Strain 3B Research Articles

Isolation of Antibiotic Resistant Bacteria from Rivers in Terengganu, Malaysia

Infectious disease cause by antibiotic resistant bacteria has been more difficult to treat because the ability of these bacteria to avoid the effect of antibiotic medication. Nowadays, water environment especially river has become the reservoir for the dissemination of antibiotic resistant bacteria due to antibiotic contamination derived from human activities. The exposure to antibiotic resistance bacteria in water increase the health risks associated with the waterborne bacteria in animals and humans. Therefore, this study was carried out to investigate the presence of antibiotic resistant bacteria from two rivers in Terengganu; Ibai River and Terengganu River. A total of 24 bacterial strains were isolated from both rivers. Antibiotic susceptibility of the bacteria isolates were assayed according to the Kirby-Bauer disk diffusion method using six types of antibiotics; ampicillin, rifampicin, gentamicin, tetracycline, ciprofloxacin and chloramphenicol. The results of this study indicate that 100% of bacterial strains were resistant to at least two antibiotics tested. The multiple antibiotic resistant (MAR) index of these bacteria was calculated and 23 bacterial strains show MAR index of more than 20%. These bacteria were selected for further characterization using 16S rRNA gene sequencing. Among the 23 isolates, a total of 10 bacterial strains (2A, 2B, 2C, 2I, 3A, 3B, 4A, 4C, 4D and 4E) were successfully identified as Chromobacterium violaceum, Enterobacter tabaci, Bacillus wiedmannii, Acinetobacter baumannii, Klebsiella quasipneumoniae subsp. quasipneumoniae, Chromobacterium aquaticum, Cupriavidus metallidurans, Weeksella massiliensis, Cupriavidus necator and Acinetobacter nosocomialis. The results from this study show that both of the rivers are exposed to high level of antibiotics compound which may lead the bacteria to develop mechanism of antibiotic resistant. This may cause a serious threat to the public health and environment.

Effect of Bacillus subtilis Strains 3B and BC4333 Starter Cultures on the Quality of Fermented Parkia biglobosa Seeds, ''iru''

Effect of Bacillus subtilis Strains 3B and BC4333 Starter Cultures on the Quality of Fermented Parkia biglobosa Seeds, ''iru''

Clinical and virological features of acute hepatitis E in Gunma prefecture, Japan between 2004 and 2015.

To evaluate the clinical and virological features of acute hepatitisE (AH-E) in Gunma prefecture and focus on the hepatitisE virus (HEV) infection in immunocompromised patients. A total of 30 patients with AH-E diagnosed at our Gunma University Hospital, and located in 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 Japan, and its affiliated hospitals from 2004 to 2015, were studied. We evaluated the detailed medical histories, laboratory examinations and virological features of these participants. Of the 30 patients, 21 patients were men, with a median age of 61years. Three of these patients had a history of recent oversea travel. A total of 14 patients had eaten raw or undercooked meat/viscera from animals, and two patients had contracted transfusion-transmitted AH-E. Eight patients were immunocompromised, including those with hematological disease, cancer receiving systemic chemotherapy and kidney transplant or connective tissue disease undergoing immunosuppressive medications. The alanine aminotransferase and total bilirubin levels were more significantly reduced in these immunocompromised patients than in the non-immunocompromised patients. Severe thrombocytopenia, an extra-hepatic manifestation of AH-E, occurred in one case. Among the 22 HEV strains whose subgenotype was determined, two were imported strains (1a and 1f), and 11 strains formed four distinct phylogenetic clusters within subgenotype3b. The remaining nine strains differed from each other by 9.8-22.4%, and were classified into four subgenotypes (3a, 3b, 3e and 3f). Markedly divergent HEV strains (3a, 3b, 3e and 3f) were found to circulate in Gunma. Although immunosuppression appears to play a crucial role in establishing chronic sequels, AH-E in eight immunocompromised patients, including transfusion-transmitted HEV infection in two patients, did not become chronic.

Sa1933 Reduced Expression of Nerve Growth Factor and Its Receptors in Scars of Healed Gastric Ulcers: A Major Mechanism Underlying Impaired Neural Plexus and Sensory Nerve Regeneration in Ulcer Scars and a Novel Causal Role in Abnormal Neovascularization?

G A A b st ra ct s clarithromycin (1μg/ml). All these 7 clarithromycin-resistant strains had the BsaI site (Figure 1). We have found mutations site at A1825G, T1834C and A2147G mutation (Figure 2). None had the new BbsI cleavage site which is characteristic of the other relatively common mutation (A2143G). The sequence of the 23S rRNA gene of the ClaR strain no 34A has been submitted and the accession No is JX827460. We introduced the PCR fragments containing the relevant mutations amplified with primer pair as listed above from the chromosomal DNA of strain 3B (ClaS strain) into H. pylori 34 and 39 (ClaR strain). The relevant mutations in both transformants were confirmed by DNA sequencing to be the same as those in the donor strain. By examining their effects in an isogenic background, it was found that the MIC of clarithromycin was the same for both transformants (4 μg/ml). Conclusion: Our results show an increasing trend of h.pylori towards dual resistance with metronidazole and clarithromycin, but maintained sensitivity towards tetracycline and amoxicillin. Clarithromycin resistance was due to mutations at site A1825G, T1834C and A2147G of 23 S rRNA gene.

New findings regarding the epidemic history and population dynamics of Japan‐indigenous genotype 3 hepatitis E virus inferred by molecular evolution

Since previous studies have investigated the population dynamics of Japan-indigenous genotype 3 hepatitis E virus (HEV) using virus sequences, more nucleotide sequences have been determined, and new techniques have been developed for such analysis. To prevent future hepatitis E epidemic in Japan, this study aimed to elucidate the cause of past HEV expansion. The epidemic history of Japan-indigenous genotype 3 HEV was determined using the coalescent analysis framework. Bayesian skyline plot (BSP) and Bayesian estimate of phylogeny with relaxed molecular clock models were calculated using Markov chain Monte Carlo sampling. Japan-indigenous strains consist of New World strains (subtype 3a), Japanese strains (3b) and European strains (3e). The oldest lineage, 3b, appeared around 1929. Lineages 3a and 3e appeared around 1960. BSPs indicated similar radical population growth of the 3a and 3b lineages from 1960 to 1980. Population dynamics of the three lineages shared some common characteristics, but had distinguishing features. The appearance of 3a and 3e lineages coincides with the increase of large-race pig importation from Europe and the USA after 1960. The epidemic phase of 3a and 3b strains from 1960 to 1980 could be related to increased opportunity for HEV infection arising from large-scale pig breeding since 1960. Our observations revealed new findings concerning the close relationship between the epidemic history of Japan-indigenous genotype 3 HEV and the improvement of the Japanese pig industry. Infection control in pig farms should be an effective method of preventing HEV infection in humans.

Genome Sequence of the Obligate Methanotroph Methylosinus trichosporium Strain OB3b

Methylosinus trichosporium OB3b (for "oddball" strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales.

Chromohalobacter beijerinckii: a psychrophilic, extremely halotolerant and enzymatically active microbe from salted food with the capacity for biogenic amine production

The species Chromohalobacter beijerinckii comb. nov. was recently proposed in order to accommodate strain Pseudomonas beijerinckii Hof 1935, which was found in salted beans, and a novel isolate, strain 3b, which was obtained from salted herrings of the Baltic sea. The species is psychrophilic, growing from 5 to 42 °C and highly salt-tolerant, growing between 0.5 and 25% NaCl. Of interest to food microbiology are several properties, which are described here, such as the enzymatic activity of Ch. beijerinckii strain 3b against fish protein, cod liver oil and butter. In addition, the strain possesses decarboxylases of lysine, tyrosine and histidine and can thus be a producer of biogenic amines, mainly histamine and tyramine. In media containing 10% NaCl, Ch. beijerinckii strain 3b is resistant to the antibiotics bacitracin, chloramphenicol, streptomycin, tetracycline and novobiocin. The potential impact of these properties, especially of the biogenic amine content in salted food, which were not known so far from other species of the genus Chromohalobacter, is discussed.

Nociceptin/orphanin FQ-induced food intake and cocaine amphetamine regulated transcript gene expression in strains derived from rats prone (WOKW) and resistant (Dark Agouti) to metabolic syndrome

In previous work, we observed that N/OFQ-induced hyperphagia is greater in DA rats, animals resistant to metabolic syndrome, than in WOKW animals, which are prone to this disease. We attributed this difference to the fact that these two strains have different Cart gene sequences and expression. As a preliminary approach to pursue this hypothesis, the present work focused on Cart gene expression by developing from DA and WOKW rats various congenic animals with exchanges of metabolic syndrome-related QTL's of different chromosomes (3, 5, 10 and 16), and analyzing their N/OFQ-induced (2.1, 4.2, and 8.4 nmol/rat) food intake in terms of their CART gene expression and N/OFQ hypothalamic immunostaining. Two groupings emerged, the first, with strains 3a, 3b, and 5a with elevated N/OFQ-induced feeding similar to that of the DA rats, and the second, with strains 16 and 10, with lower feeding, like the WOKW rats. There was a perfect correlation between Cart gene expression and N/OFQ-induced feeding data at 30 min for the strains DA, 3a, 3b, 5 in the first group, and 16 and WOKW for the second, but not for strain 10. As expected, the strains with low content of Cart gene expression had elevated N/OFQ-induced feeding, but contrary to expectations, strain 10, with the lowest Cart gene expression, exhibited low N/OFQ-induced feeding, on the order of that of the WOKW rats. A comparable trend was observed with N/OFQ hypothalamic immunostaining. This anomaly may be due to other satiety-related factors involved in N/OFQ-induced feeding.

Parenteral vaccination of domestic pigs with Brucella abortus strain RB51

To determine the immunogenicity and efficacy of Brucella abortus strain RB51 (SRB51) as a vaccine in domestic pigs. Sixty-eight 6-week-old crossbred domestic pigs and twenty-four 4-month-old gilts. In experiment 1, pigs were vaccinated IM (n = 51) with 2 x 10(10) CFUs of SRB51 or sham inoculated (17). Periodic blood samples were obtained to perform blood cultures, serologic evaluations, and cell-mediated immunity assays. Necropsies were performed at selected times between weeks 1 and 23 after vaccination to determine vaccine clearance. In experiment 2, gilts were similarly vaccinated (n = 18) or sham inoculated (8) and similar samples were obtained after vaccination. Gilts were bred and challenged conjunctivally with 5.0 x 10(7) CFUs of virulent Brucella suis strain 3B. Necropsies were performed on gilts and on fetuses or neonates after abortion or parturition, respectively. Bacterial cultures and serologic evaluations were performed on samples obtained at necropsy to determine vaccine efficacy. Humoral and cell-mediated immune responses did not differ between vaccinates and controls. After vaccination, SRB51 was not isolated from blood cultures of either group and was isolated from lymphoid tissues of 3 pigs at 2 weeks (n = 2) and 4 weeks (1) after vaccination. No differences were found in isolation of B suis or in seroconversion between vaccinated and control gilts and between their neonates or aborted fetuses. Parenteral vaccination with SRB51 does not induce humoral or cell-mediated immune responses. Vaccination with SRB51 did not protect gilts or their neonates and fetuses from virulent challenge with B suis.

Optimization of polysaccharides production by bacteria isolated from soil

Six polysaccharide-producing bacteria, isolated from soil samples and identified as Pseudomonas and Arthrobacter (Strains 3B, 4B, 7B, 21B, 18E and 21D), were tested for the yield of polysaccharides produced during growth in two culture media: one containing glucose and the other sucrose (1, 2, 3, 4 and 5%). The yield was quantified measuring the viscosity of the broth, using the Poiscuille equation. The effect of temperature and pH of the culture media was investigated. The largest polysaccharide yield was obtained when the concentration of the carbon source was lower than 2%. Glucose and sucrose stimulated the polysaccharide production in a similar way. When the initial pH of the fermentation broth was increased from 5.0 up to 7.0, there was an increase in polysaccharide production. However, higher values (pH=8.0) caused a decrease in polysaccharide production. With regard to temperature, 30oC was shown to be optimum, since higher or lower temperatures had a negative effect on saccharide production.

Reclassification of Pseudomonas beijerinckii Hof 1935 as Chromohalobacter beijerinckii comb. nov., and emended description of the species

Pseudomonas beijerinckii (type strain DSM 7218(T)=ATCC 19372(T)=NCIMB 9041(T)) was isolated from salted beans and was first described by Hof in 1935. 16S rRNA gene sequence comparisons demonstrated its close relatedness (>97-99 %) to species of the genus Chromohalobacter. A recent isolate from salted herrings originating from the Baltic Sea, strain 3b, also clustered phylogenetically within this genus. Phenotypic features, substrate utilization, fatty acid profile, quinone and polar lipid composition and whole-cell protein patterns supported the similarity of strain 3b to P. beijerinckii DSM 7218(T) and confirmed its relatedness to members of the genus Chromohalobacter. The G+C content of the DNA from strain 3b and P. beijerinckii DSM 7218(T) was 60.4 and 60.7 mol%, respectively. DNA-DNA hybridization data showed that the two strains represent the same species, but are separated from Chromohalobacter canadensis, the closest species from a phylogenetic point of view. Therefore, the reclassification of Pseudomonas beijerinckii as Chromohalobacter beijerinckii comb. nov. (type strain DSM 7218(T)=ATCC 19372(T)=NCIMB 9041(T)) is proposed. The species description has been emended considering the new data on both the type strain and strain 3b.

Purification and properties of individual collagenases fromStreptomyces sp. strain 3B

Streptomyces strain 3B constitutively secreted collagenolytic enzymes during the post-exponential growth phase. Purification is described here leading to two collagenases (I and II) with specific activity of 3350 and 3600 U/mg, respectively, the highest activity obtained as yet for any streptomycete collagenase. Analysis of the purified enzymes by the method of zymography revealed that both I and II were homogeneous, with molar mass 116 and 97 kDa, respectively. Both collagenases were identical in their pH (7.5) and temperature optimum (37 degrees C). The inhibition profile of the enzymes by EDTA and 1,10-phenanthroline confirmed these enzymes to be metalloproteinases. By testing the activity with insoluble collagen, acid soluble collagen, gelatin, casein, elastin and Pz-PLGPR it was established that I and II are very specific for insoluble collagen and gelatin, showing a high activity toward acid soluble collagen and Pz-PLGPR. However, collagenases I and II failed to hydrolyze casein and elastin; they belong to true collagenases and resemble the clostridial enzymes.

Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the delta ccr5 mutation

The alpha-chemokine receptor fusin (CXCR-4) and beta-chemokine receptor CCR5 serve as entry cofactors for T-cell (T)-tropic and macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively, when expressed with CD4 in otherwise nonpermissive cells. Some M-tropic and dual-tropic strains can also utilize other beta-chemokine receptors, such as CCR2b and CCR3. A mutation of CCR5 (delta ccr5) was recently found to be common in certain populations and appears to confer protection against HIV-1 in vivo. Here, we show that this mutation results in a protein that is expressed intracellularly but not on the cell surface. Primary CD4 T cells from delta ccr5 homozygous individuals were highly resistant to infection with prototype M-tropic HIV-1 strains, including an isolate (YU-2) that uses CCR5 and CCR3, but were permissive for both a T-tropic strain (3B) and a dual-tropic variant (89.6) that uses CXCR-4, CCR5, CCR3, or CCR2b. These cells were also resistant to M-tropic patient isolates but were readily infected by T-tropic patient isolates. Primary macrophages from delta ccr5 homozygous individuals were also resistant to infection with M-tropic strains, including YU-2, but the dual-tropic strain 89.6 was able to replicate in them even though macrophages are highly resistant to CXCR-4-dependent T-tropic isolates. These data show that CCR5 is the essential cofactor for infection of both primary macrophages and T lymphocytes by most M-tropic strains of HIV-1. They also suggest that CCR3 does not function for HIV-1 entry in primary lymphocytes or macrophages, but that a molecule(s) other than CCR5 can support entry into macrophages by certain virus isolates. These studies further define the cellular basis for the resistance to HIV-1 infection of individuals lacking functional CCR5.

Pmp27 promotes peroxisomal proliferation.

Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.

Mechanism of cytotoxic activity by hemocytes of the solitary ascidian, Ciona intestinalis

AbstractThe mechanism of cytotoxicity by the hemocytes from the solitary ascidian, Ciona intestinalis, against the target cell WEHI, a mouse myelomonocytic leukemic cell (strain 3B), was investigated in vitro. Experiments using chelators of Mg2+ and Ca2+ show that the cytotoxic activity by these hemocytes involves at least two stages distinguishable by their divalent cation requirements. In particular removal of both Ca2+ and Mg2+ by EDTA inhibits effector to target cell conjugate formation and target cell lysis, whereas selective removal of Ca2+ by EGTA inhibits target cell lysis but does not reduce the formation of effector to target cell conjugates. Evidence that cytotoxic activity is reduced by sodium azide, cytochalasin B, colchicine, vinblastine sulfate, and monensin, indicates that the response involves energy metabolism, requires an intact cyto‐skeleton, and entails active secretory processes in the effector cells. In addition, cytotoxic activity is probably not mediated by superoxide anions or hydrogen peroxide, because the antioxidant enzymes, superoxide dismutase, and catalase did not affect target cell lysis. It is therefore likely that cytotoxic activity by the hemocytes from C. intestinalis is mediated by mechanisms similar to those of vertebrate cytotoxic cells. © Wiley‐Liss, Inc.

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Papaverine hydrochloride: effects on HIV replication and T-lymphocyte cell function

Papaverine hydrochloride (PAP) has previously been shown to have a potent inhibitory effect on the replication of viruses such as cytomegalovirus (CMV) and measles. In this report the effect of PAP on human immunodeficiency virus (HIV) replication and T lymphocyte cell function were examined. MT4 cells infected with HIV strain 3b were incubated with serial dilutions of PAP (1–30 μM). At selected times postinfection HIV replication was measured by reverse transcriptase activity (RT) or HIV p24 Ag. PAP significantly inhibited HIV replication by more than 99% at doses of 30 μM with an CD50 and ED50 of 32 μM and 5.8 μM respectively. The mechanism of inhibition of HIV caused by PAP appeared independent from its ability to increase intracellular levels of cAMP and was not mediated via a direct effect on RT activity. To examine T cell function, peripheral blood mononuclear cells (PBMC) from normal donors were stimulated with phytohemagglutinin (PHA) or CMV Ag in the presence or absence of PAP (1–30 μM). At selected times proliferative response to PHA and CMV Ag were determined by [3H]thymidine uptake. In addition, interferon (IFN) gamma and interleukin 2 (IL2) response to mitogens were measured by radioimmunoassay (RIA). PAP enhanced PHA induced IFN production at doses of 1–10 μM and CMV Ag induced IFN production at doses of 1–3 μM. Higher doses were inhibitory. PAP did not affect IL-2 production or IL2 receptor expression and had an inhibitory effect on mitogenic responses.

The Crl protein activates cryptic genes for curli formation and fibronectin binding in Escherichia coli HB101.

Curli are thin, coiled, temperature-regulated fibres on fibronectin-binding Escherichia coli. The subunit protein of curli was highly homologous at its amino terminus to SEF-17, the subunit protein of thin, aggregative fimbriae of Salmonella enteritidis 27655 strain 3b, suggesting that these fibres form a novel class of surface organelles on enterobacteria. E. coli HB101 is non-curliated and unable to bind soluble, iodinated fibronectin. The phenotypically cryptic curlin subunit gene, csgA, in HB101 is transcriptionally activated by expressing the cytoplasmic Crl on a multicopy plasmid. Transcriptional activation of csgA by Crl was observed after growth at 26 degrees C but not at 37 degrees C, even though crl transcription was not thermoregulated. A deletion of the 39 carboxy-terminal residues abolished Crl activity, whereas a deletion of 10 residues at the C-terminus did not, implying that a region between residue 93 and 122 in the 132-amino-acid-residue large Crl protein is required for activating curli expression in E. coli HB101. crl is a normal housekeeping gene in E. coli and it is suggested that its gene product may either be a DNA-binding protein affecting chromatin structure as has been suggested for histone-like protein H1 or interact with specific regulatory protein(s) controlling transcription of genes required for curli formation and fibronectin binding.

Human immunodeficiency virus replication: modulation by cellular levels of cAMP.

HIV infection is associated with qualitative and functional immune deficiencies. It has been shown that the in vitro infection of CD4+ cells with HIV was associated with sustained elevation of cAMP and cGMP. In the present report the role of cAMP on HIV replication in MT-4 cells was investigated. The MT-4 cells were infected with HIV (strain 3b), in the presence or absence of agents that increase intracellular levels of cAMP, through different mechanisms. At selected times postinfection, HIV replication was measured by reverse transcriptase activity or HIV P24Ag in culture supernatants. Forskolin (FK, an activator of adenylate cyclase 1-100 microM), Isobutyl-methylxanthine (IBMX, a phosphodiesterase inhibitor, which indirectly increases intracellular levels of cAMP, 30-100 microM) and dibutyryl (db) cAMP (0.1-10 microM) enhanced HIV replication, in a dose-dependent manner. FK, IBMX, and db cAMP enhanced HIV replication by 2- to 10-fold, 4- to 7-fold, and 2- to 6-fold, respectively. Intracellular levels of cAMP were measured by radioimmunoassay and were also enhanced. Since cAMP exerts its catalytic effects through activation of protein kinase (PK) A the effect of H-8 (a specific inhibitor of the cAMP dependent PK A) on HIV replication was simultaneously examined. The H8 at doses of 0.1 to 10 microns inhibited HIV replication by 25 to 99.9%. Moreover H9 inhibited HIV replication in peripheral blood mononuclear cells by more than 90%. The replication of HIV appears to be a cAMP-dependent event, and PK A could possibly be a target for the development of anti-HIV therapies.