Mapping, Sequencing and Analysis of Legionella pneumophila Genome
Xiaoyan Qu, Gil
Segal, Sergey Kalachikov, Anthi Georghiou, Minchen Chien, Hye Y. Park, Baohui
Zhao, Jing Chen, Pieter J. de Jong, Stuart G. Fischer, Peisen Zhang, Eftihia
Cayanis, Howard A. Shuman, and James J. Russo
Genome Center and Department of Microbiology, Columbia
University, New York, NY.
Legionella
pneumophila, the causative agent of Legionnaires’ disease, is estimated to
have a genome size of ~4 Mb, and, like rickettsia, chlamydia and mycobacteria,
has an intracellular existence in eukaryotic cells. We have initiated a project to sequence the Philadelphia-1 strain
of this bacterium by a combined 3x whole genome shotgun/5x clone-based shotgun
approach. This hybrid strategy
emphasizes the advantages of both approaches, and should permit early release
of complete operons as each BAC shotgun proceeds. Since the whole genome shotgun should be completed significantly
in advance of the later clone shotguns, there will also be reasonably good
sequence selection throughout the genome at an early stage.
A nearly complete high redundancy
BAC map was constructed by hybridizing an arrayed BAC library with random
probes obtained from an initial set of whole genome sequences and known
Legionella gene sequences. Standard
approaches (ORF analysis/BLAST searches) and more advanced comparative
phylogenetic strategies are being used to suggest roles for the various genes
of the organism. Based on the initial
sequencing, gene density and number of orthologs is expected to be similar to
those found in other bacteria.
Moreover, as genes expected to play
important roles in pathogenesis or the organism’s lifestyle are identified, an
allelic exchange strategy is used to delete them in Legionella. In an initial study, a complete set of
presumed virulence genes (Agrobacterium
vir homologs) was removed and surprisingly had no effect on intracellular
growth or survival, although they did reduce conjugative DNA uptake. Future work will include microarray-based
transcriptional assays in various strains and during different growth phases.
Supported by NIH grant AI23549 to
HAS and Columbia Genome Center funds.
Dendritic Cells Transduced with an Adenoviral Vector Encoding a Model Tumor
Antigen Induce Long-term Antigen-specific Therapeutic Antitumor Immunity in
Tumor-bearing Hosts
Wenru Song1, HL Kong1, H
Carpenter1, Y. Tong1, S Rafii2, R Granstein3,
MAS Moore4 and RG Crystal1.
1Division of Pulmonary and Critical Care
Medicine, 2Division of Hematology/Oncology, 3Department
of Dermatology, The New York Hospital-Cornell Medical Center; 4Memorial
Sloan-Kettering Cancer Center, New York.
Immunization of dendritic cells
(DC) genetically modified with an adenovirus vector (Ad) expressing b-galactosidase (DC-Adbgal) confers protein against lethal IV challenge of CT26.CL25,
a Balb/c syngenic colon carcinoma line expressing bgal as a model antigen, as well as suppressing established
CT26.CL25 lung tumors (Song et al, J Exp Med 1997, 186:1247). To evaluate the
extent of immune memory in these mice, we have designed studies to challenge
mice immunized with DC-Adbgal. The data
showed that: (1) immunization of mice with a single SQ injection of DC-Adbgal effectively protected against a lethal
SQ burden of CT26.CL25, but not against a non-bgal-expressing
tumor (CT26.WT), challenged long after 240 days post DC immunization; (2)
immunization of mice with DC-Adbgal
effectively protected against a lethal IV CT26.CL25 challenge (with >50%
mice surviving >300 days post tumor challenge) and protected from a second
SQ tumor challenge by CT26.CL25 but not by CT26.WT; (3) mice with
preestablished lung tumors treated with DC-Adbgal
lived significantly longer than control mice, with some mice surviving >300
days after DC-Adbgal immunization, and
the long-term survival mice protected from a second SQ tumor challenge by
CT26.CL25 but not by CT26.WT; and (4) in adoptive CTL transfer studies, antigen
(bgal)-specific CTL protected mice
against IV or SQ tumor challenge. Interestingly, although DC-Adbgal-based tumor vaccination was effective in
most mice, some mice initially protected from a lethal SQ bgal-expressing tumor challenge for >80
days eventually succumbed to regrowth of tumors that were shown to be bgal negative, suggesting that either the
antigen was lost, or that clonal populations developed. We conclude that
genetically modified DC expressing a model tumor antigen induced long-term,
antigen-specific antitumor immunity in both naïve and tumor-bearing mice, although
long term escape from immunity is possible.
Tests of Reliability of a Chinese
Version of Quality of Life Index in New York City
Juan E.Mezzich, M.D.,
Ph.D., Jason S. Liu, M.D., Maria A.
Ruiperez, Ph.D., Gihyun Yoon, M.D., Limeng Wang, M.D., Saeed Iqbal, M.D.
Mount Sinai School of
Medicine, New York University
The
Quality of Life Index(QLI), designed by Drs. Mezzich and Cohen, is a concise
instrument for comprehensive, culture-informed and self-related assessment of
health-related quality of life. It is composed of the following ten items
covering corresponding dimension of the concept quality life, collated from the
international literature, including physical well-being,
psychological-emotional well-being, self-care and independent functioning, occupational
functioning, interpersonal functioning, social-emotional support, community and
services support, personal fulfillment, spiritual fulfillment, and global
perception of quality of life. Each item is rated by the subject according to
his/her culture-informed understanding of that concept. Each rating is made by
marking on a 10-point line, from poor to excellent.
By using a Chinese version of Quality of Life
Index, this study attempts to assess the new model of quality of life, and its
validation concerning feasibility, internal consistency, factorial structural,
reliability, and discriminant validity in Chinese population in New York city.
Time of completion has tended to oscillate between 1 and 4 minutes. The vast
majority of respondents have judged the instrument to be easy to use.
Test-retest reliability correlation coefficient of the QLI mean score was 0.82.
The discriminant validity of the QLI was documented by highly significant
differences obtained between mean scores of two sample of individual, health
professionals and psychiatric patients who were presumed to different levels of
quality of life.
Regulation of the Activity of EKLF by Histone Acetyltransferases
Wenjun Zhang, James J. Bieker
Brookdale Center for Molecular Biology, Box 1020, One
Gustave L. Levy Place, New York, NY 10029
Erythroid
Kruppel Like Factor (EKLF) is a red cell specific transcriptional factor that
is crucial for consolidating the switch to high levels of adult beta globin
expression during erythroid ontogeny. Genetic disruption of EKLF causes an
embryonic lethality due to lack of onset of beta globin expression. EKLF
interacts with histone acetyltransferase CBP and p300 in vivo. Furthermore, CBP
and p300 can acetylate EKLF and stimulate its transactivation. We report
here that EKLF is acetylated at lys 288 and 302 by CBP. In vivo labeling
experiments show that EKLF is acetylated in the erythroid cells. Moreover, both
in vitro and in vivo data demonstrate that acetylation of EKLF doesn't affect
its DNA binding activity. However, an EKLF acetylation site mutant (K288R)
shows a 50% drop in its transactivation, this mutant can not be stimulated by
CBP or p300. In addition, CBP and p300 HAT domain mutants have little effect on
EKLF transactivation, suggesting that the transactivation stimulation by CBP or
p300 is closely related to acetylation. These results establish EKLF as a
tissue specific transcription factor that undergoes post-translational
acetyaltion and suggest a mechanism by which EKLF is able to alter chromatin
structure and induce beta globin expression.
SLAP, a Dimeric Adapter Protein, Plays a Functional Role in T Cell Receptor
Signaling
Jie Tang, Sawasdikosol S, Chang JH, Burakoff SJ
Department of Pediatric Oncology, Dana-Farber Cancer
Institute and Department of Pediatrics, Harvard Medical School, 44 Binney
Street, Boston, MA 02115, USA.
Engagement of
the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine
kinases, which in turn phosphorylate downstream enzymes and adapter proteins.
Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate
TCR-mediated signal transduction, whereas others, such as Cbl, play an
inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing
a Src homology 3 and a Src homology 2 domain, was isolated from a yeast
interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts
with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent
manner. We observed that SLAP is expressed in T cells, and upon TCR activation,
SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In
transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70,
Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated
SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We
have evidence that SLAP forms homodimers through its C-terminal region. Serial
truncations and mutations in the C terminus of SLAP demonstrate that there is a
correlation between the loss of dimerization and the inhibition of nuclear
factor of activated T cells-AP1 activity. The in vivo association of SLAP with
key signaling molecules and its inhibition of T cell activation suggests that
SLAP plays an important role in TCR-mediated signal transduction.
Proteolytic Release and Nuclear Translocation of Notch-1 are Induced by
Presenilin-1 and Impaired by Pathogenic Presenilin-1 Mutations
Weihong Song*, Philip Nadeau*, Menglan Yuan*, Xudong
YangÆ,
Jie ShenÆ
and Bruce A. Yankner*
*Department of Neurology, Harvard Medical School and
Division of Neuroscience, The Children's Hospital, 300 Longwood Avenue, Boston,
MA 02115. ÆCenter
for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA 02115
The Notch
family of proteins are transmembrane receptors that play a critical role in the
determination of cell fate. Genetic
studies in C. elegans suggest that the presenilin proteins, which are
associated with familial Alzheimer's disease, regulate Notch signaling. Here we show that proteolytic release of the
Notch-1 intracellular domain (NICD), an essential step in the activation of
Notch signaling, is markedly reduced in PS1-deficient cells and is restored by
PS1 expression. Nuclear translocation
of the NICD is also markedly reduced in PS1-deficient cells, resulting in
reduced transcriptional activation.
Mutations in PS1 that are associated with familial Alzheimer's disease
impair the ability of PS1 to induce proteolytic release of the NICD and nuclear
translocation of the cleaved protein.
These results suggest that PS1 plays a central role in the proteolytic
activation of the Notch-1 signaling pathway, and that this function is impaired
by pathogenic PS1 mutations. Thus,
dysregulation of proteolytic function may underlie the mechanism by which
presenilin mutations cause Alzheimer's disease.
Role of Transcriptional Repression and Histone Deacethylation in APL
Pathogenesis: A Rationale for Transcription Therapy.
L.Z. He, F. Guidez,* R.P.
Warrell,Jr., A. Zelent, and P.P. Pandolfi.
Memorial Sloan-Kettering Cancer Center, Sloan-Kettering
Institute, New York, NY, USA. LRF Center at the Institute for Cancer Research,
London, UK.
Acute Promyelocytic Leukaemia (APL)
associated with chromosomal translocations involving the Retinoic Acid Receptor
a (RARa) gene and the PML gene is sensitive to Retinoic Acid
(RA) treatment, while APL patients harboring translocations between the RARa gene
and the PLZF gene do not respond to
RA. We have generated PML-RARa
and PLZF-RARa transgenic mice and we
show that these fusion proteins play a critical role in leukaemogenesis and in
determining responses to RA in APL, since PLZF-RARa
mice develop RA-resistant leukaemia, while PML-RARa mice are responsive to
RA treatment. In the absence of RA, RAR/RXR heterodimers can repress
transcription through histone deacetylation by recruiting nuclear receptor
co-repressors (N-CoR or SMRT), Sin3A or Sin3B, which in turn form complexes
with histone deacetylases (HDAC1 or 2), thereby resulting in nucleosome
assembly and transcriptional repression. Binding of RA to the receptor(s)
causes the dissociation of the co-repressors complex, and the recruitment of
transcriptional co-activators to the RAR/RXR complex, thus leading to the
activation of gene expression. We demonstrate that both PML-RARa
and PLZF-RARa fusion proteins can act as transcriptional repressors and are
able to interact with nuclear receptor transcriptional co-repressors such as
SMRT and N-CoR. PML-RARa
can act as a dominant negative transcriptional repressor of RARa,
through a nuclear co-repressor association that is less sensitive to RA.
PLZF-RARa can form, via its PLZF moiety, co-repressor complexes which
are insensitive to RA. Histone
deacetylase inhibitors (HDACIs) such as Trichostatin A (TSA), sodium phenylbutyrate
(PB), and suberanilohydroxamic acid (SAHA), in combination with RA, can
overcome the transcriptional repressive activity of PML-RARa and PLZF-RARa. HDACIs can also overcome the irresponsiveness of PLZF-RARa
leukaemic cells to RA. These findings
unravel a crucial role for transcriptional silencing in APL pathogenesis and
resistance to RA in APL, and suggest that HDACIs alone or in combination with
RA might be utilized for the treatment of APL.
This therapeutic approach which we termed “transcription therapy” would
represent the first example of selective pharmacological targeting of aberrant
transcriptional activity of oncogenic transcription factors. “Transcription therapy” trials in our APL
animal models with HDACIs are on going.
Results from these trials will be presented.
Combined Intratumoral
Injection of Naive Bone Marrow-Derived Dendritic Cells and Systemic
Chemotherapy Leading to Successful Cancer Treatment in Vivo
Youzhi Tong, Wenru Song and Ronald Crystal
Division of Pulmonary and Critical Care Medicine, The New
York Hospital-Cornell Medical Center, New York.
Dendritic cells
are attractive candidates for developing innovative cancer immunotherapy by
virtue of their potential to function as professional antigen-presenting cells
for initiating cellular immune responses. In this study, we evaluated the
feasibility and consequences of performing intratumoral injection of naive bone
marrow-derived dendritic cells, with or without systemic chemotherapy, for
cancer treatment in vivo. Using murine colon adenocarcinoma (CT26WT and
CT26.CL25) models, we have demonstrated that the consecutive injections of
dendritic cells at the tumor site that provides short-term contact between
dendritic cells and tumors results in partial eradication of established
subcutaneous tumors. Systemic chemotherapy using cyclophosphamide combined with
local intratumoral injection of dendritic cells leads to complete tumor
regression in the treated animals. In addition, these tumor-free animals
were able to resist repeated challenge with tumor cells, suggesting that these
animals had acquired long-term antitumor immunity. Moreover, supporting
evidence was obtained by using other two tumor models (melanoma 1316 and
sarcoma C3), indicating the broad applicability of this approach. These novel
findings raise the possibility of using this potent strategy of combined
intratumoral injection of dendritic cells and systemic
chemotherapy for cancer treatment.
Sociodemographic Data from the Minimum Data Set (MDS) Predict the Health
Status of Newly Admitted Nursing Home (NH) Residents
Zheng-Bo Huang, M.D.,
St. Vincent’s
Hospital and Medical Center, New York
Medical College; Richard Neufeld, M.D., Jewish Home and Hospital,
NYC, Mt. Sinai School of Medicine; Albert Khaski, M.D. CNR Health
Care Network, NY
Objective: To evaluate the correlation
of ethnicity and sociodemographic characteristic (SDC)
to the health status of newly admitted NH residents.
Methods: Logistic regression analyses were
used to determine the odds ratio (OR) for poor health status in 4
domains in respect with the SDC based on the initial MDS+ data from a NH in NY
City.
Results: 258 residents aged > 60
(mean 81.2+8.8) were admitted from 11/92 to 5/97. Among them were 57
White, 23 Black, 53 Hispanic, and 125 Chinese. Age, sex, and Medicaid use, were
not significant predictors for poor health status. However, married residents
were more likely to have severe cognitive impairment (SCI)
(OR=2.07, P<.05) and severe ADL dependence (SADLD) (OR=1.91,
P<.05), while less likely to have mood/behavior disturbance (MBD)
(OR=.42, P<.05). Residents living alone were less likely to have SCI (OR=.28,
P<.01) or MBD (OR=.41, P<.05). Self-pay and private
insurance users were less likely to have MBD (OR=.35,
P<0.05). When the SDC
predictors were adjusted, Chinese were less likely than White to have SCI
(OR=0.39, P<.05), MBD (OR=0.26, P<.01), or negative
psychosocial well-being (NPWB) (OR=0.45, P<.05).
Conclusions: Marital status, living arrangement,
and ethnicity are significantly associated with the health status of the newly
admitted residents.
TRACKING RPE TRANSPLANTS
LABELED BY RETROVIRAL GENE TRANSFER WITH GREEN FLUORESCENT PROTEIN
J. Kong, M.D., P. Gouras, M.D., K. Doi, Ph.D., SH
Tsang, M.D., Ph.D., Stephen P. Goff, Ph.D.
Depts. Ophthalmology and Biochemistry & Molecular
Biophysics, Howard Hughes Medical Institute, Columbia University, New York,
NY 10032
Purpose:
To determine if human RPE can be modified by retroviral mediated gene
transfer; and to monitor the human RPE cells in the subretinal space of living
rabbits with scanning laser ophthalmoscopy (SLO). Methods: Cultured HFRPE was exposed to GFP transducing retroviral
vectors, Moloney murine leukemia virus and Lentivirus. The cultured cells were
followed by fluorescence microscopy. Suspensions of GFP expressing HFRPE were
transplanted into the subretinal space of pigmented rabbits and the transplant
sites were examined by SLO for fluorescence, including fluorescein and
indocyanine green (ICG) angiography. The rabbits were euthanized at different
times after transplantation and the retinas studied histologically.
Results: Retroviral gene transfer can
introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured
RPE for at least three months. The Lentiviral vector tranduced both
non-dividing and dividing cells; the Moloney vector only transduced the latter.
GFP expressing cells can be followed in the living retina. Their changes
reflect the rejection response followed histologically. Host RPE cells
developed lipofuscin fluorescence a month or more after transplantation
surgery; this can be distinguished from GFP fluorescence by its appearance in
the retina and by its yellow color by fluorescence microscopy.
Conclusions: Cultured HFRPE could be
transduced to express GFP for long periods of time by retroviral gene
transfer. GFP allowed retinal
transplants and gene expression to be monitored in vivo. These results provide
a model for potential ex vivo gene therapy in the subretinal space.
Neuronal Apoptosis is Induced by Abnormal Zinc Homeostasis
X. Huang*, C.S. Atwood*, M.C. Russo*, C.
Konradi^, R.C. Scarpa*, J-C. A. Leveque^, R.D. Moir†, R.E. Tanzi† & A.I.
Bush*.
*Genetics and Aging Unit and Department of Psychiatry,
Massachusetts General Hospital; †Genetics and Aging Unit and Department of
Neurology, Massachusetts General Hospital; ^Department of Psychiatry,
Massachusetts General Hospital
Increasing
evidence suggests that neuronal demise in Alzheimer’s disease (AD) occurs via
apoptotic mechanisms. Although the
signals inducing neuronal apoptosis are largely undefined, AD pathology tends
to be localized to regions of the brain with abnormal zinc metabolism. Since
zinc deficiency and excess are well known triggers of apoptotic and necrotic
death in tissues and human lymphoid cell lines, we undertook to examine the
vulnerability of neurons to changes in zinc concentration. To confirm whether
zinc depletion was capable of inducing neuronal cell death, the non-toxic cell
permeable zinc chelator, N,N,N’,N’-tetrakis(2-pyridylmethyl) ethylenediamine,
was added to primary cultures of embryonic 18 d rat pup cortical neurons. Zinc
deprivation induced a rapid increase in neuronal death with features typical of
apoptosis; membrane blebbing, chromatin condensation and increased DNA
fragmentation. Conversely, zinc concentrations as low as 40 µM induced
necrosis. This narrow window of zinc tolerability compared to other cell types
emphasizes the importance of zinc as a regulator of neuronal survival.
Additionally, we found that impairment of cellular energy metabolism using the
mitochondrial inhibitor 3-nitropropionic acid (3-NP) also results in neuronal
apoptosis. Rescue of 3-NP treated neurons by addition of zinc, together with
the fact that zinc uptake into the neuron is highly energy-dependent (and
deficient in neurons in AD) suggests abnormal energy metabolism as a principle
mechanism leading to altered zinc homeostasis and neuronal death.
Supported by NRSA (1F32AG05782) to
X.H.; and NIA grant (1R29AG12686), Alliance for Aging Research (Paul Beeson
Award), Alzheimer’s Association (IIRG-94110), International Life Sciences
Institute (ILSI) to A.I.B.
Establishment of a NOD-SCID Model for Enrichment of Primitive Human
Hempatopoietic Cells Transduced with Retroviral Vectors Containing a Mutated
Dihydryfolate Reductase-green Fluorescent Frotein Fusion Gene
Li-Cheng Xu, MD,PhD, Naoko Takebe, Joseph R.
Bertino and Malcolm Moore
Memorial Sloan-Kettering
Cancer Center, NY, NY 10021
We have previously shown that
mutated DHFR (Leu22 to Phe/Phe31 to Ser, F/S) confers more than 10-fold
increase in MTX resistance in 3T3 cells. Most recently, a high titer F/S-EGFP (a fusion gene of F/S
and EGFP protein) retroviral producer cell line was generated using repetitive
infection of an amphotropic gpAm12 packaging cell line with the supernatant of
an ecotropic gpE86 FS-EGFP producer cell line. Supernatant (containing 1-2 x 107
cfu/ml) was used to transduce human peripheral blood (PB) or bone marrow (BM)
CD34+ cells twice daily for two to four days in the presence of 50 ng/ml stem
cells factor (SCF), 20 ng/ml flt-2, 100 ng/ml TPO, 20 ng/ml IL-6 and 100 ng/ml
GM-CSF on a retronectin dish. Overall transduction efficiency was 41% and 70%,
on a two- and four-day transduction protocol, respectively. Up to 39% of cells
were CD34/EGFP double positive after transduction. The transduced cells were
tested for MTX resistance using in vitro clonogenic assays and fluorescent
microscopy. Sixty per cent of CFU-C from the F/S-EGFP transduced CD34+ cells
survived at 10-6 M of MTX, whereas none of mock CFU-C survived at 5
x 10-8 M of MTX. In addition, sublethally, irradiated (350 Rad)
NOD-SCID mice were transplanted with F/S-EGFP-transduced CD34+ human
hematopoietic cells (1-2 x 106). Studies indicate that up to 2-5% PB
cells of NOD-SCID mice were positive for EGFP in the absence of MTX selection
by fluorescence activated cell sorting (FACS) analysis. Transplanted animals
are currently being treated with MTX according to a dose-escalating schedule.
Detection of EGFP expression in CFU-C assays of human hematopoietic cells from
NOD-SCID BMMN cells at week 24 post transplant suggested that we have
transduced very primitive hematopoietic cells fusing our supernatant
transduction protocol. Enrichment of highly resistant human hematopoietic cells
in the transplanted NOD-SCID mice can facilitate development of a clinically
applicable gene therapy protocol to alleviate myelosuppressive toxicities of
high dose MTX chemotherapy in refractory or relapsed IGL with poor prognosis.
Motor Axons Fail to Innervate Skeletal Muscle in Topoisomerase II Beta
Mutant mice
Xia Yang, Wei Li*, Elizabeth
Prescott, Steven J. Burden and James C. Wang*.
Skirball Institute, NYU Medical Center, 540 First Avenue, NY,
NY 10016; *Dept. of Molecular Cellular Biology, Harvard University, Cambridge,
MA 02138.
DNA topoisomerases II alpha and II
beta catalyze the transport of one duplex DNA segment through an
enzyme-operated double-strand break in a second duplex. The top2alpha gene
encoding the a isozyme is highly expressed in proliferating cells and is
thought to be required for cell survival; the top2beta gene encoding the beta
isozyme is expressed in proliferating cells as well as differentiated cells,
including neurons. To determine the role of DNA topisomerase II beta in vivo,
mutant mice lacking the top2 beta gene were generated. The top2 beta-/- mice survive through fetal
life but fail to breathe or move and die shortly after birth, suggesting a
potential defect in neuromuscular function.
Although muscle development appears normal in the mutant mice, the
diaphragm and limb muscles of mutant embryos lack motor axons. As expected for
non-innervated, developing muscle, AChR clusters are present but not restricted
to the normal endplate zone. The absence of innervation is not owing to an
absence of motor neurons, since somatic motor neurons, as well as other spinal
cord neurons are present at apparently normal numbers in the spinal cord of
mutant mice at E12.5. Motor axons extend to the diaphragm and limb muscles but
fail to grow or branch within the muscle. The number of motor neurons decreases
dramatically by E18.5, probably owing to a failure of motor axons to innervate
their muscle targets. In addition, mutant mice are also defective in their
sensory projections. The dorsal column, which contains collaterals from
proprioceptive sensory neurons, is absent.
This work has been supported by a NIH postdoctoral
fellowship to X.Y. (NS10537) and by research grants from the NIH to S.J.B.
(NS27963) and to J.C.W. (CA47958 and GM24544).
Anti-angiogenic Effect of Kringle 5: Cell Cycle Arrest and Induction
of Apoptosis in Endothelial Cells
Hua Lu, M. Dhanabal, R. Volk, M. Waterman, R.
Ramchandran, B.Knebelman and Vikas. P. Sukhatme
Dept of Medicine, BIDMC, Harvard Medical School
Angiostatin which contains the
first four kringle domains of plasminogen has been documented to be a potent
inhibitor of angiogenesis. Another kringle structure within plasminogen but
outside angiostatin, known as kringle 5 (K5) was found to inhibit endothelial
cell proliferation and migration. Both the recombinant K5 and K5 produced by
protealytic digestion of human plasminogen show dramatic anti-angiogenic effect
in vitro, and it appears to be more potent than angiostatin in those in vitro
assays. We report here the cloning , expression and purification of kringle 5
from a prokaryotic expression system. Purified soluble recombinant kringle 5
(rK5) was found to inhibit bFGF simulated endothelial cell proliferation and
migration, and these inhibitions were endothelial cell specific. More
interestingly, we show for the first time that rK5 causes cell cycle arrest and
apoptosis of endothelial cells, Shedding further insight into kringle 5
mechanism of action.
Interaction of Presenilin-1 with the ß-catenin Signal Transduction Pathway
Zhuohua Zhang, Bruce A. Yankner
Department of Neurology, Harvard Medical School, and The
Children's Hospital
The biological function of
presenilin-1 (PS1) and the pathogenic mechanism associated with PS1 mutations
are unknown. To address these issues,
we have explored an interaction of PS1 with ß-catenin and its biological
consequences. PS1 forms an
approximately 200 kD complex with ß-catenin in cell lines and in the adult
human brain, which localized to the Golgi complex. The function of this complex
may be to stabilize ß-catenin, as transfected PS1 stabilized ß-catenin in
pulse-chase experiments and PS1-deficient fibroblasts showed a pattern of
increased ß-catenin degradation products. Pathogenic PS1 mutations reduce the
ability of PS1 to stabilize ß-catenin in transgenic mice. Moreover, ß-catenin levels are significantly
reduced in the brains of AD patients with PS1 mutations. Loss of ß-catenin signaling markedly increases
neuronal vulnerability to apoptosis induced by amyloid ß protein. Thus, PS1 mutations may increase neuronal
apoptosis by altering the stability of ß-catenin, predisposing to early-onset
AD.
The Essential Role of Protein Kinase C to the the mRNA Expression of CD36,
a Class B Scavenger Receptor
Jianwei Feng*#, Jihong Han*, Roy L. Silverstein*,
Antonio M. Gotto*, David P. Hajjar*, and Andrew Nicholson*
* Center of
Vascular Biology, Cornell Medical College, NY, NY; # Department of
Medicine, New York Methodist Hospital, Brooklyn, NY
CD36, a class B scavenger receptor,
is a receptor for oxidized low density lipoprotein (OxLDL) and may play a
critical role in atherosclerotic foam cell formation. We have recently
demonstrated that OxLDL enhanced expression of CD36 (J. Biol. Chem. 19987;272:21654-9). The effect of OxLDL on CD36 is
due, in part, to its ability to activate the transcription factor, PPARg (peroxisome proliferator activated receptor-g) (Cell 1998;93:241-252).
Other PPARg ligands (15-deoxy 12,14
prostaglandin J2 (15d-PGJ2) and the thiazolidinedione class of antidiabetic
drugs) also increase CD36 expression. We evaluated signaling pathways involved
in the induction of CD36 mRNA. Treatment of RAW264.7 cells (a murine macrophage
cell line) with protein kinase C (PKC) activators (diacylglycerol (DAG) and
ingenol) up-regulated CD36 mRNA expression. Specific inhibitors of PKC reduced
CD36 mRNA expression in a time-dependent manner. In contrast, protein kinase A
(PKA) and cyclic AMP agonists had no effect on CD36 mRNA expression. PKC
inhibitors reduced basal expression of PKC inhibitors decreased both PPARg mRNA and protein expression. Treatment of
human monocytes with OxLDL, but not 15d-PGJ2, resulted in increased expression
of PKC-a as well as its translocation
from the cytosol to the plasma membrane. Finally, PKC inhibitors blocked
induction of CD36 protein surface expression by OxLDL and 15d-PGJ2 in human
monocytes, as determined by FACS. These results demonstrate that activation of
CD36 gene expression by OxLDL involves initial activation and translation of PKC with subsequent PPARg activation. Defining these signaling
pathways is critical for understanding and modulating expression of this
scavenger receptor pathway.
Transforming Growth Factor-b 1,2
Down-regulating Expression of CD36 through Blocking PPARg Signal Pathway
Jihong Han*, Jianwei Feng*#, Antonio M. Gotto*, David
P. Hajjar*, and Andrew Nicholson*
* Center of Vascular Biology, Cornell Medical College, NY,
NY; # Department of Medicine, New York Methodist Hospital, Brooklyn, NY
Uptake of oxidized LDL (OxLDL) by
macrophages is an early key event in the development of foam cells and
atherosclerotic lesions. CD36, the type B scavenger receptor, and type B
scavenger receptor and type A scavenger receptors have been identified as major
receptors that bind and internalize OxLDL. Expression of CD36 in
monocyte/macrophages is dependent both on the differentiation status and
exposure to soluble mediators (cytokines and growth factors). Transforming
growth factor-b1 (TGF-b1) and TGF-b2
are multifunctional mediators that regulate cellular growth, migration and
extracellular matrix formation, and are expressed by cells that comprise
atherosclerotic lesions. In this study, we investigated the effect of TGF- b1,2 on expression of CD36 in macrophages.
Treatment of phorbol ester-differentiated THP-1 macrophages with TGF- b1,2 significantly decreased the expression
of CD36 mRNA. TGF- b1 decreased
expression of CD36 mRNA at a broad range of concentrations (more than 0.5
ng/ml). TGF-b2 decreased the expression
of CD36 mRNA in a concentration-dependent manner. The decrease of CD36 mRNA
expression by TGF- b1,2 occurred as
early as 5 hours after treatment and progressively decreased to 16 hours.
Surface expression of CD36 protein was also decreased as demonstrated by
immunohistochemistry and FACS. Decreased 125I-OxLDL binding to TGF- b1,2 treated macrophages was the result of a
reduction in the number of binding sites without altering receptor/ligand
affinity. OxLDL and 15-deoxy-12,14 prostaglandin J2 (PGJ2), a PPARg ligand, increased macrophage expression of
CD36 mRNA and 125I-OxLDL binding. TGF-
b1,2 significantly inhibited CD36 mRNA and 125I-OxLDL binding
induced by both OxLDL and 15d-PGJ2, suggesting that the TGF- b1,2 may down-regulate CD36 expression by
blocking PPARg mediated signaling. Our
data demonstrate that TGF- b1,2 may
have physiologic or therapeutic relevance in inhibiting the binding and uptake
of oxidized lipids by macrophages by down-regulating expression of CD36.
A Newly Recognized and Treatable
Syndrome: GLUT1 Deficiency
Dong Wang, Jörg Klepper, Yuan Yuan Ho, Kevin
R. O’Driscoll, Veronica J. Hinton, Douglas R. Nordli, Pamela Kranz-Eble, Hong
Yang, Darryl C. De Vivo
Colleen Giblin Laboratories for Pediatric Neurology
Research, Columbia University, New York, NY
GLUT1 deficiency, a novel clinical syndrome, was first
described by De Vivo et al in 1991.
The syndrome is characterized by infantile seizures, acquired microcephaly,
developmental delay, low CSF/blood glucose ratio (approximately 0.33), and
decreased 3-O-methylglucose (3-OMG) uptake in erythrocytes (46±8% of
control). Glucose is the principal fuel source for brain metabolism. The GLUT1
gene encodes the major glucose transporter at the blood -brain barrier, which
transports glucose into the brain. The GLUT1 protein also is expressed
predominately in erythrocytes, which is the basis for the diagnostic 3-OMG
uptake assay. GLUT1 gene is localized to the short arm of chromosome 1
(1p31.3-35), and is about 35kb in length. It consists of 10 exons and 9 introns
and encodes a membrane-integrated protein of 492 amino acids. GLUT1 belongs to
a family of facilitative glucose transport proteins including GLUT1-5 and
GLUT7. It has been shown to be a multifunctional transporter, which can also
transport dehydroascorbic acid (DHA) and other substrates such as galactose, H2O,
and glycopeptide. Based on the above clinical findings, the diagnosis of
suspected GLUT1 deficiency cases is confirmed by a decreased rate of 3-OMG
uptake into erythrocytes (46% compared to normal control). Three patients with
hemizygous deletion mutations were identified by florescence in situ hybridization (FISH). The
remaining were screened for mutations in the GLUT1 gene by analyses of
single-stranded DNA conformational polymorphisms (SSCP) and / or by direct DNA
sequencing of PCR products amplified from the GLUT1 gene. We have identified 15
heterozygous mutations in 14 patients (one patient carried two mutations) which
include 6 missense mutations, 3 nonsense mutations, 1 insertion, 3 deletions,
and 2 splice site mutations. These
mutations are found throughout the GLUT1 gene. This GLUT1 deficiency syndrome
in a German family affected five patients in three generations. We believe that
in this family the GLUT1 deficiency was transmitted as an autosomal dominant
trait.
Western
blots were performed using membranes isolated from erythrocytes both from all
patients and their parents. In general, erythrocyte membranes from patients
with hemizygosity, nonsense, insertion, deletion and splice site mutations
contained about 50 % of the amounts of GLUT1 compared to controls, whereas,
patients with missense mutations had normal amounts of GLUT1.
The brain’s requirement for glucose is the highest
during the period of brain development. In the fasting state, the brain can use
ketone bodies as an alternative fuel. All the patients responded dramatically
to the initiation of a ketogenic diet with prompt cessation of major seizures
in each case. Anticonvulsant medications have been ineffective in managing
these patients. Based on our findings that patients with this condition have a
deficiency in transport of DHA (oxidized form of vitamin C). We speculate that
a low vitamin C level in the brain might contribute to the pathophysiology of
this condition. We therefore recommend vitamin C supplementation. Barbiturates
and caffeine are shown to inhibit glucose transport mediated by GLUT1 in our in vitro studies. The use of
barbiturates and caffeine in this condition could potentially aggravate the
existing glucose transport defect and may put these patients at increased risk.
Thioctic acid has been reported to translocate GLUT1 from intracellular pool to
the plasma membrane. This mechanism could possibly be of benefit in GLUT1
deficiency. We are constructing a mouse model for this syndrome. When this
model is available, we hope to gain a more advanced understanding of the
pathology of this syndrome. Effective treatments such as gene therapy and drugs
to upregulate the normal GLUT1 gene expression will be tested in mice.
In
conclusion: 1. GLUT1 deficiency is a newly-defined
syndrome characterized by seizures, developmental delay, microcephaly, and low
CSF/blood glucose ratio (<0.33). 2. Decreased uptake 3-OMG into erythrocytes
due to a decrease of GLUT1 transport function is an effective diagnostic test.
3. A heterozygous mutation(s) in the GLUT1 gene is often the basis of
haploinsufficiency of glucose transport. 4. GLUT1 deficiency is a treatable
inherited syndrome.
Checkpoint control and Regulation of Mitotic Proteolysis
Guowei Fang and Marc W Kirschner
Department of Cell Biology, Harvard Medical School, 240
Longwood Avenue, Boston, MA 02115
Proteolysis controls key
transitions at several points in the cell cycle. In mitosis, key regulators are
degraded in a specific temporal order through a ubiquitin-dependent pathway and
the activation of a large ubiquitin-protein ligase, the anaphase-promoting
complex (APC), is required for anaphase initiation and for exit from mitosis.
Anaphase does not initiate until all the sister chromatids are aligned at the
metaphase plate. This is accomplished by the spindle assembly checkpoint
mechanism that ensures the fidelity of sister chromatid separation. APC is the
key target for the checkpoint pathway.
We show that APC is under complex
control by a network of regulatory factors, CDC20, CDH1 and MAD2. CDC20 and
CDH1 are activators of APC; they directly bind to APC and activate its
ubiquitination activity. CDC20 activates APC at the onset of anaphase in a
destruction box (D-box) dependent manner, while CDH1 activates APC from late
anaphase through G1 with apparently a much-relaxed specificity for the D-box.
Therefore, CDC20 and CDH1 control the temporal order of activation as well as
the substrate specificity of APC. Counteracting the effect of CDC20, the
checkpoint protein MAD2 acts as an inhibitor of APC. When the spindle assembly
checkpoint is activated, MAD2 forms a ternary complex with CDC20 and APC to
prevent activation of APC, and thereby arrests cells at prometaphase. Thus, a
combination of positive and negative regulators establishes a regulatory
circuit of APC, ensuring an ordered progression of events through cell
division.
The MAD2 protein exists in two
folded states in vitro: an oligomer and a monomer. Both the oligomer and
monomer bind to CDC20, but only the oligomer inhibits activation of APC. The
checkpoint protein MAD1 acts upstream from MAD2 and induces oligomerization of
MAD2 monomer when the checkpoint is activated. Thus, the mitotic checkpoint
control involves MAD1 receiving an unknown upstream signal to induce a
structure change in MAD2, which in turn prevents activation of APC and arrests
cell cycle progression.
Opposing effects of CSB vs. INK4a on cell proliferation, apoptosis and p53
induction after UV irradiation
Hanzhou Lian1, Linda Chin2,
Ronald DePinho2, David Bregman1
Department of Pathology1, Albert Einstein College
of Medicine, Bronx, NY 10461; Department of Adult Oncology2, Dana
Farber Cancer Institute, Boston, MA
02115
The INK4a
gene encodes two distinct growth inhibitors: the cyclin-dependent kinase
inhibitor p16INK4a, which is a component of the RB pathway, and the tumor
suppressor p19ARF, which has been functionally linked to p53. Cockayne Syndrome B (CSB) is a DNA repair
disorder. CSB-deficient mouse
fibroblasts display UV sensitivity, defective resumption of transcription after
UV exposure, normal global genome repair, and a complete loss of transcription
couple repair (TCR) in the transcribed strand of an active gene. The CSB protein also enhances the
elongation rate of RNA polymerase II transcription.
Premalignant
and aberrant cells are eliminated by mechanisms involving the tumor suppressor
p53. A variety of cellular stresses,
such as damaged DNA, viral infection, and hypoxia can trigger
post-translational stabilization and activation of p53. Activated p53 suppresses cellular growth
through induction of either cell cycle arrest or apoptosis.
We
investigated apoptosis, p53 induction after UV irradiation and cell growth rate
in four different genotypes of mouse embryo fibroblast (MEF):
INK4a-ARF-/-(BBii), INK4a/CSB double knock out (bbii), CSB-/- (bbII), and wild
type (BBII). Here we show that INK4a-/-
(BBii) early passage of MEFs grow significantly faster than early passage INK4a
and CSB double knockout MEFS (bbii).
The number of colonies formed by
BBii MEFs is significantly higher than that in bbii MEFs. This result suggests
that disruption of CSB can inhibit the MEFs’ growth rate and colony formation
even in the background of INK4a -/-.
P53 induction after UV irradiation in bbII and bbii MEFs occurs at lower
UV dose (2.5 J/m2) than that in BBII and BBii MEFs (10J/m2). The p53 level and apoptosis after 2.5J/m2 of
UV irradiation is higher in bbII than that in bbii. The p53 level and apoptosis after 10J/m2 of UV is higher in BBII
MEFs than that in BBii MEFs. These
results suggest that disruption of INK4a-ARF locus can inhibit p53 induction
and apoptosis after UV irradiation. CSB-/- can potentiate p53 induction and
apoptosis after UV irradiation. Could
inactivation of CSB inhibit tumor growth? Further study is needed.
Identification of a Vertebrate Sister-Chromatid Separation Inhibitor
Involved in Transformation and Tumorigenesis
Hui Zou, Thomas J. McGarry, Teresita Bernal and Marc
W. Kirschner, Department of Cell Biology, Harvard Medical School, 240 Longwood
Avenue, Boston, MA 02115
The metaphase to anaphase
transition is the final discrete event in duplication and separation of the
genetic materials. Defects in this
process could lead to chromosome mis-segregation, consequently aneuploidy as
found in nearly all cancers. The timing
of this critical transition is regulated by the activation of the
anaphase-promoting complex (APC), which mediates selective proteolysis of
various mitotic regulators. Early
experiments in Xenopus extracts
suggested that degradation of a putative APC substrate is required for the
onset of chromatid separation. Proteins
with such an activity, known as anaphase inhibitors or securins, were
subsequently found in both budding yeast and fission yeast encoded by PDS1 and CUT2 respectively. Despite
functional similarities, these two proteins share no sequence homology with
each other, or to any sequences in the current database.
To study the regulation of
chromatid separation in vertebrates, we have identified the long-awaited
vertebrate securin (vSecurin) by functional analogy to its yeast
counterparts. Although sharing no
homology with yeast securins, we found that vSecurin associates with a putative
human separin, is degraded by APC mediated proteolysis pathway, and inhibits
chromatid separation in Xenopus
extracts. Therefore, it now appears
that there is a general model for anaphase initiation in yeast and
metazoans. More interestingly, vSecurin
is identical to a previously identified pituitary tumor transforming gene
(PTTG); its expression is high in all carcinoma cell lines tested so far and in
one case its expression level correlate with the malignancy of disease. The identification of securin as an oncogene
attests our need to understand chromatid separation and reemphasizes the
contribution of chromosome mis-segregation to cancer progression.
Reference:
Zou,
H., McGarry, T. J., Bernal, T., and Kirschner, M. W. (1999). Identification of
a vertebrate sister-chromatid separation inhibitor involved in transformation
and tumorigenesis. Science 285,
418-22.
MECHANISMS OF ALCOHOLIC FATTY LIVER
Xujun Wu and Alfred Leong*
Department of Medicine, Columbia University; *Department of
Medicine, New York Methodist Hospital
Alcoholic cirrhosis is a major
health problem world-wide. One of the fundamental consequences of alcohol
consumption is fatty liver. We and
others have reported that alcohol induces fatty liver partially by increasing
triglyceride (TG) synthesis. However,
from the present study we conclude that alcohol also promotes net TG
accumulation in cytoplasm independent of TG synthetic rate. Rat hepatocytes
were incubated with [3H]glycerol in the presence of BFA, a specific
inhibitor of transportation from endoplastic reticulum (ER)to Golgi for 2
hours, allowing [3H]TG to form.
[3H]glycerol was then removed and cells were then divided
into two groups: the first group was incubated in control medium (C), and the
second group was incubated in control medium with 20 mM ethanol (E). The amounts
of [3H]TG in these two groups were the same. The incubation was
continued for 1 hour, and [3H]TG activity in the cytoplasm and the
ER was measured. In the C group the ratio of cytoplastic TG to ER TG was 4:1,
whereas in the E group this ratio was increased to 7:1. Since cytoplasmic TG is responsible for the
formation of fatty liver, our results, together with previous findings, suggest
that alcohol induces fatty liver by two mechanisms: (1) increasing TG synthesis
and (2) promoting net cytoplasmic TG accumulation, independent of TG synthetic
rate.
Investigation of MMTV-like LTR in Human Breast Cancer
Yue Wang, James Holland and Beatriz G.T.Pogo
Department of Medicine, Division of Medical Oncology, Mount
Sinai Medical
Center, New York, NY 10029
The mouse
mammary tumor virus (MMTV) has been regarded as a potential model for human
breast cancer (BC). We have previously
reported that a 660 bp MMTV-like env gene sequence was present in about 38%
human BC, but not in the normal breast and normal tissues. We also found that 66% of the positive
samples expressed this gene. The 3'
long terminal repeats (LTR) sequence of the MMTV plays a very important role in
mouse mammary tumorigenesis, since it contains several important elements, like
enhancers, promoters, glucorticoid responsive element (GRE) and an open reading
frame (ORF) for potential superantigen, which have critical functions during
the MMTV infection. Therefore, we have
focused our research on LTR identification and expression. By using PCR, cloning and sequencing, a 630
bp fragment was first amplified from 32 human BC and breast cancer cell
lines. It showed over 90% homology to
MMTV LTR, but very low homology to any other known sequences in the
genebank. Using an extra long PCR and a
nested PCR, a longer 1.2 Kb sequence was detected, and complete ORF and GRE
elements have been identified. The
results from the preliminary experiments also indicated that the LTR gene is
expressed in BC. This human MMTV-like
LTR has been cloned into a bacterial and a mammalian expression vectors to be
used for detection in human tissues and functional assays. The results obtained from this research will
help to understand the molecular mechanism(s) of human breast carcinogenesis,
and to identify new genetic markers possibly useful for diagnosis, prognosis and
therapy.
Characterization of an activation protein-1 binding site in the murine interleukin-12
p40 promoter: Demonstration of novel functional cis-acting elements by a
reductionist approach
Chen Zhu1, Khatuna Gagnidze1, James H.
M. Gemberling2, Scott E. Plevy1, 3
1Immunobiology
Center, Box 1630, The Mount Sinai School of Medicine,1 Gustave L. Levy Place, New
York, NY 10029-6574; 2Howard Hughes Medical
Institute, 6-730 MRL, 675 Circle Drive South, UCLA School of
Medicine, Los Angeles, CA
90095-1662
Interleukin (IL)-12 is a
heterodimeric cytokine produced by macrophages in response to intracellular
pathogens and bacterial products. IL-12
provides an obligatory signal for the differentiation of T-helper-1 (Th1)
cells. We have recently reported an
analysis of the IL-12 p40 promoter in RAW 264.7 macrophages. Multiple control elements were involved in
activation of transcription by bacterial products. The most important control
element, located between -96 and -88, interacts with C/EBP family members. However, mutations in several other elements
had functional effects on promoter activation.
In the current study, using a strategy to demonstrate functional
activity in a minimal promoter context, at least three novel cis-acting
elements are demonstrated to have an important role in IL-12 p40 promoter
activation by lipopolysaccharide (LPS).
One of these elements is characterized in detail. Mutations from -79 to -74 in the murine
IL-12 p40 promoter significantly reduce LPS-induced promoter activity. Electrophoretic
mobility shift assays demonstrate binding of AP-1 family members to this
region. Spacing between the C/EBP and
AP-1 site is important for promoter activation, suggesting cooperativity
between these two elements. Finally,
expression of a dominant negative AP-1 protein, A-Fos, in RAW 264.7 cells
inhibits p40 promoter activation by LPS.
SPECIAL PRESENTATION:
Introduction of Columbia Genome
Center
Xiaoyan Qu, M.D.,
James J. Russo, PhD.
Columbia Genome Center, Columbia University, New York, NY
10032, U.S.A
The Columbia Genome Center (CGC) is
an outgrowth of the National Institute of Health’s (NIH) Human Genome
Initiative, which seeks to map and sequence all 23 pairs of human chromosomes.
In 1990, as part of that project, researchers at Columbia University’s College
of Physicians and Surgeons started work on chromosome 13 and have nearly
completed a fine annotated map of this chromosome. The CGC is a consortium of
most of those scientists and their laboratories, brought together in 1996,
under the directorship of Isidore Edelman, M.D., for the purpose of gene
identification. In late 1997 Conrad Gilliam, PhD was named co-director of the
center. CGC is a university-wide interdepartmental undertaking that will
examine DNA from a variety of sources, although the main thrust is human
disease conditions (URL: http://genome4.cpmc.columbia.edu).
