Faculty Bibliography

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.

Ephrin/Eph signalling is crucial for axonal pathfinding in vertebrates and invertebrates. We identified the Drosophila ephrin orthologue, Dephrin, and describe for the first time the role of ephrin/Eph signalling in the embryonic central nervous system (CNS). Dephrin is a transmembrane ephrin with a unique N terminus and an ephrinB-like cytoplasmic tail. Dephrin binds and interacts with DEph, the Drosophila Eph-like receptor, and Dephrin and DEph are confined to different neuronal compartments. Loss of Dephrin or DEph causes the abberant exit of interneuronal axons from the CNS, whereas ectopic expression of Dephrin halts axonal growth. We propose that the longitudinal tracts in the Drosophila CNS are moulded by a repulsive outer border of Dephrin expression.

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region

The study was to observe the therapeutic effect of HJ-1 NO--HFJV respirator on treating pulmonary edema caused by seawater drowning. Seawater was infused into the rabbit's lung to establish the animal model of pulmonary edema caused by seawater drowning(PE-SWD). Then the animals were divided into three groups: simple PE-SWD model as control group, treat group(animal model treated with HFJV respirator and four medicines) and HFJV respiratior plus NO group. Pao2, Sao2 and pH were measured by the blood-gas analyzer. The survival time and seawater drowing-respiratiory distress syndrom(SW-RDS) were observed. The results showed that Pao2, Sao2 in NO group were remarkably higher than that of PE-SWD control group, and the survival time was longer than that of medicine treated group and the incidence of SW-RDS decreased to zero. We assume that HJ-1 NO-HFJV respirator is efficient on treating pulmonary edema

The atmospheric concentration of NO2 in Lima, Peru was measured through 1 year using passive samplers. The concentration was stable and evident seasonal change was not observed. Also, the distribution of NO2 concentration in entire Lima was monitored twice in different seasons. The average NO2 concentrations at 33 and 27 sites of these monitoring were 17.1 and 15.3 ppb, respectively. NO2 distribution was high in the downtown area and decreased gradually with distance from there. The wind that almost always blew from the ocean had a great influence on it. High NO2 level and the change of CO concentration suggest that the residential area in the northeast side forms a topological channel among hills to blow out the pollutants from the downtown area.

OBJECTIVE: Corticotropin-releasing hormone deficient mice (CRH-KO) serve as an interesting model to understand the role of CRH in the regulation of adrenomedullary system. The aim of this study was to compare tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) on the levels of gene expression and protein in adrenal medulla of CRH-KO mice, their CRH (+/+) mates and Sprague-Dawley (SD) rats. METHODS: Levels of TH and PNMT mRNA were determined by reverse transcription with subsequent polymerase chain reaction (RT-PCR) and quantified relatively to the housekeeper glyceraldehyde-3-phosphate dehydrogenase. The amount of TH and PNMT protein was determined by Western blot analysis and visualized by enhanced chemiluminiscence. RESULTS: We detected a clear signal of 645 bp for TH mRNA and of 260 bp for PNMT mRNA in adrenal medulla of rats and CRH (+/+) mice, with higher concentration of TH and PNMT mRNA in rat adrenal medulla. Subsequently, TH and PNMT immunoprotein was measured and we found significantly higher amount of TH and also PNMT protein in the rat compared to CRH (+/+) mice. On the other hand, the amount of TH and PNMT immunoprotein in adrenal medulla of CRH-KO mice was significantly lower compared to CRH (+/+) mice. CONCLUSIONS: Our results indicate the lower production of adrenomedullary TH and PNMT protein in CRH (+/+) mice compared to rats, which reflects the lower gene expression of these enzymes in adrenal medulla of mice. We also demonstrated the differences in TH and PNMT protein levels between CRH (+/+) and CRH-KO (-/-) mice.

OBJECTIVE: The emergence of Candida glabrata infections among patients with compromised immunity has become a serious concern, especially at centers caring for individuals with cancer. METHODS: During a prospective evaluation of Candida species associated with either clinically significant colonization or infection, 26.9% of C. glabrata isolates showed in vitro resistance to fluconazole (MIC of > or = 64 microg/ml). RESULTS: Antifungal susceptibility profiles and genetic fingerprinting analysis performed by randomly amplified polymorphic DNA (RAPD) techniques confirmed low-probability of phenotypic and genotypic relatedness among nosocomial C. glabrata isolates. CONCLUSIONS: Presence of polyclonal strains of C. glabrata in patients at our hospital was probably related to selection of resistant yeasts from environmental pool rather than monoclonal expansion or clustering of multi-drug resistant C. glabrata in high-risk patients

Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane

OBJECTIVE: To explore the effect of human endostatin expressed by host cells on the growth of human liver carcinoma in vivo. METHODS: Human endostain gene was transferred into SMMC7721 cells by retroviral pLncx to build endostatin-transfected cell line. PCR, immunohistochemistry and Western blot analysis were applied to examine the transfection, expression and secretion of endostatin. Endothelial cell proliferation assay was used to determine the biological activity of expressed endostatin. The in vivo and in vitro growth rates of the endostatin-transfected and control SMMC7721 cells were also observed. RESULTS: PCR proved that the genome of endostatin-transfected SMMC7721 cells contained a 550 bp specific fragment of endostatin. The expression and secretion of human endostatin from endostatin-transfected SMMC7721 cells were confirmed by immunohistochemistry and Western blot analysis. Endostatin expressed by host cells could inhibit the proliferation of human umbilical vein endothelial cells by 48% (P < 0.01). In vitro proliferation assay showed that endostatin-transfected SMMC7721 cells had no change in proliferation rate compared to control SMMC7721 cells. In comparison with control group, however, tumor growth rate in vivo from endostatin-transfected SMMC7721 cells was inhibited greatly by 94.5%, 22 days after inoculation into nude mice (P < 0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit greatly the growth of human liver carcinoma SMMC7721 in vivo