HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals.

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.

Apoptotic effects in primary human umbilical vein endothelial cell cultures caused by exposure to virion-associated and cell membrane-associated HIV-1 gp120.

During the course of HIV-1 infection, free virus, infected cells, and free HIV-1 proteins circulate within the host, exposing the host endothelium to these viral factors. We have previously presented evidence showing that soluble HIV-1 gp120 protein interacts with chemokine receptors on primary human endothelium and (through those interactions) induces apoptosis as well as other intracellular effects. The current study examines the effect of exposure of vascular endothelium to gp120 IIIb expressed on the surface of Jurkat cells and in the context of viral particles. Apoptosis was observed in human umbilical vein endothelial cell (HUVEC) cultures exposed to gp160-transfected Jurkat cells as well as to virion particles with gp120 on their surface. Additional experiments show that this apoptotic effect was caused by gp120 protein acting through chemokine receptors on the HUVEC surface, primarily the CXCR4 receptor. At higher concentrations of gp120, this lymphotrophic variant, which has been shown to interact predominantly with CXCR4, seems to interact with and induce apoptosis through the CCR5 receptor. Finally, this apoptotic effect in HUVEC cultures occurs at low levels of the inducing agent, gp120, on cell membranes or on virion particles. These results demonstrate that HIV-1 gp120 is capable of interacting with and killing vascular endothelial cells in multiple in vivo contexts.

Internet resources for Tibetan medicine.

Tibetan medicine has a history of over one thousand years. With the recent fascination with Tibet and Tibetan culture, Tibetan medicine is receiving greater attention from the public, scholars, and the media. In the past few years, researchers and practitioners of Tibetan medicine have established a presence on the Internet, evident through Web sites and discussion groups. This paper presents a sampling of valuable resources about Tibetan medicine readily accessible on the Internet. The selected sites were evaluated on the basis of quality and quantity of information, authoritativeness, currency of material, quality of links, and navigability.

Effects of extracellular human immunodeficiency virus type 1 vpr protein in primary rat cortical cell cultures.

Recent evidence suggests that HIV-1 Vpr exists in soluble form in the serum and cerebrospinal fluid (CSF). Further, its abundance in the bloodstream, and the CSF, and its activity on other cell types suggest that it could have an effect on brain activity. Using mixed embryonic rat brain cultures as a model to examine the effects of physiological concentrations of extracellular Vpr protein, Vpr-induced cell death was observed. We also observed similar Vpr-induced effects in enriched primary cortical rat astrocytes, as well as in the C6 glioma cell line. Vpr-induced cell death observed in the astrocytic cells appeared to be caused primarily by a necrotic mechanism, although a few apoptotic nuclei were also present. We did not observe Vpr-induced effects on any primary cortical neurons, although we did observe Vpr-induced cell death in hippocampal neurons and astrocytes. Finally, we observed no cell cycle effects due to extracellular Vpr protein. This data points out that different cell types are affected by the toxic effects of extracellular Vpr protein, and that differential toxic effects of extracellular Vpr protein are observed in similar cell types.

Involvement of protein kinase C in HIV-1 gp120-induced apoptosis in primary endothelium.

We previously showed that HIV-1 gp120-induced apoptosis in primary human umbilical vein endothelial cell cultures (HUVEC), through CCR5 and CXCR4. Here, we have found that agonists of protein kinase C (PKC), basic fibroblast growth factor (bFGF), and short exposure to low concentrations of phorbol esters were found to block gp120-induced apoptosis in HUVEC cultures. PKC antagonists, sphingosine, H7, and extended exposure of cultures to high concentrations of phorbol esters were also found to block gp120-induced apoptosis in HUVEC cultures. A significant increase in the total amount of cellular PKC enzymatic activity was observed on exposure of HUVEC to gp120. No increase in total PKC activity was observed on exposure of HUVECs to the natural ligands SDF-1alpha, or regulated-on-activation normal T-expressed and secreted (RANTES) cells, and gp120-induced PKC induction was found to be totally blocked by CXCR4 antibodies and partially blocked by the caspase 3 inhibitor, DEVD-CHO. Alternatively, CXCR4 antibodies and DEVD-CHO totally blocked apoptosis. Finally, gp120-induced effects were found to be insensitive to pertussis toxin. Accumulated evidence suggests PKC involvement at multiple points in the gp120-induced apoptotic pathway; also suggests involvement of the CXCR4 receptor internalization pathway, and potentially suggests different downstream effects of gp120-receptor interactions and natural ligand-receptor interactions.

Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures.

During the course of an HIV-1 infection, free infectious and noninfectious virus particles, and free HIV-1 proteins, circulate within the host, exposing the host endothelium to these viral factors, even if the endothelium is not infected. This suggests that extracellular HIV-1 proteins could influence endothelial cell function, leading to pathogenesis. In light of this, we have used primary cultured human vascular endothelial cells (HUVECs) to screen for effects of the HIV-1 protein gp120 on endothelial cell function. The results of this study show that short exposure of HUVEC cultures to this protein causes significant levels of cytotoxicity. Further, using several different assays, we have shown that this cytotoxic effect on HUVECs appears to be due to induction of an apoptotic program. The biphasic nature of gp120 titration curves suggests that multiple cellular factors are mediating these gp120-induced effects. Competition studies appear to confirm this by showing that the apoptotic effect is mediated through two cell surface receptors on HUVECs, CCR5 and CXCR4. Alternatively, competition studies examining CD4 receptors suggests that CD4 played no role in gp12O-induced effects on HUVECs.

[Separation of pharmaceutical enantiomers on column and thin-layer plate of cellulose triacetate].

Microcrystalline cellulose triacetate (CTA) was prepared by heterogeneous acetylation of microcrystalline cellulose and used as liquid chromatographic chiral stationary phase and chiral thin layer plate to separate pharmaceutical enantiomers. The racemic Troegor's base, mathaqualone, chlormezanone and chloroquine were separated on the CTA chiral column and on the CTA chiral plate. 95% ethanol and the mixtures of 95% ethanol and water with different ratio and different pH values were used as mobile phase. The influences of eluent composition, pH and temperature on the chiral separation were discussed. The experimental results indicate that the CTA chiral plate was more sensitive to eluent composition and pH than chiral column possibly because the chiral adsorbent was not swollen on the plate.

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression.

The HIV-1 encoded regulatory Rev protein acts to selectively increase the cytoplasmic concentration of incompletely spliced viral mRNAs through interaction with the Rev responsive element (RRE). In addition, the Rev activation domain, believed to be a nuclear export sequence, has been shown to modulate the export of non-RRE containing RNAs (e.g. 5S rRNA, splicesomal U snRNAs). Recent evidence suggests Rev activity depends on interactions with cellular cofactors, leading to speculation that Rev utilizes a cellular RNA and/or a protein export pathway. Rev interactions with cellular cofactors could lead to sequestration of those cofactors from normal cellular activities, suggesting potential Rev effects on cellular gene products and their resultant activity. We have examined the role of Rev in modulating the expression of cellular gene products. Through transient cotransfection assays, we observed a consistent and significant decrease in the levels of luciferase and B-galactosidase activity in the presence of a Rev expressing construct. Cell fractionation studies demonstrated the nuclear retention of the luciferase gene transcripts. Surprisingly, similar effects were observed on constitutively expressed RNAs such as gamma-actin transcripts, and the 18S and 28S rRNAs. These results suggest Rev can disrupt the nuclear export of multiple classes of RNAs.

Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae.

We developed PCR primers specific for the blaTEM and blaROB ampicillin resistance genes. The specificity of the primers was confirmed by testing a series of Escherichia coli isolates containing a variety of ampicillin resistance genes and a series of ampicillin-resistant and ampicillin-susceptible Haemophilus influenzae isolates. There was a perfect correlation between ampicillin MICs, the presence of beta-lactamase (as determined by the nitrocefin test), and the results with the blaTEM and blaROB primers. Isolates of H. influenzae and Streptococcus pneumoniae obtained from 25 frozen cerebrospinal fluid (CSF) specimens were also tested. Four of 14 H. influenzae isolates were positive with the blaTEM primers; none were positive with the blaROB primers. Ampicillin MICs were determined for the H. influenzae isolates, and penicillin MICs were determined for the S. pneumoniae isolates. Only the four PCR-positive H. influenzae isolates had elevated MICs of ampicillin and were beta-lactamase positive. None of the H. influenzae isolates contained the blaROB gene, and none of the S. pneumoniae isolates produced positive reactions with either primer set. We then used universal primers directed to conserved regions of rRNA and a Haemophilus detection probe to identify which of the 25 frozen samples of CSF contained H. influenzae. Fourteen of the 25 CSF specimens were positive for H. influenzae, which correlated with the number of organisms obtained by culture of the CSF samples. Four of the CSF samples were positive with the blaTEM primer set, and these correlated with the four H. influenzae isolates that were positive when tested directly by PCR. The blaTEM assay required the use of native Taq polymerase because Amplitaq preparations were contaminated with vector DNA that contained the blaTEM-1 gene.

Optimizing testing of methicillin-resistant Staphylococcus species.

Selection of the appropriate NaCl concentration for test medium for oxacillin susceptibility testing of Staphylococcus aureus and coagulase-negative staphylococci has been problematic when using different antimicrobial susceptibility testing methods. Broth microdilution, using cation-adjusted Mueller-Hinton broth + 2% NaCl, is the currently recommended reference method. There is currently no recommendation for the addition of NaCl to agar for dilution susceptibility tests when Staphylococcus species are tested with oxacillin. We examined the effects of adding 0, 2%, 4%, and 5% NaCl to Mueller-Hinton agar and broth for agar dilution, Etest, and broth microdilution tests. The results of these tests were compared with the reference broth microdilution results and with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive and had oxacillin minimum inhibitory concentrations (MICs) > or = 4 micrograms/ml. Seven strains of S. aureus were mec probe negative but were oxacillin resistant. Seven coagulase-negative strains (three S. epidermidis, one S. haemolyticus, and three S. simulans) were mec probe positive and were oxacillin susceptible. The MICs for oxacillin-resistant strains increased two- to fourfold with the addition of 2% NaCl, but the MICs for oxacillin-susceptible strains were unchanged. Major and very major interpretative rates ranged from 18.2% to 20.2% for agar dilution and Etest without NaCl added to the medium, and these rates decreased to < 1% with the addition of 2% NaCl to the medium. The addition of 4% or 5% NaCl caused major error rates of > 17% for all test methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods.

The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of < or = 2 micrograms/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of > or = 4 micrograms/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence of added salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were > 17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.9%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of > or = 18.3%. We recommended the addition of 2% NaC1 to Mueller-Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaC1 should not be added for the disk diffusion test.

Accuracy of the E test for determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial agents.

We compared the results of the E test MIC method with the results of agar dilution susceptibility testing for 18 antimicrobial agents against 324 strains of gram-positive and gram-negative bacteria, including 99 strains of staphylococci, 101 strains of antimicrobial-resistant gram-negative bacteria, 40 strains of enterococci, and 84 isolates of Campylobacter jejuni. Overall agreement of MICs (+/- 1 log2 dilution) was 97.3% for staphylococci, 94.6% for gram-negative bacilli, and 100.0% for enterococci. The MIC results for C. jejuni showed an overall agreement of only 82.9%. This was due primarily to a number of offscale values that limited the number of comparisons with clindamycin, trimethoprim-sulfamethoxazole, and tetracycline. Interpretative criteria for the results of the two test methods, however, were similar. Overall, the E test produced MIC results comparable to those of agar dilution when multiresistant organisms were tested. However, it was necessary to add 2% NaCl to the agar when testing oxacillin against staphylococci for both the E test and agar dilution to obtain results comparable to those of the broth microdilution method.