Faculty Bibliography

Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40 degrees C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40 degrees C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40 degrees C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40 degrees C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4',6'-diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40 degrees C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.

The cerebral deposition of amyloid beta-peptide, a central event in Alzheimer's disease (AD) pathogenesis, begins several years before the onset of clinical symptoms. Noninvasive detection of AD pathology at this initial stage would facilitate intervention and enhance treatment success. In this study, high-field MRI was used to detect changes in regional brain MR relaxation times in three types of mice: 1). transgenic mice (PS/APP) carrying both mutant genes for amyloid precursor protein (APP) and presenilin (PS), which have high levels and clear accumulation of beta-amyloid in several brain regions, starting from 10 weeks of age; 2). transgenic mice (PS) carrying only a mutant gene for presenilin (PS), which show subtly elevated levels of Abeta-peptide without beta-amyloid deposition; and 3). nontransgenic (NTg) littermates as controls. The transverse relaxation time T(2), an intrinsic MR parameter thought to reflect impaired cell physiology, was significantly reduced in the hippocampus, cingulate, and retrosplenial cortex, but not the corpus callosum, of PS-APP mice compared to NTg. No differences in T(1) values or proton density were detected between any groups of mice. These results indicate that T(2) may be a sensitive marker of abnormalities in this transgenic mouse model of AD

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells

Abstract The enzyme gamma-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid beta-peptide from the beta-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature gamma-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores gamma-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another gamma-secretase complex component. Analysis of gamma-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on gamma-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional gamma-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly

BACKGROUND: Five patients with DiGeorge syndrome presented with infections, skin rashes, and lymphadenopathy after the newborn period. T-cell counts and function varied greatly in each patient. Initial laboratory testing did not suggest athymia in these patients. OBJECTIVE: The purpose of this study was to determine whether the patients had significant immunodeficiency. METHODS: Research testing of peripheral blood included immunoscope evaluation of T-cell receptor beta variable gene segment repertoire diversity, quantification of T-cell receptor rearrangement excision circles, and detection of naive T cells (expressing CD45RA and CD62L). RESULTS: The patients were classified as having DiGeorge syndrome on the basis of syndromic associations and heart, parathyroid, and immune abnormalities. Immunoscope evaluation revealed that the T-cell repertoires were strikingly oligoclonal in all patients. There were few recent thymic emigrants, as indicated by the very low numbers of naive T cells (<50/mm(3)) and the absence of T-cell receptor rearrangement excision circles. These studies showed that all 5 patients were athymic. Two patients died, one from infection. No thymus was found during the complete autopsy performed on one patient. CONCLUSION: Patients with DiGeorge syndrome, skin rash, and lymphadenopathy should undergo analysis of naive T-cell numbers and of T-cell receptor beta variability segment repertoire to determine whether they are athymic, even if they have T cells with mitogen responsiveness. It is important for physicians to realize that patients with complete DiGeorge syndrome remain profoundly immunodeficient after development of these atypical features (rash, lymphadenopathy, and oligoclonal T cells). Prompt diagnosis is necessary for appropriate management.

The epidermal growth factor receptor (EGFR) is overexpressed and/or constitutively activated in a variety of human malignancies. Detection of increased expression levels of EGFR in cancer and the association between overexpression and decreased patient survival has led to the development of several therapeutic strategies to target this receptor. The results of early-phase clinical trials to date suggest that targeting EGFR alone may not be sufficient to eradicate established tumors. This limited antitumor efficacy as monotherapy has led to combining EGFR inhibitors with chemotherapy or radiation therapy for advanced disease, or incorporating EGFR inhibition to cancer prevention approaches. This review will discuss the role of EGFR signaling in carcinogenesis and the rationale for EGFR inhibition as a clinical prevention and treatment strategy.

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis

OBJECTIVE: To establish of acute respiratory distress syndrome (ARDS) model in canine after inhalation of perfluoroisobutylene (PFIB), and to observe the progressing of lung injury, and to study the mechanisms of injury. METHODS: A device of inhalation of PFIB for canine was made. The concentration of PFIB was 0.30 - 0.32 mg/L. Serum IL-6 and IL-8 were dynamically measured. Clinical manifestations, pathology of organs in canine were observed. RESULTS: (1) During inhalation, the concentration of PFIB remained stable; (2) After inhalation, blood arterial oxygen partial pressure fell gradually, and eventually met the criteria for diagnosing ARDS; (3) The level of IL-8 in serum rises significantly after inhalation (P < 0.05), whereas that of IL-6 was not obviously altered (P > 0.05); (4) Within 6 hours after inhalation, no abnormality in canine was observed, but afterwards symptoms gradually appeared, and typical breath of ARDS, such as high frequency and lower level could be seen in later phase; (5) Pathological examination showed severe congestion, edema and atelectasis in most part of both lungs, and signs of anoxia in other organs. CONCLUSIONS: (1) The device designed is capable of ensuring control of inhalation of PFIB; (2) Exposure to PFIB for 30 mins, canines all met the criteria for diagnosing ARDS 22 hours after inhalation, therefore the modeling is successful; (3) PFIB specifically damages the lung by causing excessive inflammation.

The horizontal gene transfer (HGT) being inferred within prokaryotic genomes appears to be sufficiently massive that many scientists think it may have effectively obscured much of the history of life recorded in DNA. Here, we demonstrate that the tree of life can be reconstructed even in the presence of extensive HGT, provided the processes of genome evolution are properly modeled. We show that the dynamic deletions and insertions of genes that occur during genome evolution, including those introduced by HGT, may be modeled using techniques similar to those used to model nucleotide substitutions that occur during sequence evolution. In particular, we show that appropriately designed general Markov models are reasonable tools for reconstructing genome evolution. These studies indicate that, provided genomes contain sufficiently many genes and that the Markov assumptions are met, it is possible to reconstruct the tree of life. We also consider the fusion of genomes, a process not encountered in gene sequence evolution, and derive a method for the identification and reconstruction of genome fusion events. Genomic reconstructions of a well-defined classical four-genome problem, the root of the multicellular animals, show that the method, when used in conjunction with paralinear/logdet distances, performs remarkably well and is relatively unaffected by the recently discovered big genome artifact.

Talin and RapL are components of molecular pathways that regulate the avidity of the integrin lymphocyte function-associated antigen 1 (LFA-1) for its ligand, intercellular adhesion molecule 1. In this review, we discuss recent advances in our understanding of LFA-1 affinity regulation and signaling and discuss a scenario for how Talin and Rap1 might act in synergy to achieve regulation of LFA-1 that is tailored to the specific functional requirements of different situations. Speedy delivery of signals may be crucial, and membrane trafficking from endosomes and the Golgi apparatus seem to be essential in delivering the messages from spatially segregated surface receptors