Search

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP[psi(CH2N) indicating a reduced peptide bond], which binds the cell surface-expressed nucleolin, is a potent inhibitor of HIV infection. Here, by using primary T lymphocyte cultures and an experimental cell model to monitor HIV entry, we show that HB-19 inhibits in a dose-dependent manner both T lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at high concentrations. These observations, and the fact that HB-19 has no effect on SIV-mac and HIV-1 pseudotyped with VSV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with beta-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells a few hours after HIV entry. On the other hand, in HB-19-pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop. In view of its specific mode of action on various HIV isolates, HB-19 represents a potential anti-HIV drug.

Cytolysis by natural killer (NK) cells is impaired in HIV infection. We investigated whether the expression of zeta (zeta) molecules, essential elements of signalling initiated upon ligation of, e.g., CD16, is reduced and if so, whether this reduction could be involved in defective cytolysis. FACS analysis revealed significantly lower levels of zeta in NK cells from AIDS patients compared to cells from patients without AIDS and healthy controls. CD16-dependent cytolysis by NK cells correlated with expression of zeta molecules and CD16, the latter possibly related to zeta expression. No correlation was observed between CD16-independent cytolysis and zeta expression. Reduced expression of zeta molecules by NK cells from HIV-infected patients thus correlates with disease progression and may, in part, explain the defective cytolysis by these cells.

Objective: To assess the patterns of HIV phenotypic cross-resistance to protease inhibitors (PI) in patients experiencing viral load rebound on combination therapy including a PI., Methods: Phenotypic analysis of sensitivity to indinavir, nelfinavir, saquinavir, ritonavir and amprenavir was carried out using a single-cycle recombinant virus assay. Viral protease was sequenced by automated dideoxynucleotide chain termination., Results: Of the 108 patients studied, 68 had received indinavir, 50 ritonavir, 25 saquinavir and eight nelfinavir. The majority (71%) had received only one PI. The incidence of cross-resistance between indinavir, nelfinavir, ritonavir and saquinavir was high (60-90%). Cross-resistance to amprenavir was less frequent (37-40%). However there was some correlation between levels of sensitivity to amprenavir and indinavir (r2 = 0.34; P < 0.01). Conversely, the correlation between levels of sensitivity to indinavir and saquinavir was poor (r2 = 0.25), particularly for patients who had not received saquinavir. The degree of cross-resistance correlated with the level of resistance and with the total number of mutations in the protease gene (P < 0.05, chi square test) but could not be significantly correlated to any one particular mutation or combination of mutations. Mutation 184V was significantly associated with cross-resistance to amprenavir, with no mutations at codon 50 observed, while mutations associated with cross-resistance to saquinavir differed according to the treatment received., Conclusions: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.

In human immunodeficiency virus type 1 (HIV-1) infection, functional activities of T lymphocytes are impaired. Analogous to tumor-infiltrating T lymphocytes from cancer patients, in whom poor proliferative responses are associated with fewer zeta molecules, this study compared expression of CD3zeta molecules by T lymphocytes from HIV-infected persons and healthy controls. Flow cytometry and immunoblotting revealed significantly diminished zeta expression by CD3, CD4, and CD8 T lymphocytes from AIDS patients but not from persons without AIDS. zeta-mRNA levels were also decreased in cells from AIDS patients. CD3zeta expression correlated significantly with CD4 cell counts and HIV-1 RNA levels; impaired expression of CD3zeta molecules appeared to be reversible upon virus load reduction following highly active antiretroviral treatment (HAART). Thus, reduced expression of CD3zeta molecules by T lymphocytes from HIV-infected persons correlates with disease status. Investigations into CD3zeta expression by subpopulations of peripheral T lymphocytes and by T lymphocytes in lymphoid tissues will contribute to the understanding of immune reconstitution of HIV-infected patients following HAART.

Newborns are susceptible to hemorrhages (hemorrhagic disease of the newborn or HDN) due to vitamin K deficiency. Induction of cytochrome P450 in the fetal liver by maternal anticonvulsant therapy such as phenobarbital or phenytoin is considered to be a major cause. An observed increase in late hemorrhagic disease (LHD) in breast fed neonates gave rise to the hypothesis that PCBs and dioxins, P450-inducing contaminants present in human milk, might effect vitamin K-dependent blood coagulation. This hypothesis was studied in rats. Administration of a single oral dose of 0.003, 0.03, 0.3, 3 or 30 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per kg bodyweight or 0.75, 4, 20, 100 or 500 micromol 2,2',4,4',5,5'-hexachlorobiphenyl/kg bw (HxCB) to female and male rats resulted in dose-related reductions of the vitamin K-dependent coagulation factor VII. The highest factor VII reduction in female rats was 44%, observed after TCDD exposure. The Lowest Observed Adverse Effect Level (LOAEL) of TCDD on female factor VII levels was 0.3 nmol/kg bw (96 ng/kg). There was a significant inverse correlation between Factor VII levels and induction of hepatic ethoxyresorufin O-deethylating (EROD) activity, reflecting CYP1A1, and total P450 content. HxCB had no effect on female coagulation factors. In contrast, in male rats only exposure to HxCB, which induces mainly CYP2B1 and 2B2, decreased both coagulation factors dramatically up to 88%. The LOAEL of HxCB on factor VII in male rats was 100 micromol/kg bw (36 mg/kg). In general, effects on coagulation factors in male rats exceeded those in females. In addition, sex-dependent differences of TCDD and HxCB were observed on the hepatic vitamin K cycle enzyme activities in female and male rats. Vitamin K-dependent (gamma-glutamyl carboxylase activity was mainly induced in female rats; 2.3-fold in the highest dose group of TCDD. In male rats only vitamin K 2,3-epoxide reductase (KO-reductase) activity was induced 1.7-fold by the highest dose of HxCB. KO-reductase activity in female rats was also increased by TCDD, however, less pronounced than the carboxylase activity. Concluding, the hepatic vitamin K cycle still functions and is not blocked by TCDD or HxCB, thus explaining the observed reduction in factor VII. Finally, the possible role of P450 in vitamin K deficiency is discussed. Based on these results it is suggested to investigate the possible role of PCBs and dioxin-like compounds in LHD in more detail.

Diurnal and nocturnal trunk and limb motor activity of 20 healthy individuals was evaluated by actimetry for 45 consecutive hours. Sleep was assessed by sleep logs. Overall, motor activity significantly (p < .05) decreased in the order wrist, ankle, and trunk. There was significantly more motor activity in the dominant wrist during the diurnal period. Motor activity was significantly affected by the 24-hr sleep-wake cycle, with lower levels and prolonged immobility during the night. Time series analyses revealed different but significant correlations between motor activity at all sites. These data imply that (a) motor activity should be recorded at the dominant wrist when the highest level of motor activity is of importance, (b) recordings at the nondominant wrist are better indicators of trunk movement than are dominant wrist recordings, and (c) sites other than the conventional nondominant wrist recording site should be evaluated to improve the validity of motor activity-based sleep-wake scoring.

The simian retrovirus-1 (SRV-1) gag-pro frameshift signal was identified in previous work, and the overall structure of the pseudoknot involved was confirmed (ten Dam E, Brierley I, Inglis S, Pleij C, 1994, Nucleic Acids Res 22:2304-2310). Here we report on the importance of specific elements within the pseudoknot. Some mutations in stem S1 that maintain base pairing have reduced frameshift efficiencies. This indicates that base pairing in itself is not sufficient. In contrast, frameshifting correlates qualitatively with the calculated stability of mutations in S2. The stems thus play different roles in the frameshift event. The nature of the base in L1 has little influence on frameshift efficiency. It is however required to bridge S2; deleting it lowers frameshifting from 23 to 9%. In L2, frameshift efficiency was not affected in a mutant that changed 10 to 12 bases. This makes it unlikely that the primary sequence of L2 plays a role in -1 frameshifting, in contrast to readthrough in Moloney murine leukemia virus (Wills N, Gesteland R, Atkins J, 1994, EMBO J 13:4137-4144). Deletions of 2 and 3 bases gave more frameshifting than the wild type, probably reflecting the increased stability of the pseudoknot due to a shorter loop L2. Deleting even more bases reduces frameshifting compared to wild-type levels. At this point, stress will build up in L2, and this will reduce overall pseudoknot stability.

The pro and pol genes of simian retrovirus-1 (SRV-1) are expressed as parts of a fusion protein generated by -1 ribosomal frameshifting. To investigate the requirements for frameshifting at the gag-pro overlap, we have inserted a stretch of 58 nucleotides containing the proposed frameshift signal into a plasmid that allows monitoring of translation in all three reading frames. In vitro translation of mRNAs derived from this plasmid indicated that the 58 nucleotides from the SRV-1 gag-pro overlap were sufficient to induce an efficient -1 shift in a heterologous context. Mutational analysis demonstrated that the slip site is formed at the heptanucleotide G GGA AAC. The frameshift efficiency of the wild type sequence in rabbit reticulocyte lysate was 23%. A second component of the frameshift signal is formed by a pseudoknot seven bases downstream of the slip site. The presence of this pseudoknot was confirmed by mutational analysis, employing complementary and compensatory base changes, and by probing the structure of short RNA transcripts containing the frameshift signal. Adding increasing amounts of an SRV-1 pseudoknot containing RNA transcript to a translation reaction programmed with an SRV-1 frameshift reporter mRNA had no effect on the frameshift efficiency, arguing against the role of a specific pseudoknot-recognising factor in the frameshifting process.

The antiarrhythmic drug, amiodarone, induces thyroid dysfunction, which is potentially dangerous in cardiac patients. After discontinuation of the drug it takes several months before euthyroidism is restored. The potent antithyroid drug, potassium perchlorate (KClO4), is used successfully to treat amiodarone-induced thyrotoxicosis, but it is less well known as potential treatment in amiodarone-induced hypothyroidism. In this case report we describe the successful use of two courses of KClO4 treatment in a cardiac patient with severe amiodarone-induced hypothyroidism. The mechanisms responsible for the amiodarone-induced hypothyroidism and rationale for the use of KClO4 in this condition are discussed.

The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine. The latter protecting group can be removed under mild conditions with fluoride ion.

Ribosomal frameshifting on retroviral RNAs has been proposed to be mediated by slippage of two adjacent tRNAs into the -1 direction at a specific heptanucleotide sequence. Here we report a computer-aided analysis of the structure around the established or putative frameshift sites in a number of retroviral, coronaviral, toroviral, and luteoviral RNAs and two dsRNA yeast viruses. In almost all cases a stable hairpin was predicted four to nine nucleotides downstream of the shifty heptanucleotide. More than half of the resulting hairpin loops give rise to potential pseudoknotting with sequences downstream of this hairpin. Especially in the case of the shifty heptanucleotides U UUA AAC and G GGA AAC, stable downstream pseudoknots are present. Indications were also found for the presence of pseudoknots downstream of amber stop condons at readthrough sites in some retroviral RNAs.