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1.  B7H Costimulates Clonal Expansion of, and Cognate Destruction of Tumor Cells by, CD8+ T Lymphocytes In Vivo 
The Journal of Experimental Medicine  2001;194(9):1339-1348.
B7H/B7RP (hereby called B7H) is a new member of the B7 family of costimulatory molecules and interacts with inducible costimulatory molecule (ICOS). Its function for CD8 T cells has not been reported. We report here that expression of B7H on the tumor cells reduced tumorigenicity and induced immunity to subsequent challenge with parental tumor cells. The immune protection correlates with an enhanced cytotoxic T lymphocyte (CTL) response against P1A, the major tumor antigen expressed in the J558 tumor. To understand the mechanism of immune protection, we adoptively transferred transgenic T cells specific for tumor antigen P1A into mice that bore P1A-expressing tumors. We found that while the transgenic T cells divided faster in mice bearing the B7H+ tumors, optimal B7H-induced clonal expansion of P1CTL required costimulation by B7–1 and B7–2 on the endogenous host antigen-presenting cells (APCs). Interestingly, when B7H+ and B7H− tumors were coinjected, P1CTL selectively eliminated the B7H+ tumor cells. Moreover, B7H expressed on the tumor cells made them highly susceptible to destruction by CTL in vivo, even if the CTL was administrated into mice with large tumor burdens. Tumors that recurred in the P1CTL-treated mice lost transfected B7H and/or H-2Ld, the class I molecule that presents the P1A peptide. Taken together, our results reveal that B7H costimulates clonal expansion of, and cognate destruction by CD8+ T lymphocytes in vivo.
PMCID: PMC2195972  PMID: 11696598
cytotoxic T lymphocytes; tumor immunity; B7H; effector function; clonal expansion
2.  Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures 
The Journal of Cell Biology  2001;155(1):157-166.
Targeted disruption of core binding factor α1 (Cbfa1) showed that Cbfa1 is an essential transcription factor in osteoblast differentiation and bone formation. Furthermore, both in vitro and in vivo studies showed that Cbfa1 plays important roles in matrix production and mineralization. However, it remains to be clarified how Cbfa1 controls osteoblast differentiation, bone formation, and bone remodelling. To understand fully the physiological functions of Cbfa1, we generated transgenic mice that overexpressed Cbfa1 in osteoblasts using type I collagen promoter. Unexpectedly, Cbfa1 transgenic mice showed osteopenia with multiple fractures. Cortical bone, which was thin, porous, and enriched with osteopontin, was invaded by osteoclasts, despite the absence of acceleration of osteoclastogenesis. Although the number of neonatal osteoblasts was increased, their function was impaired in matrix production and mineralization. Furthermore, terminally differentiated osteoblasts, which strongly express osteocalcin, and osteocytes were diminished greatly, whereas less mature osteoblasts expressing osteopontin accumulated in adult bone. These data indicate that immature organization of cortical bone, which was caused by the maturational blockage of osteoblasts, led to osteopenia and fragility in transgenic mice, demonstrating that Cbfa1 inhibits osteoblast differentiation at a late stage.
doi:10.1083/jcb.200105052
PMCID: PMC2150799  PMID: 11581292
Cbfa1; osteoblast; osteocyte; transgenic mice; osteopenia
3.  Characterization of Bovidae sex-determining gene SRY 
In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3' regions.
doi:10.1186/1297-9686-33-6-687
PMCID: PMC2705398  PMID: 11742636
sex determination; SRY; evolution; Bovidae
4.  RhoB Is Dispensable for Mouse Development, but It Modifies Susceptibility to Tumor Formation as Well as Cell Adhesion and Growth Factor Signaling in Transformed Cells 
Molecular and Cellular Biology  2001;21(20):6906-6912.
RhoB is an endosomal small GTPase that is implicated in the response to growth factors, genotoxic stress, and farnesyltransferase inhibitors. To gain insight into its physiological functions we examined the consequences of homozygous gene deletion in the mouse. Loss of RhoB did not adversely affect mouse development, fertility, or wound healing. However, embryo fibroblasts cultured in vitro exhibited a defect in motility, suggesting that RhoB has a role in this process that is conditional on cell stress. Neoplastic transformation by adenovirus E1A and mutant Ras yielded differences in cell attachment and spreading that were not apparent in primary cells. In addition, transformed −/− cells displayed altered actin and proliferative responses to transforming growth factor β. A negative modifier role in transformation was suggested by the increased susceptibility of −/− mice to 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis and by the increased efficiency of intraperitoneal tumor formation by −/− cells. Our findings suggest that RhoB is a negative regulator of integrin and growth factor signals that are involved in neoplastic transformation and possibly other stress or disease states.
doi:10.1128/MCB.21.20.6906-6912.2001
PMCID: PMC99867  PMID: 11564874
5.  Role of CD28/B7 costimulation and IL-12/IL-10 interaction in the radiation-induced immune changes 
BMC Immunology  2001;2:8.
Background
The present paper aims at studying the role of B7/CD28 interaction and related cytokine production in the immunological changes after exposure to different doses of ionizing radiation.
Results
The stimulatory effect of low dose radiation (LDR) on the proliferative response of lymphocytes to Con A was found to require the presence of APCs. The addition of APCs obtained from both low- and high-dose-irradiated mice to splenic lymphocytes separated from low-dose-irradiated mice caused stimulation of lymphocyte proliferation. B7-1/2 expression on APCs was up-regulated after both low and high doses of radiation. There was up-regulation of CD28 expression on splenic and thymic lymphocytes after LDR and its suppression after high dose radiation (HDR), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression showed changes in the opposite direction. IL-12 secretion by macrophages was stimulated after both low and high doses of radiation, but IL-10 synthesis by splenocytes was suppressed by low dose radiation and up-regulated by high dose radiation.
Conclusion
The status of CD28/CTLA-4 expression on T lymphocytes in the presence of up-regulated B7 expression on APCs determined the outcome of the immune changes in response to radiation, i.e., up-regulation of CD28 after LDR resulted in immunoenhancement, and up-regulation of CTLA-4 associated with down-regulation of CD28 after HDR led to immunosuppression. Both low and high doses of radiation up-regulated B7-1/2 expression on APCs. After LDR, the stimulated proliferative effect of increased IL-12 secretion by APCs, reinforced by the suppressed secretion of IL-10, further strengthened the intracellular signaling induced by B7-CD28 interaction.
doi:10.1186/1471-2172-2-8
PMCID: PMC48143  PMID: 11532194
6.  Induction of Caspase-Dependent Apoptosis in Cultured Cells by the Avian Coronavirus Infectious Bronchitis Virus 
Journal of Virology  2001;75(14):6402-6409.
Avian coronavirus infectious bronchitis virus (IBV) is the causative agent of chicken infectious bronchitis, an acute, highly contagious viral respiratory disease. Replication of IBV in Vero cells causes extensive cytopathic effects (CPE), leading to destruction of the entire monolayer and the death of infected cells. In this study, we investigated the cell death processes during acute IBV infection and the underlying mechanisms. The results show that both necrosis and apoptosis may contribute to the death of infected cells in lytic IBV infection. Caspase-dependent apoptosis, as characterized by chromosomal condensation, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase degradation, was detected in IBV-infected Vero cells. Addition of the general caspase inhibitor z-VAD-FMK to the culture media showed inhibition of the hallmarks of apoptosis and increase of the release of virus to the culture media at 16 h postinfection. However, neither the necrotic process nor the productive replication of IBV in Vero cells was severely affected by the inhibition of apoptosis. Screening of 11 IBV-encoded proteins suggested that a 58-kDa mature cleavage product could induce apoptotic changes in cells transiently expressing the protein. This study adds one more example to the growing list of animal viruses that induce apoptosis during their replication cycles.
doi:10.1128/JVI.75.14.6402-6409.2001
PMCID: PMC114363  PMID: 11413307
7.  Inhibitory Effects of Dimeric Copper(II) bis(o-acetoxybenzoate) on Platelet-neutrophil adhesion and Thrombosis 
Metal-Based Drugs  2001;8(2):103-105.
Antithrombotic effect of the copper-aspirin complex (dimeric copper(II) bis(o-acetoxybenzoate) was evaluated in the model of venous thrombosis; its effects on platelet-neutrophil adhesion were investigated by use of rosette assay. The results showed that the intragastrically administered copper-aspirin complex (5, 7, and 10 mg kg-1) dose-dependently lowered the wet and dry thrombus weight; it significantly decreased the binding of arachidonic acid-activated platelets to neutrophils with an IC50 value of 41.5 μmol L-1. The results suggested that copper aspirinate inhibited platelet-neutrophil adhesion and resulted in a more potent antithrombotic activity.
doi:10.1155/MBD.2001.103
PMCID: PMC2365252  PMID: 18475982
8.  Vitamin E Suppression of Microglial Activation Is Neuroprotective 
Journal of neuroscience research  2001;66(2):163-170.
Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer’s disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NFκB), with consequent increases in interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1α and TNFα expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFκB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFκB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.
PMCID: PMC3903400  PMID: 11592111
Alzheimer’s disease; interleukin-1; NFκB; nitric oxide; p38 mitogen-activated protein kinase; tumor necrosis factor; vitamin E
9.  Elucidation of CYP2E1 5′ regulatory RsaI/Pstl allelic variants and their role in risk for oral cancer 
Oral oncology  2001;37(5):437-445.
The CYP2E1 gene, whose protein product plays an important role in the metabolism of various carcinogens, exhibits two polymorphisms recognized by the restriction enzymes RsaI and PstI in its transcriptional regulatory region that have been previously implicated in cancer susceptibility. In this study, we have examined these polymorphisms to elucidate CYP2E1 allelic haplotype, examining the prevalence of these CYP2E1 alleles in Caucasians and African Americans and their potential role in risk for oral cancer. In addition to the cl (RsaI[+]/PstI[−]) and c2 (RsaI[−]/PstI[+]) alleles reported in previous studies, we have identified two new alleles, c3 (RsaI[+]/PstI[+]) and c4 (RsaI[−]/PstI[−]). The prevalence of the c2 and c3 alleles differs between racial groups, with African Americans exhibiting a lower prevalence of the c2 allele (0.003) but a higher prevalence of the c3 allele (0.049) than Caucasians (0.031 for c2 and 0.004 for c3). Of the 570 subjects screened in this study, the c4 allele was observed in one subject, a Caucasian case with the (c4/c4) genotype. A significant increase in the CYP2E1 (cl/cl) genotype was observed in oral cancer cases as compared to frequency-matched controls in subjects who smoked ≤ 24 pack-years (P = 0.033). No association was observed between CYP2E1 genotype and risk for oral cancer in the heavy-smoking group (i.e. > 24 pack-years). Similar trends were observed for both Caucasians and African Americans. These data suggest that the cl allele may contribute to increased risk for oral cancer.
PMCID: PMC3715306  PMID: 11377232
CYP2E1; Cytochrome P450; Oral cancer risk; Genetic susceptibility; Polymorphisms
10.  Structure-Function Relationships of the Raloxifene-Estrogen Receptor-α Complex for Regulating Transforming Growth Factor-α Expression in Breast Cancer Cells* 
The Journal of biological chemistry  2001;277(11):9189-9198.
Amino acid Asp-351 in the ligand binding domain of estrogen receptor α (ERα) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERα complexes. 4-Hydroxyta-moxifen is a full agonist at a transforming growth factor a target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERα. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERα allosterically silences activation function-1 activity in the 4-hydroxytamox-ifen-ERα complex with the complete loss of estrogenlike activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERα expression vectors. Profound differences in the agonist and antagonist actions of Ral-ERα complexes were noted only in stable transfec-tants. The agonist activity of the Ral-ERα complex was enhanced with D351E and D351Y ERα, but raloxifene lost its agonist activity with D351F ERα. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERα. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERα complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERα complex.
doi:10.1074/jbc.M108335200
PMCID: PMC3696956  PMID: 11751902
11.  Identification of biochemical pathways for the metabolism of oxidized low-density lipoprotein derived aldehyde-4-hydroxy trans-2-nonenal in vascular smooth muscle cells 
Atherosclerosis  2001;158(2):339-350.
Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [3H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60–65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography–chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25–30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.
PMCID: PMC3469324  PMID: 11583712
Lipid peroxidation; 4-Hydroxy-trans-2-nonenal; Glutathione conjugates; Aldose reductase; Vascular smooth muscle cells; Atherosclerosis
12.  Structure and Topology of a Peptide Segment of the 6th Transmembrane Domain of the Saccharomyces cerevisae α-Factor Receptor in Phospholipid Bilayers 
Biopolymers  2001;59(4):243-256.
A detailed analysis of the structure of an 18-residue peptide AQSLLVPSIIFILAYSLK [M6(252–269, C252A)] in 1,2-dimyristoyl-sn-glycero-phosphocholine bilayers was carried out using solid state NMR and attenuated total reflection Fourier transform infrared spectroscopy. The peptide corresponds to a portion of the 6th transmembrane domain of the α-factor receptor of Saccharomyces cerevisiae. Ten homologs of M6(252–269, C252A) were synthesized in which individual residues were labeled with 15N. One- and two-dimensional solid state NMR experiments were used to determine the chemical shifts and 1H–15N dipolar coupling constants for the 15N-labeled peptides in oriented dimyristoylphosphatidylcholine bilayers on stacked glass plates. These parameters were used to calculate the structure and orientation of M6(252–269, C252A) in the bilayers. The results indicate that the carboxyl terminal residues (9–14) are α-helical and oriented with an angle of about 8° with respect to the bilayer normal. Independently, an attenuated total reflection Fourier transform infrared spectroscopy analysis on M6(252–269, C252A) in a 1,2-dimyristoyl-sn-glycero-phosphocholine bilayer concluded that the helix tilt angle was about 12.5°. The results on the structure of M6(252–269, C252A) in bilayers are in good agreement with the structure determined in trifluoroethanol/water solutions (B. Arshava et al. Biopolymers, 1998, Vol. 46, pp. 343–357) . The present study shows that solid state NMR spectroscopy can provide high resolution information on the structure of transmembrane domains of a G protein-coupled receptor.
doi:10.1002/1097-0282(20011005)59:4<243::AID-BIP1021>3.0.CO;2-H
PMCID: PMC3282060  PMID: 11473349
6th transmembrane peptide; Saccharomyces cerevisae; α-factor receptor; phospholipid bilayers; ATR-FTIR; solid state NMR
13.  Identification of a Novel Structural Variant of the α6 Integrin* 
The Journal of biological chemistry  2001;276(28):26099-26106.
The α6 integrin is a 140-kDa (nonreduced) laminin receptor. We have identified a novel 70-kDa (nonreduced) form of the α6 integrin called α6p for the latin word parvus, meaning small. The variant was immunoprecipitated from human cells using four different α6-specific monoclonal antibodies but not with α3 or α5 antibodies. The α6p integrin contained identical amino acid sequences within exons 13–25, corresponding to the extracellular “stalk region” and the cytoplasmic tail of the α6 integrin. The light chains of α6 and α6p were identical as judged by α6A-specific antibodies and electrophoretic properties. The α6p variant paired with either β1 or β4 subunits and was retained on the cell surface three times longer than α6. Reverse transcription/polymerase chain reaction analysis revealed a single polymerase chain reaction product. The α6p variant was found in human prostate (DU145H, LnCaP, PC3) and colon (SW480) cancer cell lines but not in normal prostate (PrEC), breast cancer (MCF-7), or lung cancer (H69) cell lines or a variant of a prostate carcinoma cell line (PC3-N). Protein levels of α6p increased 3-fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. A novel form of the α6 integrin exists on cell surfaces that contains a dramatically altered extracellular domain.
doi:10.1074/jbc.M102811200
PMCID: PMC2824502  PMID: 11359780
15.  RESTORATION OF ENDOGENOUS WILD-TYPE p53 ACTIVITY IN A GLIOBLASTOMA CELL LINE WITH INTRINSIC TEMPERATURE-SENSITIVE p53 INDUCES GROWTH ARREST BUT NOT APOPTOSIS 
p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37°C and as a functional wild-type p53 below 34°C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1p21 and transforming growth factor-α, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G1/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages.
doi:10.1002/ijc.1431
PMCID: PMC2819747  PMID: 11668476
p53; temperature-sensitive mutant; cell cycle; growth arrest
16.  Neuronal NOS Inhibitor That Reduces Oxidative DNA Lesions and Neuronal Sensitivity Increases the Expression of Intact c-fos Transcripts after Brain Injury 
Journal of biomedical science  2001;8(4):336-341.
In response to oxidative stress, the ischemic brain induces immediate early genes when its nuclear genes contain gene damage. Antioxidant that reduces gene damage also reduces cell death. To study the mechanism of neuronal sensitivity, we investigated the transcription of the c-fos gene after brain injury of the ischemia-reperfusion type using focal cerebral ischemia-reperfusion in Long-Evans hooded rats. We observed a significant (p < 0.01) increase in c-fos mRNA in the ischemic cortex immediately after brain injury. However, the c-fos transcript was sensitive to RNase A protection assay (RPA) upon reperfusion. The transcript became significantly resistant to RPA (42%, p < 0.03) when 3-bromo-7-nitroindazole (25 mg/kg, i.p.), known to abolish nitric oxide, gene damage and neuronal sensitivity, was injected. Our data suggest that neuronal nitric oxide synthase and aberrant mRNA from genes with oxidative damage could be associated with neuronal sensitivity.
PMCID: PMC2727053  PMID: 11455196
Gene expression; Neuroregeneration; Oxidative stress; Stroke; Transcription
17.  Ischemic injury and faulty gene transcripts in the brain 
Trends in neurosciences  2001;24(10):581-588.
The brain has the highest metabolic rate of all organs and depends predominantly on oxidative metabolism as a source of energy. Oxidative metabolism generates reactive oxygen species, which can damage all cellular components, including protein, lipids and nucleic acids. The processes of DNA repair normally remove spontaneous gene damage with few errors. However, cerebral ischemia followed by reperfusion leads to elevated oxidative stress and damage to genes in brain tissue despite a functional mechanism of DNA repair. These critical events occur at the same time as the expression of immediate early genes, the products of which trans-activate late effector genes that are important for sustaining neuronal viability. These findings open the possibility of applying genetic tools to identify molecular mechanisms of gene repair and to derive new therapies for stroke and brain injury.
PMCID: PMC2726704  PMID: 11576672
18.  Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons 
Development (Cambridge, England)  2001;128(13):2421-2432.
SUMMARY
The POU domain transcription factors Brn3a, Brn3b and Brn3c are required for the proper development of sensory ganglia, retinal ganglion cells, and inner ear hair cells, respectively. We have investigated the roles of Brn3a in neuronal differentiation and target innervation in the facial-statoacoustic ganglion. We show that absence of Brn3a results in a substantial reduction in neuronal size, abnormal neuronal migration and downregulation of gene expression, including that of the neurotrophin receptor TrkC, parvalbumin and Brn3b. Selective loss of TrkC neurons in the spiral ganglion of Brn3a−/− cochlea leads to an innervation defect similar to that of TrkC−/− mice. Most remarkably, our results uncover a novel role for Brn3a in regulating axon pathfinding and target field innervation by spiral and vestibular ganglion neurons. Loss of Brn3a results in severe retardation in development of the axon projections to the cochlea and the posterior vertical canal as early as E13.5. In addition, efferent axons that use the afferent fibers as a scaffold during pathfinding also show severe misrouting. Interestingly, despite the well-established roles of ephrins and EphB receptors in axon pathfinding, expression of these molecules does not appear to be affected in Brn3a−/− mice. Thus, Brn3a must control additional downstream genes that are required for axon pathfinding.
PMCID: PMC2710107  PMID: 11493560
Brn3a; POU domain; Transcription factor; Spiral ganglion; Vestibular ganglion; Innervation; Axon pathfinding; Mouse
19.  Autosomal Dominant Retinal Degeneration and Bone Loss in Patients with a 12-bp Deletion in the CRX Gene 
Purpose
To define the phenotypic expression of a deletion in the gene encoding the transcription factor CRX in a large, seven-generation, white family.
Methods
Fourteen affected individuals, all heterozygous for the Leu146del12 mutation in the cone-rod homeobox gene (CRX), and four nonaffected relatives from the same family were examined with visual function tests, and 10 underwent bone mineral density (BMD) measurement.
Results
The ability of the mutated CRX protein to transactivate rhodopsin promoter was decreased by approximately 25%, and its ability to react synergistically with neural retinal leucine zipper (NRL) was reduced by more than 30%. The affected members of the family had an autosomal dominant ocular condition most closely resembling Leber congenital amaurosis (LCA) with severe visual impairment at an early age. Depending on age, affected members showed varying degrees of significant visual acuity loss, elevated dark-adaptation thresholds, significantly reduced cone and rod electroretinogram (ERG) amplitudes, and progressive constriction of the visual fields, in most cases leading to complete blindness. Six affected members had reduced levels of BMD in the spine and the hip (osteopenia). Four affected female members who were receiving long-term hormonal replacement therapy (HRT) demonstrated normal values of BMD.
Conclusions
This large deletion of the CRX gene is associated with a severe form of autosomal dominant retinal degeneration. Affected members not receiving HRT showed reduced BMD (osteopenia). This phenotype may reflect the abnormal influence of mutant CRX on both retinal and pineal development.
PMCID: PMC2581450  PMID: 11328746
20.  Transduction of an IL-2 Gene into Human Melanoma-Reactive Lymphocytes Results in Their Continued Growth in the Absence of Exogenous IL-2 and Maintenance of Specific Antitumor Activity 
IL-2-dependent activated cells undergo apoptotic death when IL-2 is withdrawn either in vitro or after in vivo cell transfer. To attempt to sustain their survival after IL-2 withdrawal, melanoma-reactive human T lymphocytes were retrovirally transduced with an exogenous human IL-2 gene. Transduced PBMC and cloned CD8+ T cells produced IL-2 and maintained viability after IL-2 withdrawal. Upon restimulation, IL-2 transductants proliferated in the absence of exogenous IL-2 and could be actively grown, and their survival could be maintained without added IL-2 for over 8 wk. PBMCs similarly transduced with a control vector did not produce IL-2 and failed to proliferate in the absence of IL-2. A CD8+ T cell clone, when transduced with an IL-2 gene, manifested the same phenotypes as PBMCs in the absence of exogenous IL-2. Furthermore, an Ab reactive with the α-chain of IL-2R complex reduced the viability mediated by IL-2 secretion of the IL-2 transductants. Moreover, transduction of an IL-2 gene did not affect the high degree of recognition and specificity of transductants against melanoma targets. These tumor-reactive IL-2 transductants may be valuable for in vitro studies and for improved adoptive transfer therapies for patients with metastatic melanoma.
PMCID: PMC2430884  PMID: 11714800
21.  Sustained Expression of Naked Plasmid DNA Encoding Hepatocyte Growth Factor in Mice Promotes Liver and Overall Body Growth 
Hepatology (Baltimore, Md.)  2001;33(4):848-859.
To understand the physiological functions of exogenous hepatocyte growth factor (HGF) on normal adult animals, we delivered human HGF gene into mice by a hydrodynamics-based in vivo gene transfection approach using a naked plasmid vector. Systemic administration of naked plasmid containing HGF cDNA driven under cytomegalovirus promoter (pCMV-HGF) by rapid injection via the tail vein produced a remarkable level of human HGF protein in the circulation, beginning to appear at 4 hours and peaking at 12 hours following injection. Tissue distribution studies identified the liver as the organ with the highest level of transgene expression. Through weekly repeated injections of plasmid vector, we achieved sustained, long-term, high levels of exogenous HGF expression in mice for 8 weeks. Increases of more than 31% and 16% in liver and body weights were found, respectively, in the mice that received pCMV-HGF plasmid compared with that given the control vector for 8 weeks. Expression of exogenous HGF in vivo activated mitogen-activated protein kinases and induced proliferating cell nuclear antigen expression in normal adult liver and kidneys. These data suggest that systemic administration of naked plasmid vector is a convenient, safe, and highly efficient approach to introduce and maintain exogenous HGF gene expression in vivo in a controllable fashion. Our results also indicate that long-term expression of human HGF in mice markedly activates growth-related signal transduction events, promotes cell proliferation, and leads to liver and overall body growth in whole adult animals.
doi:10.1053/jhep.2001.23438
PMCID: PMC1821076  PMID: 11283849
22.  Ligation of CD40 Stimulates the Induction of Nitric-oxide Synthase in Microglial Cells* 
The Journal of biological chemistry  2001;276(48):44527-44533.
The present study was undertaken to investigate the role of CD40 ligation in the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 microglial cells and primary microglia. Ligation of CD40 alone by either cross-linking antibodies against CD40 or a recombinant CD40 ligand (CD154) was unable to induce the production of NO in BV-2 microglial cells. The absence of induction of NO production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggests that a signal transduced by the ligation of CD40 alone is not sufficient to induce NO production. However, CD40 ligation markedly stimulated interferon-γ (IFN-γ)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-γ-induced production of NO. This stimulation of NO production was accompanied by stimulation of the iNOS protein and mRNA. In addition to BV-2 glial cells, CD40 ligation also stimulated IFN-γ-mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mechanism of induction/stimulation of iNOS, we investigated the roles of nuclear factor κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ), transcription factors responsible for the induction of iNOS. IFN-γ alone was able to induce the activation of NF-κB as well as C/EBPβ. However, CD40 ligation alone induced the activation of only NF-κB but not of C/EBPβ, suggesting that the activation of NF-κB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPβ is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Δp65) and C/EBPβ (ΔC/EBPβ) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-γ and CD40 ligand. Stimulation of IFN-γ-mediated activation of NF-κB but not of C/EBPβ by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-γ-treated BV-2 microglial cells through the stimulation of NF-κB activation. This study illustrates a novel role for CD40 ligation in stimulating the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.
doi:10.1074/jbc.M106771200
PMCID: PMC2041871  PMID: 11551948
23.  Sodium homeostasis with chronic sodium loading in preascitic cirrhosis 
Gut  2001;49(6):847-851.
BACKGROUND—Preascitic cirrhotic patients receiving 200 mmol of sodium daily for seven days remain in positive sodium balance. Thereafter, sodium handling is unknown.
AIM—To assess renal sodium handling in preascitic cirrhosis on a high sodium diet for five weeks.
METHODS—Sixteen biopsy proven preascitic cirrhotics were assessed at weekly intervals for five weeks on a diet of 200 mmol sodium/day using a daily weight diary and weekly 24 hour urinary sodium estimations. Fasting supine neurohormone levels were measured at baseline and weekly for five weeks while haemodynamics were measured at baseline and at five weeks.
RESULTS—The daily diet of 200 mmol of sodium resulted in weight gain and a positive sodium balance for three weeks, associated with significant suppression of plasma renin activity and aldosterone levels, and a significant rise in plasma atrial natriuretic peptide levels (p<0.05). Patients' weights plateaued during week 4, associated with complete sodium balance and significant suppression of plasma noradrenaline levels (p<0.05). This was followed by a negative sodium balance and weight loss, and finally complete sodium balance, again despite a mean net gain of 2.3 (0.3) kg, associated with a return of plasma renin activity and aldosterone levels to within normal ranges. The lack of increase in central blood volume in addition to the persistent increase in plasma atrial natriuretic peptide levels indicated that residual volume expansion, consequent to persistent weight gain, was distributed on the venous side of the circulation. No free fluid was seen on repeat abdominal ultrasound after five weeks.
CONCLUSION—Preascitic cirrhotics have a natriuretic "escape" after three weeks on high sodium dietary intake, associated with elevated plasma atrial natriuretic peptide levels and suppression of the renin-angiotensin-aldosterone system. With continued suppressed sympathetic activity, preascitics re-establish complete sodium balance but with a net weight gain and presumed increased intravascular volume, but without ascites. This further elucidates the compensated sodium retaining abnormality that characterises preascitic cirrhosis.


Keywords: preascitic cirrhosis; sodium handling; renin-angiotensin-aldosterone system
doi:10.1136/gut.49.6.847
PMCID: PMC1728551  PMID: 11709521
24.  Pulsatile ocular blood flow in asymmetric exudative age related macular degeneration 
The British Journal of Ophthalmology  2001;85(12):1411-1415.
BACKGROUND/AIMS—Decreased perfusion or increased vascular resistance of the choroidal vessels had been proposed as the vascular pathogenesis for age related macular degeneration (AMD). This study planned to answer the question whether pulsatile ocular blood flow (POBF) was different in patients with asymmetric exudative AMD between eyes with drusen, choroidal neovascularisation (CNV), or disciform scar.
METHODS—37 patients with asymmetric exudative AMD were enrolled in this observational case series study. POBF were measured in both eyes of each subject. Eyes with high myopia, anisometropia, recent laser treatment, and glaucoma were excluded.
RESULTS—After adjusting for ocular perfusion pressure, intraocular pressure, and pulse rate, multivariate regression analysis with generalised estimating equation showed POBF was significantly higher in eyes with CNV (1217 (SD 476) µl/min) than the contralateral eyes with drusen (1028 (385) µl/min) (p = 0.024). Eyes with disciform scar had lower POBF than the contralateral eyes with drusen (999 (262) µl/min and 1278 (341) µl/min, respectively, p<0.001). There was no significant correlation between the POBF and the lesion size of the CNV.
CONCLUSION—The POBF in eyes with drusen was lower than their fellow eyes with CNV, but higher than their fellow eyes with disciform scar. This finding suggests that haemodynamic differences between fellow eyes in individuals are relevant to the development of CNV and the formation of disciform scar. Further studies on the follow up patients might shed light on the pathogenesis of exudative AMD.


doi:10.1136/bjo.85.12.1411
PMCID: PMC1723809  PMID: 11734510
25.  Is it time for a new attitude to "simultaneous" bilateral cataract surgery? 
The British Journal of Ophthalmology  2001;85(12):1489-1496.
doi:10.1136/bjo.85.12.1489
PMCID: PMC1723798  PMID: 11734526

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