease susceptibility including malaria,
  AIDS, hepatitis B/C, and so on.
• A web server, INDELSCAN (http//140.109.55.72/Inde
  lScan/), for identification of HS/non-HS indels
  is also provided.

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It has been suggested that insertions and
deletions (indels) have contributed to the
sequence divergence between the human and
chimpanzee genomes more than do nucleotide
changes (3 vs. 1.2). Because the chimpanzee
genome draft is not sufficiently accurate, we
inferred only human-specific indels using
multiple sequence alignments of mammalian
genomes. Here we identified more than 840,000
indels, which affect more than 7,000
UCSC-annotated human genes (gt11,000 transcripts).
These indels, however, amount to only 0.21
sequence change in the human lineage for the
regions compared, whereas in pseudogenes indels
contribute to a sequence divergence of 1.40,
suggesting that most of the indels that occurred
in genic regions have been eliminated. Functional
analysis reveals that the genes whose coding
exons have been affected by human-specific indels
are enriched in transcription and translation
regulatory activities and virus infection, but
are underrepresented in catalytic and transporter
activities, cellular and physiological processes,
and extracellular region/matrix. This functional
bias suggests that human-specific indels might
have contributed to human unique traits by
causing changes at the RNA and protein level and
Homo-Pan difference in disease susceptibility
(e.g., malaria, AIDS, and hepatitis B/C).
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High throughput combinatroy synthesis of
compounds in microplates for inhibitor screening
in situ
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During 2003 to 2004, SARS has caused the global
crisis and over 800 people to die. At the same
time, because there is no effective medicine and
this situation made people fear deeper. The team
of 16 Taiwaness researchers led by Dr. Wong, the
president of Academia Sinica, has successfully
identified 15 compounds that are potent against
the SARS by screening over 10000 compounds. This
result has been published in PNAS in 2004 and the
notable science magazine Nature Review,
Microbiology has quoted this achievement on the
September 2004.In order to develop the effective
medicine, scientist has already found out in SARS
virus itself, there is a protease named as 3CLpro
which plays a major role in the viral
replication, and thus become an attractive target
for the development of SARS therapeutics. Wongs
team has been developing a strategy named
Combinatorial reaction in microtiter plates
followed by screening in situ. By using this
method, they screened over 5000 compounds and
found the most potent anti-SARS 3CLpro agent in
the world.