Nuclear magnetic resonance analysis of solution conformations in C4-V3 hybrid peptides derived from human immunodeficiency virus (HIV) type 1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.     

Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents

ClC-4 and ClC-5, together with ClC-3, form a distinct branch of the CLC chloride channel family. Although ClC-5 was shown to be mainly expressed in endocytotic vesicles, expression of ClC-5 in Xenopus oocytes elicited chloride currents. We now show that ClC-4 also gives rise to strongly outwardly rectifying anion currents when expressed in oocytes. They closely resemble ClC-5 currents with which they share a NO3- > Cl- > Br- > I- conductance sequence that differs from that reported for the highly homologous ClC-3. Both ClC-4 and ClC-5 currents are reduced by lowering extracellular pH. We could measure similar currents after expressing either channel in HEK293 cells. To demonstrate that these currents are directly mediated by the channel proteins, we introduced several point mutations that change channel characteristics. In ClC-5, several point mutations alter the kinetics of activation but leave macroscopic rectification and ion selectivity unchanged. A mutation (N565K) equivalent to a mutation reported to have profound effects on ClC-3 does not have similar effects on ClC-5. Moreover, a mutation at the end of D2 (S168T in ClC-5) changes ion selectivity, and a mutation at the end of D3 (E211A in ClC-5 and E224A in ClC-4) changes voltage dependence and ion selectivity. This shows that ClC-4 and ClC-5 can directly mediate plasma membrane currents.     

Contribution of NMDA and non-NMDA receptors to synaptic transmission from the brachium of the inferior colliculus to the medial subdivision of the medial geniculate nucleus in the rabbit

Previous work from this laboratory has demonstrated that monosynaptic inputs from the brachium of the inferior colliculus (BIC) to the medial subdivision of the medial geniculate nucleus (mMG) strengthen as a result of associative conditioning with an acoustic conditioned stimulus (i.e., fear conditioning). One model that has been proposed to underlie certain types of neuronal plasticity involves the recruitment of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors. The purpose of the present study was to examine the relative contributions of glutamatergic NMDA and non-NMDA receptors to synaptic transmission within this pathway. Individual contributions of the specific receptor types were assessed through the use of 2-amino-5-phosphonovaleric acid (AP5), a selective NMDA receptor antagonist, and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. Bipolar stimulating electrodes were stereotaxically implanted in BIC and recording electrodes (attached to dual 32-gauge cannulae for delivery of drug) were positioned in mMG of New Zealand albino rabbits. Single pulses (150 micros, 100-350 microA) delivered to BIC resulted in short-latency (<4 ms) responses in mMG. BIC-evoked single-unit activity was recorded from mMG before, during, and at several intervals after injection of AP5, CNQX, and/or artificial cerebrospinal fluid (ACSF). Injection of either AP5 or CNQX, but not ACSF, significantly attenuated the short-latency BIC-evoked responses in the vast majority of cells tested. These findings suggest that the monosynaptic pathway from BIC to mMG is glutamatergic and that this pathway frequently employs NMDA-type receptors during electrically stimulated synaptic transmission. Due to the NMDA receptors' proposed role in plasticity (e.g., long-term potentiation), these results may have implications for understanding the mechanisms of synaptic plasticity observed at this synapse during associative learning.     

Comparison of methods to enumerate bacteria in dental unit water lines

Millipore HPC samplers are simple, self-contained test devices that can be used by personnel in dental offices who do not have microbiologic training to easily and economically monitor dental unit water quality without laboratory support. This study evaluated the correlation of HPC samplers to R2A agar for enumerating planktonic bacteria in dental unit treatment water. Eight different dental units were sampled. Five replicates were performed for each media at each dilution. The Pearson correlation coefficient between the R2A agar and HPC sampler is 0.89. These data suggest HPC samplers correlate with conventional laboratory-based R2A culture techniques for determining dental unit water line contamination.     

Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand

PATIENTS AND METHODS: The role of cerebral magnetic resonance imaging (MRI) in the diagnosis of growth hormone (GH) deficiency in children has been studied in 100 children. The diagnosis of GH deficiency was assessed at a mean age of 6.7 +/- 4.1 years: morphological abnormalities of the hypothalamic-pituitary (HP) region have been studied in three different groups: in the first group (70 cases), the neurohypophysis was present and normally located; in the second group (ten cases) it was missing; in the third group (20 cases) the neurohypophysis was ectopic (truncated stalk syndrome with ectopic neurohypophysis, small antehypophysis, thin or non-visualized stalk). RESULTS: In the majority of cases, children presenting with only one morphological abnormality of the HP region (ectopic neurohypophysis or small antehypophysis or non-visualized or thin stalk) had an isolated GH deficiency. When multiple morphological abnormalities were present, anterior pituitary deficiency was multiple in more than half the cases. Cerebral midline anomalies (above all Chiari I malformation and basipharyngeal canal) had been observed in 20% of the children presenting with GH deficiency. In the majority of cases (95%), these anomalies were associated with one or more abnormalities of the HP region. A familial case is reported: morphological anomalies of the HP region were different for both siblings. Genetic factors are evoked. CONCLUSION: The severity of the hormone deficiency is correlated to the ectopic location of the neurohypophysis, the thin appearance or non visibility of the pituitary stalk and the associated midline anomalies.     

Excision of products of oxidative DNA base damage by human NTH1 protein

A functional human homologue of Escherichia coli endonuclease III (Nth-Eco protein) has recently been cloned and characterized [Aspinwall, R., Rothwell, D. G., Roldan-Arjona, T., Anselmino, C., Ward, C. J., Cheadle, J. P., Sampson, J. R., Lindahl, T., Harris, P. C., and Hickson, I. D. (1997) Proc. Natl. Acad. Sci. U.S.A., 94, 109-114]. This enzyme, designated hNTH1 protein, shares an extensive sequence similarity with Nth-Eco protein and a related enzyme from Schizosaccharomyces pombe (Nth-Spo protein). We investigated the substrate specificity of this human enzyme for oxidative DNA base damage, using the technique of gas chromatography/isotope-dilution mass spectrometry. Four different DNA substrates damaged by various free radical-generating systems were used. 5-Hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil were substrates of hNTH1 protein among 17 lesions found in DNA substrates. The substrate specificity and excision kinetics of the human enzyme were found to be significantly different from those of Nth-Spo and Nth-Eco proteins.     

Assessing medical care of dying residents in nursing homes

A rational approach to hemodialysis access in the patient with renal failure requires an understanding of many factors. The initial approach to such patients and subsequent decisions have a significant impact on quality of life as well as the morbidity of access choices. Autogenous vascular access remains the gold standard in the care of these patients. Alternatives are less desirable but are necessary in an increasing number of situations. Careful thought before embarking on a procedure will provide better long-term function and preserve the patient's options for the future.     

Differential effects of deoxycholic acid on proliferation of neoplastic and differentiated colonocytes in vitro

The secondary bile acid deoxycholic acid is believed to be a promoter of large bowel cancer, in part by inducing colonic epithelial proliferation. The effects of deoxycholic acid on [3H]thymidine incorporation by the human colon cancer cell line HT29 and two differentiated subclones were measured and compared. The subclone HT29-C1 has features of mature absorptive cells and HT29-N2 cells secrete mucus under cholinergic control. The three cell lines were treated with deoxycholic acid (DCA) at concentrations of 0, 5, 10, 50, 100, 150, and 300 microM for 3, 6, 9, 15, 24, and 48 hr. A significant increase in proliferation was noted in HT29 cells only at 6 hr with 5 and 10 microM deoxycholic acid. Neither the subclone HT29-C1, nor HT29-N2 cells exhibited significant change in [3H]thymidine incorporation with DCA at these concentrations or time points. Higher doses of deoxycholic acid above 50 microM and duration of exposure greater than 24 hr were cytotoxic to all three cell lines. The proliferative effects of DCA in HT29 cells were not paralleled by changes in protein kinase C activity or protein kinase C isoform expression. Quantitative and qualitative differences in PKC isoform expression were not noted in the three cell lines used in this study. The proliferative effects of DCA on HT29 cells appear to be independent of the PKC signal transduction pathway.