Multilevel samplers as microcosms to assess microbial response to biostimulation.

Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used as in situ microcosms in conjunction with a push-pull biostimulation experiment designed to promote biological U(VI) and Tc(VII) reduction. MLS were deployed at 24 elevations in the injection well and two downgradient wells to investigate the spatial variability in microbial community composition and growth prior to and following biostimulation. The microbial community was characterized by real-time quantitative polymerase chain reaction (Q-PCR) quantification of bacteria, NO(3)(-)-reducing bacteria (nirS and nirK), delta-proteobacteria, Geobacter sp., and methanogens (mcrA). Pretest cell densities were low overall but varied substantially with significantly greater bacterial populations detected at circumneutral pH (t-test, alpha= 0.05), suggesting carbon substrate and low pH limitations of microbial activity. Although pretest cell densities were low, denitrifying bacteria were dominant members of the microbial community. Biostimulation with an ethanol-amended ground water resulted in concurrent NO(3)(-) and Tc(VII) reduction, followed by U(VI) reduction. Q-PCR analysis of MLS revealed significant (1 to 2 orders of magnitude, Mann-Whitney U-test, alpha= 0.05) increases in cell densities of bacteria, denitrifiers, delta-proteobacteria, Geobacter sp., and methanogens in response to biostimulation. Traditionally, characterization of sediment samples has been used to investigate the microbial community response to biostimulation; however, collection of sediment samples is expensive and not conducive to deep aquifers or temporal studies. The results presented demonstrate that push-pull tests with passive MLS provide an inexpensive approach to determine the effect of biostimulation on contaminant concentrations, geochemical conditions, and the microbial community composition and function.

Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease.

Evidence is continuing to accumulate that the FMS-like tyrosine kinase 3 (FLT3) receptor plays an important role in acute leukemias. Acute myeloid leukemia patients often express constitutive active mutant forms of the receptor in their leukemic cells. A t(12;13)(p13;q12) translocation between Tel and the FLT3 receptor was recently described in a patient with myeloproliferative disease (MPD). Here a Tel-FLT3 construct mimicking this fusion protein was used to generate transgenic mice. The fusion protein was previously found to constitutively activate FLT3 signaling and transform Ba/F3 cells. Expression of the fusion protein in the transgenic mice was found in all tissues assayed including spleen, bone marrow (BM), thymus and liver. These mice developed splenomegaly and had a high incidence of MPD with extramedullary hematopoiesis in the liver and lymph nodes. Spleens also had increased dendritic and natural killer cell populations. In vitro analysis of the hematopoietic progenitor cells derived from Tel-FLT3 transgenic mice showed a significant increase in the number of CFU-GM in the BM, and CFU-GM, BFU-E and CFU-GEMM in the spleen. BM also showed significant increases of in vivo CFU-S colonies. Thus, transgenic mice expressing constitutively activated Tel-FLT3 develop MPD with a long latency and also result in the expansion of the hematopoietic stem/progenitor cells.

Broad substrate specificity of naphthalene- and biphenyl-utilizing bacteria.

Although aromatic compounds are most often present in the environment as components of complex mixtures, biodegradation studies commonly focus on the degradation of individual compounds. The present study was performed to investigate the range of aromatic substrates utilized by biphenyl- and naphthalene-degrading environmental isolates and to ascertain the effects of co-occurring substrates during the degradation of mono-aromatic compounds. Bacterial strains were isolated on the basis of their ability to utilize either biphenyl or naphthalene as a sole source of carbon. Growth and transformation assays were conducted on each isolate to determine the range of substrates degraded. One isolate, Pseudomonas putida BP18, was tested for the ability to biodegrade benzene, toluene, ethylbenzene and xylene isomers (BTEX) individually and as components of mixtures. Overall, the results indicate that organisms capable of growth on multi-ring aromatic compounds may be particularly versatile in terms of aromatic hydrocarbon biodegradation. Furthermore, growth and transformation assays performed with strain BP18 suggest that the biodegradation of BTEX and biphenyl by this strain is linked to a catabolic pathway with overlapping specificities. The broad substrate specificity of these environmental isolates has important implications for bioremediation efforts in the field.

Cloning and epitope mapping of a functional partial fusion receptor for human cytomegalovirus gH.

A cDNA clone encoding a partial putative human cytomegalovirus (HCMV) gH fusion receptor (CMVFR) was previously identified. In this report, the cDNA sequence of CMVFR was determined and the role of this CMVFR in HCMV/cell fusion was confirmed by rendering fusion-incompetent MOLT-4 cells susceptible to fusion following transfection with receptor cDNA. Blocking experiments using recombinant gH or either of two MAbs (against recombinant gH or purified viral gH:gL) provided additional evidence for the role of gH binding to this protein in virus fusion. An HCMV-binding domain of 12 aa in the middle hydrophilic region of CMVFR was identified by fusion blocking studies using synthetic receptor peptides. The 1368 bp cDNA of CMVFR contained a predicted ORF of 345 aa with two potential membrane-spanning domains and several possible nuclear localization signals. A search of sequence databases indicated that CMVFR is a novel protein. Further characterization of this cell membrane protein that confers susceptibility to fusion with the viral envelope should provide important information about the mechanism by which HCMV infects cells.

Polymerase chain reaction amplification of bacterial 16s rRNA genes in prostate biopsies from men without chronic prostatitis.

OBJECTIVES: A previously reported study using nested polymerase chain reaction (PCR) analysis indicated the presence of DNA from a variety of prokaryotic microorganisms in 77% of transperineal prostate biopsies from patients with chronic nonbacterial prostatitis. Because that study did not include a control group, we investigated whether microbial DNA could also be found in transperineal prostate biopsies obtained from men who did not have a history of prostatitis. METHODS: Transperineal biopsies of both lobes of the prostate were obtained under ultrasound guidance from 9 patients with localized adenocarcinoma of the prostate. DNA was extracted from the prostatic tissue and two-round amplification performed using nested primers from a highly conserved region of the bacterial 16s rRNA gene. Amplified DNA was purified and sequenced, and sequences obtained were compared to bacterial rRNA genes recorded in GenBank. Results. Eleven of 18 biopsy specimens from 8 of 9 patients were positive for bacterial DNA by PCR. Sequence data indicated a predominant organism in 8 of 11 specimens, with greater than 95% homology to DNA from several different genera of bacteria, including Escherichia and Bacteroides. All 9 control samples from the instruments before biopsy were negative. CONCLUSIONS: The presence of bacterial 16s rRNA genes in prostatic tissue is not specific for chronic prostatitis and occurred in most of our patients with localized prostate cancer. Whether the presence of such bacteria is related to the development of prostatic diseases such as prostatitis or prostatic cancer will require carefully controlled trials, including appropriate control groups examined identically.

Polymerase chain reaction amplification of bacterial 16S rRNA genes from cold-cup biopsy forceps.

PURPOSE: In looking for a possible infectious cause for interstitial cystitis (IC), we previously determined that bladder tissue specimens from both IC patients and controls were uniformly positive by polymerase chain reaction assay (PCR) for bacterial 16S ribosomal RNA genes from various genera including Escherichia, Propionobacterium, Acinetobacter, and Salmonella. We therefore determined whether the biopsy forceps might be contaminated with bacterial DNA. MATERIALS AND METHODS: A total of 23 samples were obtained following disinfection of 6 cold-cup bladder biopsy forceps (2 to 5 specimens from each forceps over a period of 19 months). DNA was extracted from each sample, and PCR performed using nested primers from a highly conserved region of the bacterial 16S rRNA gene. Amplified DNA was purified and sequenced, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. RESULTS: Thirteen of 23 forceps specimens were positive by PCR for bacterial DNA, including at least one rinse from each of the 6 forceps. In comparison, none of 9 negative control specimens (sterile distilled water put into tubes and processed in the same manner as forceps rinses) had detectable bacterial DNA. Sequence data indicated the presence of a predominant organism in 12 of the 13 positive specimens, with >95% homology to DNA from several different genera of bacteria including Escherichia, Propionobacterium, Stenotrophomonas and Pseudomonas. CONCLUSIONS: These data indicate that reusable bladder biopsy forceps are frequently contaminated with bacterial DNA. Tissue specimens procured with such instruments therefore are inappropriate sources to look for the presence of bacterial pathogens by PCR.

Polymerase chain reaction amplification of bacterial 16S rRNA genes in interstitial cystitis and control patient bladder biopsies.

PURPOSE: Several characteristics of the chronic bladder disease called interstitial cystitis (IC) suggest an infectious etiology. However, a single causative organism has not been convincingly cultured in vitro, and DNA for a variety of microorganisms has been found inconsistently in bladder biopsies from IC patients. We therefore looked for a possible bacterial cause for IC by using a sensitive nested PCR assay on cystoscopic bladder biopsy specimens obtained from IC patients and controls. MATERIALS AND METHODS: Bladder biopsies were obtained at cystoscopy from 6 IC patients and 6 controls. DNA was extracted from these specimens and PCR with 2-round amplification performed using nested primers from a highly conserved region of the bacterial 16s rRNA gene. Amplified DNA was purified and sequenced using the Sequenase PCR Product Sequencing Kit, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. RESULTS: Biopsy specimens from all 6 patients and 6 controls were positive by PCR for DNA encoding bacterial 16s rRNA. Sequence data indicated a predominant microorganism in 10 of the 12 specimens, with > 95% homology to DNA from several different genera of bacteria including Acinetobacter, Propionobacterium, Salmonella, and Escherichia. None of the organisms identified by PCR had been cultured from tissue or urine obtained simultaneously from these persons, using sensitive culture techniques. CONCLUSIONS: These data indicate no difference between IC patients and controls in the proportion of bladder biopsies with PCR positivity or the type(s) of organism present, providing additional evidence that IC is not associated with infection by a particular type of bacterium.

Evidence for the role of cell protein phosphorylation in human cytomegalovirus/host cell fusion.

The mechanism by which human cytomegalovirus (HCMV) enters cells is unknown. We sought evidence that protein phosphorylation plays a role in HCMV infection in two ways: (1) by determining whether the degree of phosphorylation of a constitutively phosphorylated 92.5 kDa putative cell membrane receptor for HCMV gH is changed following exposure to HCMV or monoclonal anti-idiotype antibodies (MAb2) that antigenically mimic HCMV gH, and (2) by studying the effects of protein kinase inhibition on receptor phosphorylation and HCMV adsorption or fusion. Phosphorylation of the 92.5 kDa cell membrane protein was specifically increased within 10 min of incubation with HCMV or MAb2 that had been crosslinked by goat anti-mouse antibodies. In addition, fusion of viral envelope with the cell membrane was inhibited by certain protein kinase inhibitors which also inhibited receptor phosphorylation, while the adsorption of [3H]HCMV to human embryonic lung cells was not affected. Tyrosine kinase inhibitors inhibited virus/cell fusion to a greater extent than protein kinase C (PKC) inhibitors, and an inhibitor which primarily affects cAMP and cGMP kinases had little effect. In addition, fusion was stimulated by preincubating cells with agents that stimulated receptor phosphorylation including a phorbol ester, tyrosine phosphatase inhibitor and serine/threonine phosphatase inhibitor. These data indicate that increased phosphorylation of a 92.5 kDa putative cell membrane protein receptor for gH is an early event in response to HCMV, and that cell protein phosphorylation by tyrosine kinase(s) and PKC may facilitate HCMV/cell membrane fusion.

Molecular cloning and expression of receptor peptides that block human cytomegalovirus/cell fusion.

Clones expressing a partial human cytomegalovirus putative fusion receptor were selected by binding specifically to monoclonal anti-idiotypic antibodies that mimic glycoprotein H. cDNA was isolated from 2 of the clones (131 and 611) and fused in frame with the glutathione S-transferase gene in a pGEX-4T-1 vector. Two purified peptides (FR131 and FR611) were produced: both were shown to bind specifically to the monoclonal anti-idiotypic antibodies and inhibit virus/cell fusion and viral plaque formation in a specific and dose-dependent manner. This is the first demonstration of cloned peptides encoding a putative cell membrane receptor that are able to block cytomegalovirus infection.

Increases in [Ca2+]i mediated by the 92.5-kDa putative cell membrane receptor for HCMV gp86.

We determined that changes in intracellular Ca2+ concentration ([Ca2+]i) occur in human fibroblasts within the first hour of human cytomegalovirus (HCMV) infection when viral adsorption and fusion take place, and we investigated whether such changes also occur in response to monoclonal anti-idiotype antibodies (MAb2) that mimic HCMV gp86 and bind to a 92.5-kDa putative cell membrane receptor for gp86. Digitized image analysis of fura 2-loaded human embryonic lung fibroblasts indicated specific transient increases in [Ca2+]i beginning in some cells within the first 5 min of incubation with cross-linked MAb2 (70-750 nM, P < 0.01), which were similar in timing and intracellular distribution to those induced by HCMV. A primary source of Ca2+ appeared to be intracellular Ca2+ stores, since prior depletion of these stores with 30 nM thapsigargin inhibited the response (91.7 +/- 8.6%, P < 0.01); influx of Ca2+ from the extracellular medium was apparently necessary to maintain the intracellular Ca2+ stores. Transient increases in inositol trisphosphate (IP3) occurred in response to MAb2 (up to 3,329 +/- 84%, P < 0.001) or HCMV (92.8 +/- 19%, P < 0.01) during this same time period. These data suggest that the 92.5-kDa receptor for HCMV gp86 mediates an increase in IP3 and subsequent release of Ca2+ from intracellular stores.