Comparison of envelope 2 CD81 binding regions in PBMC-derived versus serum-derived hepatitis C virus isolates: higher conservation of CD81 region 2 in PBMC isolates.

UNLABELLED: The aim of the present study was to investigate the variability of hepatitis C virus (HCV) CD81 binding regions (CD81-1/2) in peripheral blood mononuclear cells (PBMC)-derived and serum-derived HCV-RNA samples. HCV-RNA was isolated from PBMC (104 cells) and serum samples from 37 patients chronically infected with HCV genotype 1a/1b (n=21/16). The hypervariable regions 1/2 (amino acid 384-410, amino acid 474-482) and regions CD81-1/2 (amino acid 474-494, amino acid 522-551) were analysed. Mutational frequency of amino acid sequences was compared between PBMC-derived and serum-derived HCV variants as well as local accumulation of mutations. Furthermore, CD81 was quantified on PBMC. Mutational frequency was not different between PBMC-derived and serum-derived HCV variants. A trend to lower mutational frequency in genotype 1a PBMC variants compared with serum-derived variants was observed in region CD81-2 (5%vs 10%). Smoothed mutational frequency analysis showed a significantly lower variability within genotype 1a CD81-2 in PBMC-derived compared to serum-derived HCV-RNA (P=0.026). CD81 expression on PBMC was not correlated with the number of mutations within the CD81 binding regions. CONCLUSION: A higher conservation was observed in region CD81-2 in PBMC-derived versus serum-derived HCV-RNA indicating selection of HCV variants on PBMC. The variability in the CD81 binding regions appeared to be independent from CD81 expression.

Triclosan-tolerant bacteria: changes in susceptibility to antibiotics.

There is no clear consensus regarding the effect of biocide tolerance on antibiotic susceptibility. In this work, triclosan-tolerant strains of Escherichia coli, Staphylococcus aureus and Acinetobacter johnsonii were compared with sensitive strains in order to ascertain their susceptibility to a range of antibiotics. The minimum inhibitory concentrations of triclosan were measured using broth- and agar-dilution techniques. Antibiotic susceptibilities were determined using the British Society for Antimicrobial Chemotherapy guidelines. No triclosan-tolerant strains were resistant to antibiotics, and there was no overall tendency for triclosan-tolerant strains to have significantly smaller zones of inhibition compared with counterpart strains. Triclosan-tolerant strains of E. coli were significantly more susceptible to aminoglycoside antibiotics. The mechanism by which E. coli develops tolerance to triclosan appears to be linked to aminoglycoside susceptibility. It is proposed that changes in outer membrane, or the loss of plasmids, may be responsible for this relationship.

Triclosan-bacteria interactions: single or multiple target sites?

AIMS: To investigate the inhibitory and lethal effects of triclosan against several micro-organisms at different stages of their phase of population growth. METHODS AND RESULTS: Triclosan minimum inhibitory concentrations against several test organisms were determined in broth and agar using standard protocols. The bisphenol effect on bacterial population growth kinetics was studied using the Bioscreen C microbial growth analyser. Finally, the efficacy of triclosan on phases of bacterial growth was determined using a standard suspension test. The duration of the lag phase for all micro-organisms tested was increased by bisphenol in a concentration-dependent manner. The population growth kinetics of the micro-organisms was also altered after biocide exposure. At higher concentrations, triclosan was bactericidal regardless of their phase of population growth, although population in stationary phase and particularly, washed suspensions, were more resilient to the lethality of triclosan. This lethal activity was concentration and contact time dependent, and in some instances, bactericidal activity of bisphenol was observed within 15 s. CONCLUSIONS: Low concentrations of triclosan affected the growth of several bacteria, while higher concentrations were bactericidal regardless of the bacterial phase of population growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Here, we presented clear evidence that the interaction of triclosan with the bacterial cell is complex and its lethality cannot be explained solely by the inhibition of metabolic pathways such as the enoyl acyl-reductase. However, the inhibition of such pathways cannot be ruled out as part of the lethal mechanism of the bisphenol at a low bactericidal concentration.

Triclosan inhibition of fatty acid synthesis and its effect on growth of Escherichia coli and Pseudomonas aeruginosa.

OBJECTIVES: To assess the effect of triclosan on fatty acid synthesis and to relate the inhibition of enoyl reductase to bacterial viability. METHODS: The effect of triclosan on fatty acid synthesis in a triclosan-resistant Escherichia coli and its sensitive counterpart and in Pseudomonas aeruginosa was investigated by measuring acetate incorporation into total lipid followed by analysis of fatty acid methyl esters by gas chromatography. Concurrently, the bactericidal effect of triclosan against these bacterial strains was assessed. RESULTS: Triclosan inhibited fatty acid biosynthesis in all the strains tested. However, for triclosan-resistant E. coli (MIC > 1000 mg/L) the concentration required to achieve inhibition was higher than that required for the susceptible counterpart. These concentrations did not significantly affect cell survival in any of the strains tested. CONCLUSIONS: This study shows that the inhibition of fatty acid biosynthesis by the bisphenol might be involved in its growth-inhibitory action and that other mechanisms are involved in its lethal effect. In addition, although microorganisms with a high triclosan MIC were still susceptible to the inhibitory effect of the bisphenol on fatty acid biosynthesis, a higher concentration of the compound was required. This suggested that triclosan bioavailability was different in these strains.

Long-term outcome study of growth factor-treated pressure ulcers.

BACKGROUND: Exogenous application of growth factors have been reported in an attempt to accelerate healing of chronic wounds. Most of the trials were of brief duration with short to no follow-up periods. Long-term outcome studies are sparse for pressure ulcer therapies with success rates around 30% for both operative and nonoperative treatments. METHODS: Follow-up evaluations were performed serially up to 12 months for patients completing a 35 day blinded, placebo-controlled cytokine clinical trial of pressure ulcers. RESULTS: Fifty-four of 61 patients completed the follow-up period with 68.5% of the patients (37 of 54) being healed after 1 year. Of patients healing > or =85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed <85% during treatment (P <0.05). CONCLUSION: Long-term outcome was better in this growth factor trial than with surgical or standard nonoperative treatment of pressure ulcers. Since only patients receiving exogenously applied cytokines achieved >85% closure during the treatment phase of the trial, the excellent long-term outcome appears attributable to the cytokine therapy.

Sequential cytokine therapy for pressure ulcers: clinical and mechanistic response.

OBJECTIVE: To compare the healing response of sequential topically applied cytokines to that of each cytokine alone and to a placebo in pressure ulcers, and to evaluate the molecular and cellular responses. SUMMARY BACKGROUND DATA: Because of a deficiency of cytokine growth factors in chronic wounds and the reversal of impaired healing in animal models, pressure ulcer trials have been performed with several exogenously applied growth factors. Because single-factor therapy has not been uniformly successful, combination or sequential cytokine therapy has been proposed. Laboratory data have suggested that sequential treatment with granulocyte-macrophage/colony-stimulating factor (GM-CSF)/basic fibroblast growth factor (bFGF) might augment the previously reported effect of bFGF alone. METHODS: A masked, randomized pressure ulcer trial was performed comparing sequential GM-CSF/bFGF therapy with that of each cytokine alone and with placebo during a 35-day period. The primary measure was wound volume decrease over time. Cytokine wound levels and mRNA levels were serially determined. Fibroblast-populated collagen lattices (FPCLs) were constructed from serial fibroblast biopsies. Cellular ultrastructure was evaluated by electron microscopy. Changes in ease of surgical closure and its relative cost were determined. RESULTS: Ulcers treated with cytokines had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Patients treated with GM-CSF or bFGF had higher levels of their respective cytokine after treatment. Patients with the greatest amount of healing showed higher levels of platelet-derived growth factor (PDGF) on day 10 and transforming growth factor beta (TGFbeta1) on day 36. Message for the bFGF gene was upregulated after treatment with exogenous bFGF, suggesting autoinduction of the cytokine. FPCLs did not mimic the wound responses. Ultrastructure of wound biopsies showed response to bFGF. Treatment with any of the cytokines improved the wound by allowing easier wound closure. This was most marked for the bFGF-alone treatment, with a cost savings of $9,000 to $9,200. CONCLUSIONS: Treatment with bFGF resulted in significantly greater healing than the other treatments in this trial. The clinical response appeared to be related to upregulation of the bFGF message and to increased levels of PDGF-AB, bFGF, and TGFbeta1 in the wounds and changes in ultrastructure. The resultant improvements could be correlated with cost savings.

Regulatory sequences and a novel gene in the msp (GP63) gene cluster of Leishmania chagasi.

The surface protease GP63 of Leishmania chagasi is encoded by a cluster of more than 18 tandem major surface protease (msp) genes belonging to three classes (mspL, mspS, mspC). mspL and mspS transcripts are differentially expressed during parasite growth. RNAs from mspS genes predominate during stationary phase, the time when parasite virulence and GP63 expression are maximal. We hypothesized that the unique regions downstream of mspS genes contain signals important for gene expression. The 2.8 kb region between tandem mspS genes was found to contain an 882 bp open reading frame designated mag. Copies of mag were found downstream of all mspS genes in the cluster. mag hybridized faintly to bands on Northern blots and a fully processed mag cDNA was identified in a promastigote cDNA library, providing evidence that mag genes are expressed at low levels. Similar to mspS RNAs, the abundance of mag RNAs was greater in stationary phase than logarithmic phase organisms, although mag RNAs were less abundant than mspS RNAs throughout growth. Northern blots and enzyme assays of promastigotes containing plasmid constructs in which the beta-galactosidase gene was followed by sequences between mspS coding regions, either with or without mag and its downstream sequences, suggest these regions have several regulatory effects accounting for the growth-associated changes in mspS expression.

Total synthesis and biological activities of (+)- and (-)-boscialin and their 1'-epimers.

Natural (-)-boscialin [(-)-1] has recently been described as one of the constituents of various medicinal plants. To obtain more material for investigations of its biological activities, we carried out the synthesis of (-)-1 and its isomers. Starting from the chiral building block 2, the key steps of the synthesis involved a regioselective reduction and a nucleophilic addition. The enantiomer of the natural product, (+)-boscialin [(+)-1], could be obtained via acid-catalyzed epimerization of hydroxyketone 4 to (+)-3. Starting the synthesis with (-)-3 led to (-)-boscialin [(-)-1] with the natural absolute configuration. In addition to (+)- and (-)-boscialin, the corresponding 1'-epimers (+)- and (-)-epiboscialin were also obtained. In vitro assays with (-)-boscialin [(-)-1] and its three stereoisomers were carried out to test for activity against microbes, parasites, and human fibroblasts. The investigations revealed activity against various microbes and against Trypanosoma brucei rhodesiense and also revealed cytotoxicity against human cancer cells.

Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors.

Determination of the rate of Trypanosoma cruzi infection in its triatomine vectors is an element in control programmes directed at reducing transmission of the organism to humans. Traditionally, T. cruzi has been detected in these insects by microscopical examination of intestinal contents or excreta. The sensitivity of this laborious process has not been defined because of the lack of a bench-mark method against which microscopical examination could be compared. The purpose of this study was to compare the sensitivity of a polymerase chain reaction (PCR) assay with that of microscopical examination for detecting T. cruzi in Triatoma infestans nymphs that had fed on patients with chronic Chagas disease. To this end, we analysed 54 pairs of samples, each containing 2 groups of 10 insects, obtained by feedings on 19 patients with chronic T. cruzi infection, 17 of whom were fed upon 3 times. One group of insects in each pair was analysed by PCR and the other by microscopical examination of excreta. Overall, the PCR assay gave positive results in 32 of 54 groups of insects examined (59%), whereas only 7 of 54 groups (13%) were positive by microscopical examination (P = 0.038). These results demonstrate that the PCR assay is significantly more sensitive for the detection of T. cruzi in triatomine vectors than is microscopical examination, and suggest that the PCR assay could be a useful tool in epizootiological studies.

Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi.

The diagnosis of acute infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is generally made by detecting parasites by microscopic examination of fresh blood. Although highly specific, this approach often lacks sensitivity. Several years ago, PCR assays for the detection of T. cruzi were described, but the sensitivities and specificities of these tests have not yet been defined precisely. In the present study, we first compared the sensitivities of PCR methods that differ in sample processing as well as in the target sequences that are amplified. Then, we challenged eight mice with T. cruzi, and on 31 days over a 380-day period, we compared the ability of the PCR method with the highest sensitivity to detect parasites in blood with that of microscopic examination. During the acute phase of the infections, parasites were detected on average 3.9 days earlier by the PCR method than by microscopy. Furthermore, the infected mice were consistently positive by the PCR method during the chronic phase, while parasites were intermittently detected by microscopic examination during that period. Overall, among the 248 comparisons, in 84 the PCR method was positive and no parasites were seen by microscopic examination, whereas the reverse was true in only 1 case, a difference that is highly significant. These findings suggest that this approach should be in patients suspected of having acute Chagas' disease. Moreover, the higher sensitivity of the PCR method observed in both the acute and chronic phases of the T. cruzi infections in the mice that we studied indicates that this approach should be useful in evaluating experimental drugs in T. cruzi-infected laboratory animals.

Postmortem diagnosis of autochthonous acute chagasic myocarditis by polymerase chain reaction amplification of a species-specific DNA sequence of Trypanosoma cruzi.

We report a fatal case of vector-transmitted acute Chagas' myocarditis in a seven-month-old child in south Texas. This diagnosis was not suspected during the three days of hospitalization that preceded the child's death, which was caused by heart failure. A diagnosis of acute myocarditis, probably of viral origin, was listed as the cause of death after cardiac tissue was examined microscopically at autopsy. One year after the death of the patient, a diagnosis of Trypanosoma cruzi myocarditis, based solely on morphological grounds, was made after newly prepared slides of cardiac tissue were examined. Seven years later, we confirmed the diagnosis of T. cruzi infection by using the polymerase chain reaction to amplify a species-specific genomic repetitive DNA sequence of the parasite from fixed cardiac tissue.

Maxicircle genomic organization and editing of an ATPase subunit 6 RNA in Trypanosoma cruzi.

The DNA sequence of a 5736-nucleotide (nt) Trypanosoma cruzi maxicircle fragment was determined. Sequence comparisons indicate that its 5' terminus is the homologue of the downstream portion of the NADH dehydrogenase subunit 7 gene and that its 3' region is homologous to the maxicircle unidentified reading frame II gene. The region between these two gene segments contains six additional genes that encode mitochondrial proteins, including ATPase subunit 6 (A6). Comparison of the A6 maxicircle DNA sequence with that of an A6 cDNA indicates that the A6 RNA is extensively edited throughout its length. A 49-nt sequence that could serve as template for transcription of a guide RNA for editing a segment of the A6 RNA was found in one of 24 minicircle variable regions sequenced. Moreover, the presence of an RNA having this sequence was demonstrated in an RNAse protection assay. This is the first identification of a guide RNA template in a T. cruzi minicircle. Taken together, our findings suggest that T. cruzi and Trypanosoma brucei brucei are phylogenetically closer to each other than they are to Leishmania tarentolae, despite the relative similarity of the life cycles of the latter and T. cruzi.

Large-scale preparation and biological activity of recombinant human parathyroid hormone.

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-beta-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.

Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.

Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius.

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.

Lack of formyl-methionyl-leucyl-phenylalanine receptors on porcine neutrophils.

The response of blood neutrophils to the chemotactic peptide formyl-methyl-leucyl-phenylalanine varies among species. Our results indicate that this peptide does not activate the respiratory burst of porcine neutrophils. Specifically, concentrations less than or equal to 10(-6) M did not cause production of either superoxide or hydrogen peroxide. Studies designed to delineate the biochemical deficit responsible for these results indicated that these cells do not express specific chemotactic peptide receptors on the external surface of the plasma membrane. Although these data do not rule out the possibility that internal stores of chemotactic peptide receptor exist, attempts to induce expression of the receptor by priming the cells with either lipopolysaccharide or calcium ionophore were unsuccessful.

Silastic prosthesis plombage for right postpneumonectomy syndrome.

In rare instances, right pneumonectomy can produce progressive exertional dyspnea and reduce ventilatory reserve because of extreme mediastinal shift (right postpneumonectomy syndrome). The diagnosis can be made by bronchoscopy and computed tomography. We report a case of a 43-year-old patient in whom plombage with two Silastic breast implants produced mediastinal derotation and symptomatic relief of this syndrome.

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.