Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor‐expressing neurons and glia

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene‐carrying retroviral vectors is described. Use of a vector encoding the oncogene v‐myc results in lines of nestin‐positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate. In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin‐positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).     

Periodontal health in 200 HIV-positive patients

Two hundred HIV-positive subjects were surveyed to determine their periodontal health status. Particular attention was given to the occurrence of a severe and rapidly progressing form of periodontal disease designated "HIV-associated periodontitis", which has been reported as being unique to AIDS patients. Among the subjects comprising the cohort, 85 subjects had good gingival health, 59 subjects exhibited gingivitis, 49 cases of adult periodontitis were observed, 5 subjects presented with advance adult periodontitis, and 2 cases of necrotizing ulcerative periodontitis (NUP) were found within the group. The periodontitis of the patients in this survey did not have unique or pathognomonic characteristics which could set their periodontal disease apart from the periodontal disease seen in HIV negative population.     

Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii

Summary Genetic analysis of 25 nuclear mutants defective in the chlorophyll-protein complex CP1 was undertaken. The mutants belong to 13 complementation groups scattered throughout the nuclear genome. All these mutants lack the apoprotein of CP1 and, in addition, a specific set of six low molecular weight thylakoid polypeptides. System I particles obtained by treating WT thylakoid membranes with detergent specifically contain those polypeptides which the mutants lack. These observations suggest that a particular sub-structure of the thylakoid membrane associated with the photosystem I activity is missing from all 25 mutants studied, and that this general phenotype can result from mutation at any one of several unlinked Mendelian loci.     

Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein

YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic Yersinia species. YopB is required for Yersinia spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed. Yersinia pseudotuberculosis strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.     

Interstitial deletion of 10q: clinical features and literature review.

We report on a patient with interstitial deletion of 10q and compare her to 8 previously described patients, 2 of whom have chromosomal breakpoints similar to our patient. Minor anomalies including broad forehead, hypertelorism, strabismus, prominent philtrum, and "dysplastic" pinnae are present in our patient. Psychomotor retardation and hypotonia are universal findings in 10q interstitial deletion. Growth retardation, not present in our patient, is seen in some. These clinical findings are sufficiently distinct to suggest early chromosome studies.     

Cognitive and motor skills in achondroplastic infants: Neurologic and respiratory correlates

Thirteen infants with achondroplasia underwent psychometric testing as part of a comprehensive neurologic assessment. As a group, mental development was average and motor development was delayed, although a wide range of scores was obtained. Foramen magnum measurements were correlated with respiratory dysfunction, abnormal so-matosensory evoked potentials, and delayed motor development. Abnormal polysomno-gram outcome was associated with reduced mental capacity. In light of the reported increased frequency of respiratory dysfunction in achondroplasia, these findings warrant careful attention and further study.