Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis.

OBJECTIVES: Polyisoprenylated acylphloroglucinols have recently emerged as antitumoral agents. This study aims at elucidating the antiretroviral activity of two such compounds which were isolated from Caribbean propolis: 7-epi-nemorosone and plukenetione A, the structure of which is based on an adamantane moiety. Plukenetione A is for the first time shown to have antiretroviral activity. MATERIAL AND METHODS: The isolation of both small molecules was carried out using RP-HPLC. Their antiretroviral activity was studied based on lentiviral particles produced in HEK293T cells from the SIV-based vector VLDBH; their cytotoxicity was monitored by MTT proliferation assay. The antiviral activity of 7-epi-nemorosone was studied in CEMx174-SEAP infected with the HIV-1-strain pNL4.3wt. Reverse transcriptase inhibition was determined by a standard two-step RT-PCR using MMLV RT. RESULTS: 7-epi-nemorosone and plukenetione A were found to be potent antilentiviral agents in the employed system, inhibiting viral infection at concentrations below 1 µM/2 µM, respectively. Whereas 7-epi-nemorosone was not able to inhibit the reverse transcriptase in vitro (IC50 > 25 µM), plukenetione A effectively inhibited its enzymatic activity at an IC50 of 1.75 µM. CONCLUSIONS: Despite 7-epi-nemorosone and plukenetione A sharing some structural core elements, the mechanism of action involved in their antiretroviral activity seems to be different. We propose that 7-epi-nemorosone inhibits the viral replication by interrupting the Akt/PKB signaling cascade, as was demonstrated previously in various cell lines. Since plukenetione A effectively inhibits the enzymatic activity of MMLV reverse transcriptase at concentrations that show antilentiviral activity, we suggest that this small molecule acts by interfering with the enzyme's catalytic site.

Implications of and perspectives on HIV surveillance using a serological method to measure recent HIV infections in newly diagnosed individuals: results from a pilot study in Berlin, Germany, in 2005-2007.

OBJECTIVES: This cross-sectional study was designed to pilot the analysis of clinical data, knowledge about and attitudes towards HIV/AIDS, and prevention and risk behaviour in persons recently infected with HIV. METHODS: Blood samples and demographic, laboratory, clinical and behavioural data were collected from patients with newly diagnosed HIV infections. The BED IgG-capture ELISA (BED-CEIA) was used to determine the recency of infection. RESULTS: Recent HIV infections contributed 54% [95% confidence interval (CI) 45; 64%] of infections in men who have sex with men (MSM) and 16% (95% CI 0; 39%) of infections in patients with other transmission risks (P=0.041). Recently infected MSM were characterized by younger age and higher viral load as compared with MSM who had longstanding infections (P=0.011 and 0.005, respectively). Symptoms during primary infection and patients' assumptions with regard to time of infection were significantly correlated with test results indicating whether or not the HIV infection was recently acquired (P<0.001). CONCLUSIONS: Cross-sectional surveillance of recent HIV infections proved to be relevant to the identification of current risks for acquiring HIV infection. The high proportion of recent HIV infections in MSM and the even higher proportion in MSM younger than 30 years indicate ongoing HIV transmission in this group. The method will be used in future national HIV surveillance in Germany.

Country-wide HIV incidence study complementing HIV surveillance in Germany.

Serological methods exist that allow differentiating between recent and long-standing infections in persons infected with HIV. During a pilot study in Berlin between 2005 and 2007 methodologies have been evaluated. In a cross-sectional study blood samples, demographic, laboratory, clinical and behavioural data based on a KABP survey were collected from patients with newly diagnosed HIV infections. The BED-CEIA was used to determine recency of infection. Recent HIV infections contributed 54% (CI [95%]: 45; 64) in MSM and 16% (CI [95%]: 0; 39) in patients with other transmission risks (p=0.041). Proportions of recent infections were significantly higher in MSM

A functional toll-like receptor 8 variant is associated with HIV disease restriction.

BACKGROUND: Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS: We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS: This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.

Impact of transmission of drug-resistant HIV on the course of infection and the treatment success. Data from the German HIV-1 Seroconverter Study.

BACKGROUND: Data on the clinical course of infection in patients with transmitted drug-resistant HIV before and after initiation of treatment are scarce. PATIENTS AND METHODS: Genotypic resistance was analysed in 504 therapy-naïve individuals with a known date of infection. Resistance was predicted using the Stanford algorithm. Clinical parameters for 80 individuals with transmitted drug-resistant HIV and for 424 patients with susceptible virus were analysed. RESULTS: In 16% of the individuals transmitted drug-resistant HIV was found. Detection of drug-resistant HIV was more likely in individuals with acute primary HIV infection [odds ratio (OR)=1.529; 95% confidence interval (95% CI) 1.001; 2.236]. At the time of infection patients with an acute infection with resistant HIV had lower viral loads. CD4 cell counts tended to be higher and the CD4 cell loss more pronounced in the group with resistant HIV. Suppression of the viral load below the detection limit was achieved in 64% of the group with resistant HIV and in 85% of the group with susceptible HIV 6 months after initiation of therapy (P=0.199). The majority of the group with resistant HIV (74%) received at least one compromised drug. CONCLUSION: First-line treatment including drugs with predicted resistance can impair virological success in some patients. Factors influencing the decision to include compromised drugs need to be investigated.

Frequency of genotypic and phenotypic drug-resistant HIV-1 among therapy-naive patients of the German Seroconverter Study.

Genotypic and phenotypic resistance of viral reverse transcriptase (RT) and protease (PR) was determined for 64 therapy-naive, HIV-1-infected seroconverters of the German Seroconverter Study coordinated by the Robert Koch-Institut, Berlin. The date of seroconversion of patients and the laboratory, clinical, and therapeutic follow-up data were documented. Samples were collected between 1996 and 1999. Phenotypic resistant HIV-1 were found in 8 (13%) seroconverters; in most cases resistance was weak and mainly directed against RT inhibitors (4 nucleoside reverse transcriptase inhibitors [NRTIs], 2 nonnucleoside reverse transcriptase inhibitors [NNRTIs], 1 combination NRTI/NNRTI). Only one infection with a weak PR inhibitor resistance was identified. Transmission of multidrug-resistant HIV-1 has not yet been observed. Frequently at least one or more amino acid mutations associated with antiretroviral drug resistance were detected by genotypic analysis. The mean number of resistance-associated mutations in the RT of the transmitted virus has increased significantly since 1996. Studies have shown the improved benefit of initial antiretroviral therapy if based on genotypic resistance data. In view of the considerably high level of transmission of resistant HIV-1 in Germany, which is also seen in other studies in Europe and the United States, we suggest determining the genotypic resistance pattern before starting therapy of newly HIV-1-infected patients.

A multicentre quality assessment study to monitor the performance of HIV-1 PCR.

Eleven German laboratories and one Swiss laboratory initiated a quality assessment study to evaluate the specificity and sensitivity of their polymerase chain reaction (PCR) for detection of HIV-1 DNA. Following its own PCR protocols, each laboratory tested a panel of ten coded samples consisting of cell pellets containing 0, 0.1, 1, 10, 10(2), 10(3) and 10(4) ACH-2 cells per 1.5 x 10(5) uninfected peripheral blood mononuclear cells. Of the twelve participating laboratories, three reported correct results for the dilution series as well as for uninfected specimens. One or more classification errors were recorded for 12% of the samples for which the diagnosis was expected to be positive or negative. Samples containing 10 copies of the target template were correctly reported by eleven of the twelve participants. The average sensitivity was 97%. The results of the study revealed no significant differences between the Amplicor kit and in-house procedures. Most of the classification errors occurred in specimens from HIV-negative samples. Out of 36 negative samples, 5 were reported false positive, showing that contamination remains a problem for some laboratories, regardless of the PCR test performed. Careful laboratory techniques and internal as well as external quality control procedures will help avoiding carryover contamination.

Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization.

Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV-1 Nef non-covalently associates with actin forming a high-molecular-mass complex of 150-300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N-terminal myristoylation of Nef, whereas the SH3-binding proline motif of Nef was not involved. Despite being myristoylated, HIV-2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell-fractionation experiments revealed that HIV-1 Nef was virtually exclusively localized in the cytoskeletal (detergent-insoluble) fraction whereas HIV-2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV-1-infected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV-infected cells. This novel interaction of HIV-1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV-1 and HIV-2.

Evolutionarily conserved positive and negative cis-acting elements control the blastoderm-specific expression of the Drosophila serendipity alpha cellularisation gene.

The serendipity alpha (sry alpha) cellularisation gene is only transcribed at the blastoderm stage, when this morphogenetic event takes place. We show that a 95 bp sry alpha upstream region is sufficient for blastoderm-specific expression of a lacZ reporter gene. This region encompasses four nucleotide motifs (I-IV, 5' to 3') conserved at similar relative positions in several Drosophila species. Removal of motif I leads to ectopic expression of lacZ in precursor cells of the PNS. Deletion of motif IV decreases the level of lacZ transcripts and modifies their banded pattern of accumulation late in cycle 14, whereas deletion of motifs II and III abolishes the sry alpha promoter activity. Motif III includes a consensus recognition site for b-HLH proteins. A point mutation in this E-box both severely reduces lacZ expression at blastoderm and prevents its ectopic expression in the PNS upon deleting motif I. These two effects depend upon da+ activity, suggesting that daughterless may positively control sry alpha transcription.

[Idiopathic CD4 lymphocytopenia with lethal Salmonella typhimurium sepsis]. / Idiopathische CD4-Lymphozytopenie mit letaler Salmonella-typhimurium-Sepsis.

In April 1991, a then 43-year-old woman fell ill with a systemic cryptococcal infection which responded well to antimycotic treatment. Four months later she was found to have a T-helper cell deficiency (48/microliters, 15%). Since August 1992 she noted an increased tendency towards infections and had recurrent fever bouts over 4 weeks, which led to her hospitalization in January 1993. Repeated HIV tests were negative. During the first 4 days she was free of infection, but developed a temperature of 39.1 degrees C on the fifth day, and within a few hours a fulminant septicaemia with cardiorespiratory failure developed ending fatally the same day. Blood cultures drawn during fever bouts grew Salmonella typhimurium by the time of her death. Lymphocyte differentiation revealed absolute and relative reduction in the number of CD4-lymphocytes (158/microliters; 18%), retrospectively providing the diagnosis of idiopathic CD4-lymphocytopenia.--In cases such as this there is the need of including opportunistic and masked disease entities in the differential diagnosis, even in the absence of HIV infections, and start early treatment.

Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts.

Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli. Between 10% and 20% of asnC transcripts reached oriC in vivo. Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping. Only about 10% of the transcripts terminated at the asnC terminator in vivo. DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein. In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts. We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.

AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC.

The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system. This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping. From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level. gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control. However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA. The gidA promoter was mapped by deletion analysis and by S1 mapping. We defined two regions that affect promoter activity negatively. Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region. These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.

Transcripts within the replication origin, oriC, of Escherichia coli.

Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.

Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively.

We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh). The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions. In E. coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed. The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription. Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription.

Regulation of transcription of the chromosomal dnaA gene of Escherichia coli.

By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription. Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription. Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC. The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.

dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes.

Both initiation of replication and initiation of transcription are influenced by dnaA protein, when minichromosomes are assayed in vitro for dnaA protein complementation. This dnaA protein effect is seen only if minichromosomes are used containing the 16-kd promoter, from which transcription is directed into the minimal origin. Determination of the 16-kd promoter activity both in vivo and in vitro showed that this strong promoter is specifically repressed by dnaA protein. The 16-kd promoter is thus an integral regulatory region of oriC.

The occurrence of two ribosomal ribonucleases depending on growth phase in yeast. Induction of ribonuclease in glucose-starved cells.

There are two latent ribonucleases associated with the 40 S subunits of yeast ribosomes which differ in their digestion products, pH optimum, molecular weight, and in their activity during growth phase. The 3'-nucleotide-producing enzyme is active only in the late logarithmic or stationary growth phase, whereas the ribonuclease which produces 5'-nucleotides is present at all growth phases. The enzymes were separated by affinity chromatography and were partially characterized. By changing growth conditions--i.e. decreasing and increasing the glucose concentration in the medium--the activity of the 3'-ribonuclease could be induced or reduced.

The induction of a ribosomal ribonuclease in Saccharomyces cerevisiae.

The ribosomal ribonuclease of yeast is induced after a withdrawal of glucose. Cycloheximide inhibits the synthesis of the enzyme under conditions of induction but antimycin shows no effect. Hence, the increase of the ribonuclease activity during growth of yeast is due to newly synthesized enzyme, and the induction does not depend on respiration.