Bone density and asymmetry are not related to DDT in House Sparrows: Insights from micro-focus X-ray computed tomography.

In organisms, DDT (Dichlorodiphenyltrichloroethane) and its metabolites, DDE (Dichlorodiphenyldichloroethylene) and DDD (Dichlorobischlorophenylethane) are endocrine mimics. They can influence bone density and other bone structural features. This study was conducted on House Sparrows (Passer domesticus) caught from the Free State - and the Limpopo Provinces of South Africa (SA). The sites were chosen based on spraying patterns of DDT for malaria control or non-spraying. The bone mineral densities of the femurs as well as the lengths of the left- and right leg bones were determined using micro-focus X-ray computed tomography (µ-XCT). The concentrations of DDT and its metabolites in the liver were determined with gas-chromatography mass-spectrometry to provide baseline concentrations of DDT in the body, allowing comparison of the various groups of birds. There was no asymmetry between the lengths of the bones of the left- and the right legs. DDT concentrations in the liver did not correlate with bone lengths. In addition, there were no significant differences between the relative densities of the left- and right leg bones with increase of concentrations of DDT. The concentrations of DDT and its metabolites did not have a significant effect on the measured bone parameters of House Sparrows. It is possible that the concentrations of DDT and its metabolites in the environments were too low to be injurious to the birds and/or tolerance to the insecticide has developed in the birds over more than six decades of almost continuous application of DDT.

Associations between DDT and egg parameters of the House Sparrow Passer domesticus from the Thohoyandou area of South Africa.

This study investigated whether the pesticide DDT (Dichlorodiphenyltrichloroethane) and its metabolites, DDE (Dichlorodiphenyldichloroethylene) and DDD (Dichlorobischlorophenylethane) were associated with adverse effects on multiple endpoints of the eggs of House Sparrows from the Thohoyandou area in South Africa, where DDT is used for malaria control. Eggshell thickness, pore numbers, pore shapes, and volume densities of the pores were measured to test possible adverse effects. Analysis was done using a scanning electron microscope and the concentrations of the pesticides were determined with the aid of gas chromatography-mass spectrometry. The highest concentrations recorded was p,p'-DDE at 0.84 µg/g wm (wet mass) in the eggs collected from Mangondi (a site last sprayed five years before sampling). Overall, the concentrations of total DDT recorded in this study were lower than reported by most other studies conducted in the same area. The association between DDT concentrations and House Sparrows eggshells were noticeable in the eggshell thicknesses, with significant differences between the eggs collected from Muledane (a site last sprayed 30 years before sampling) and Makula (a site sprayed both years of sampling) (P < 0.0022). Limited differences were found between the pore numbers and pore density of eggshells from the various sites. It may be that the limited effect on the pore numbers and volume densities of the pores are associated with low concentrations of DDT in the House Sparrow eggs.

Comparison of the growth kinetics and proteolytic activities of Chryseobacterium species and Pseudomonas fluorescens.

The effect of temperature on the growth kinetics and proteolytic activity of Chryseobacterium joostei and Chryseobacterium bovis was determined during this study. The results were compared with the activities of Pseudomonas fluorescens, which is regarded to be a major food spoilage psychrotolerant microorganism. For the growth studies, cultures were incubated in nutrient broth in a temperature gradient incubator (from 9 to 50 °C) and separately at 4 °C, and the optical density was measured at different time intervals. Growth temperature profiles for each organism were constructed. For determination of proteolytic activity, the cultures were incubated in fat-free ultra-high temperature processed milk in the temperature gradient incubator for 72 h (temperature range as above). Cell-free extracts were used to determine the proteolytic activity using the azocasein method. Results of the growth studies showed that C. joostei had the ability to grow over a wider temperature range than C. bovis and P. fluorescens without being affected by changes in the temperature. For the proteolytic activity, C. joostei had significantly (p < 0.001) higher activity per milligram of protein at 15.5 °C, followed by C. bovis and P. fluorescens. The results showed that C. joostei potentially has an even greater spoilage capacity in milk on the basis of growth rate and proteolytic activity than did P. fluorescens.

Growth phase-associated changes in protein expression in Mycobacterium smegmatis identify a new low molecular weight heat shock protein.

De novo protein synthesis and the heat-shock response during different stages of bacterial culture of Mycobacterium smegmatis LR222 were investigated. A discontinuance in the increase in number of colony forming units occurred at mid-exponential phase of growth. This coincided with a plateau in the ATP content of the culture, a reduction in the synthesis of exponential phase proteins (58, 30.5, and 20 kDa), a transitory synthesis of a 32 kDa protein and the induction of stationary-phase proteins (48, 46, 31, 25, and 20 kDa). The response to heat shock showed a growth-phase dependency, with the highest fold-induction of the 75 kDa (DnaK) protein occurring during the transitory cessation in the increase in CFU, while the greatest increase of the 95 kDa, 66 kDa (GroEL), and approximately 17 kDa (a doublet) proteins occurred during stationary phase. The approximately 17 kDa doublet was resolved into four polypeptides by two-dimensional electrophoresis. Mass spectrometric analysis of the sequence of one polypeptide (named Hsp17-2, 16.8 kDa) revealed significant homology to a conserved, 16.2 kDa, hypothetical protein of unknown function in Mycobacterium tuberculosis H37Rv. The increased synthesis of Hsp17-2 in response to heat shock suggests that it may represent a new low molecular weight heat shock protein.

The cold-shock stress response in Mycobacterium smegmatis induces the expression of a histone-like protein.

The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37 degrees C to 10 degrees C having the most widespread effect on growth, metabolism and protein composition. This 27 degrees C temperature reduction was associated with a lag period of 21-24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9-12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.

Ischemic heart disease in black South African stroke patients.

BACKGROUND AND PURPOSE: Stroke patients in western countries frequently have coronary artery disease (CAD). In black Africans, CAD has been reported as being rare in both stroke patients and the general population. In this study, an attempt has been made to determine the prevalence of CAD in a black South African stroke population. METHODS: The prevalence of CAD was determined by indicators identified through a series of 5 observational studies in black patients diagnosed with stroke. CAD indicators included (1) bedside diagnosis in 741 patients; (2) resting ECG in 555 consecutively admitted patients; (3) a combination of clinical examination, cardiac ultrasound, radionuclide scintigraphy, and multigated blood pool studies in 102 consecutively admitted patients; (4) thallium scintigraphy in 60 patients; and (5) necropsy in 23 patients. RESULTS: On bedside questioning, only 0.7% complained of previous angina. There was no history given of myocardial infarction (MI), but documentation of this was found in the clinical notes of 0.7% of the patients. In the resting ECG study, evidence of myocardial ischemia was present in 14.6% and MI in 2.1%. In the combined study, cardiac ischemia was documented on ECG in 12.7% of patients and evidence of previous MI in 5.8%. Cardiac scintigraphic studies revealed changes of myocardial ischemia in 31.7% and MI in 13.3% of the 60 patients studied. Four (17.4%) of 23 patients in the necropsy study had histological evidence of previous MI, and 50% of all patients had evidence of >50% atherosclerotic stenosis in 1, 2, or 3 coronary arteries. CONCLUSIONS: The prevalence of CAD in black African stroke patients is significantly higher than has been documented in the general nonstroke black population as well as in stroke patients. Black stroke patients may have a risk for CAD similar to that of their white counterparts.

Thalidomide-induced antigen-specific immune stimulation in patients with human immunodeficiency virus type 1 and tuberculosis.

Thalidomide, which inhibits monocyte tumor necrosis factor (TNF)-alpha production and costimulates T cells, was tested for immune modulation in patients with human immunodeficiency virus (HIV) infection and tuberculosis (TB) in a placebo-controlled study. Thalidomide therapy resulted in increased levels of plasma interleukin (IL)-2 receptor, soluble CD8, interferon-gamma, and IL-12, indicating immune stimulation. TNF-alpha levels were not reduced. Thalidomide treatment increased CD4+ and CD8+ T cell counts and lymphocyte proliferation to purified protein derivative. Immune stimulation was not associated with an increase in plasma HIV levels. In vivo, a thalidomide dose-dependent costimulatory effect on T cell proliferation and HIV replication was observed after stimulation with antigens or anti-CD3, respectively. Thalidomide-induced increased viral replication in CD4+ T cells was abrogated by adding back autologous CD8+ T cells. Thus, in the presence of thalidomide, antigen-specific immune responses in vitro and in patients with HIV/TB were enhanced.

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF.

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.

Characterization of a Mycobacterium tuberculosis homologue of the Streptomyces coelicolor whiB gene.

SETTING: Molecular Research Laboratory, Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital. OBJECTIVE: Characterize Mycobacterium tuberculosis homologue of the Streptomyces coelicolor, sporulation specific, whiB regulatory gene. DESIGN: The M. tuberculosis whiB3 gene was isolated by enriched cloning of a 2.8 kb BamHl fragment to which the S. coelicolor whiB gene hybridized. Expression of the gene was analysed by S1 nuclease analysis and promoter studies. RESULTS: An open reading frame within the 2.8 kb BamHl fragment was identified as the M. tuberculosis whiB3 gene, one of four whiB homologues in the M. tuberculosis genome. The deduced amino acid sequence has a 92% identity with a M. leprae protein, and 32% identity with the S. coelicolor WhiB protein. S1 nuclease analysis showed that the M. tuberculosis whiB3 gene is constitutively expressed by the cells in liquid culture. Primer extension analysis revealed three transcriptional start sites. Expression from the three potential promoters is growth phase-dependent. CONCLUSION: The M. tuberculosis whiB3 gene is expressed throughout growth, but expression from the individual promoters is growth phase dependent.

Novel method for rapid measurement of growth of mycobacteria in detergent-free media.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Selective increase in plasma tumor necrosis factor-alpha and concomitant clinical deterioration after initiating therapy in patients with severe tuberculosis.

The initiation of antituberculosis treatment in patients with severe tuberculosis may be accompanied by clinical deterioration and even death before any improvement occurs. To investigate this phenomenon, newly diagnosed human immunodeficiency virus-negative adults with severe tuberculosis were followed for the first 42 days of standard short-course therapy. Clinical status, serum lactate, plasma cytokine, and plasma cytokine receptor levels were monitored on days 0, 3, and 7 and then weekly for up to 42 days. Following 7 days of antituberculosis therapy, a significant transient decrease in mean Karnofsky score (P < .001), a concomitant increase in serum lactate (P = .06), a decrease in patient weight (P = .02), and an increase in plasma tumor necrosis factor-alpha (TNF-alpha) concentrations (P = .04) were observed. Plasma levels of soluble interleukin-2 receptor, interferon-gamma, interleukin-6, and TNF-alpha receptor decreased over the 42-day study period. These observations suggest that increases in plasma TNF-alpha levels may be associated with clinical deterioration observed early in the treatment of severe tuberculosis.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

rhuIL-2 adjunctive therapy in multidrug resistant tuberculosis: a comparison of two treatment regimens and placebo.

SETTING: Low-dose recombinant human interleukin 2 (rhuIL-2) adjunctive immunotherapy in multidrug resistant tuberculosis (MDR-TB) patients. OBJECTIVE: Evaluation of the effects of daily versus pulse-administered rhuIL-2 compared to placebo. DESIGN: MDR-TB patients on best available antituberculous chemotherapy received rhuIL-2 for 30 consecutive days (daily therapy), or for 5 days followed by a 9-day 'rest', for three cycles (pulse therapy). Placebo control patients received diluent. The cumulative total dose of rhuIL-2 given to each patient in either rhuIL-2 treatment group was the same. Patient immunologic, microbiologic, and radiologic responses were compared. RESULTS: The three treatment schedules induced different results. Immune activation was documented in patients receiving daily rhuIL-2 therapy. Numbers of CD25+ and CD56+ cells in the peripheral blood were increased in these patients, but not in patients receiving pulse rhuIL-2 or placebo. In addition, 5/8 (62%) patients receiving daily rhuIL-2 demonstrated reduced or cleared sputum bacterial load while only 2/7 (28%) pulse rhuIL-2 treated and 2/8 (25%) controls showed bacillary clearance. Chest radiographs of 7/12 (58%) patients receiving daily rhuIL-2 indicated significant improvement over 6 weeks. Only 2/9 (22%) pulse rhuIL-2-treated patients and 5/12(42%) placebo controls showed radiologic improvement. CONCLUSION: Daily low dose rhuIL-2 adjunctive treatment stimulates immune activation and may enhance the antimicrobial response in MDR-TB.

Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.

16S rRNA sequence analysis of an isolate of Mycobacterium haemophilum from a heart transplant patient.

Biopsy samples from a heart transplant patient with cellulitis and bursitis yielded an isolate of Mycobacterium haemophilum. The isolate was identified on the basis of a growth requirement for haemin or ferric ammonium citrate, growth at 30 degrees C but not at 37 degrees C, negative catalase test, intracellular growth in McCoy fibroblasts and sequence identify with a portion of the 16S rRNA sequence of the type strain. In comparisons with known 16S rRNA sequences, M. haemophilum grouped with other pathogenic, slow-growing mycobacteria, showing close sequence similarity to M. marinum (98.8%) and lower similarity to M. ulcerans and M. tuberculosis complex organisms. M. haemophilum and M. marium share other features including optimal growth at 30 degrees C and the ability to cause superficial skin lesions in man.

High level kanamycin resistance associated with the hyperproduction of AAC(3)II and a generalised reduction in the accumulation of aminoglycosides in Acinetobacter spp.

The clinical isolate of Acinetobacter baumannii strain SAK contains an actively transcribed aacC2 gene and a latent aadB gene that encode the aminoglycoside-modifying enzymes, AAC(3)II and AAD(2"), respectively. In an attempt to activate the aadB gene, the strain was cultured in the presence of kanamycin which is a substrate for AAD(2"). Although it was possible to isolate kanamycin resistant derivatives these were not associated with detectable AAD(2") activity. Instead, there was a marked increase in the level of AAC(3)II activity which was associated with amplification of the aacC2 gene.