Biomedical subjects

K L Martin

Publications and source records attributed to K L Martin.

At least 37 records · Page 2Linked to original sources

PubMed1993

A role for the primary care physician in counseling young African-American men about homicide prevention.

Homicide is the leading cause of death in African-American men aged 15-34 years, yet physicians rarely discuss homicide prevention with patients. The authors propose that physicians have a role in preventing homicide similar to their role in other preventive medicine issues. This study evaluated patients' responsiveness to a physician's counseling about firearms and homicide. While being treated for unrelated problems at a walk-in ambulatory clinic, 53 African-American men patients received brief counseling by the physician about six preventive medicine topics, including firearms. A postvisit interview demonstrated that the discussion of firearms was well received and recalled more than any other preventive medicine issue discussed.

Adolescent↗

PubMed1993

Activity of enzymes of energy metabolism in single human preimplantation embryos.

A method was developed to measure the activities of enzymes in extracts from single human preimplantation embryos. The method permits the analysis of two enzymes plus appropriate controls in an extract from a single embryo, and was used to investigate the control of energy metabolism during the development of human embryos from the two-cell to the blastocyst stage. Hexokinase (HK), 6-phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-diphosphate aldolase (ALD), glucose phosphate isomerase (GPI), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH) and 2-oxoglutarate dehydrogenase (ODH) were all detectable, whereas glycogen phosphorylase (GP) was not. The enzyme activities of ODH, PFK, LDH, PK, GPI and G6PDH, averaged over all stages of development from the two-cell to blastocyst stage (days 2-6 after insemination), were 3.5, 6.6, 15, 69, 73 and 87 times greater than HK, respectively. The activity of ALD was very similar to that of HK. The activities of ALD, GPI, PFK, PK and LDH showed no significant variation with stage of development, although the activity of GPI fell significantly from the four-eight cell to the eight-sixteen cell stage (P < 0.05). HK activity decreased from the two-eight cell to the eight-sixteen cell (P < 0.05), and increased significantly from the eight-sixteen cell to the blastocyst stage (P < 0.01). The overall relationship between hexokinase activity and stage approached significance (P = 0.059, one-way analysis of variance). The activity of G6PDH decreased significantly with development (P < 0.001, one way analysis of variance).(ABSTRACT TRUNCATED AT 250 WORDS)

Blastocyst↗

PubMed1992

The relative sensitivity of competing hydropsychid species to fluoride toxicity in the Cache la Poudre River (Colorado).

The influence of increased fluoride concentrations generated by a wastewater treatment plant on the spatial distribution and abundance of benthic larvae of Hydropsychidae (Insecta, Trichoptera) species in the Cache la Poudre River (Colorado) was examined. Acute lethal concentrations of fluoride ion (F-) to these species were determined in soft water (average value of hardness 40.2 mg CaCO3/L) by static toxicity bioassays. The wastewater treatment plant caused a significant (P less than 0.05) increase in the fluoride concentration at three downstream sampling sites (mean values 1.17, 0.84, and 0.56 mg F-/L at 0.1, 1.6, and 9.2 km downstream sites, respectively) compared with the upstream reference station (0.31 mg F-/L). The 48, 72, 96, 120, and 144-h LC50s (mg F-/L) were 52.6, 25.8, 17.0, 13.4, and 11.5 for Hydropsyche bronta Ross, 102.0, 53.5, 34.7, 27.0, and 21.4 for Hydropsyche occidentalis Banks, and 128.0, 73.2, 42.5, 31.9, and 24.2 for Cheumatopsyche pettiti (Banks). LC50 values for H. bronta were significantly (P less than 0.05) lower than LC50 values for the other two test species. Abundance and biomass of all hydropsychid species were significantly (P less than 0.05) lower at the 0.1 km downstream site than at the upstream reference site. H. occidentalis was usually the most abundant species at the reference and 9.2 km downstream stations, with highest abundances at the 9.2 km downstream site. C. pettiti was dominant at 0.1 and 1.6 km downstream sites, showing higher abundances at the 1.6 km downstream site than at the upstream reference site. H. bronta was never collected at 0.1 and 1.6 km downstream sites, but was abundant at the upstream reference site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.

When Azotobacter vinelandii is grown under nitrogen-fixing conditions, the mean cell volume fluctuates from 2.7 to 6.6 microns 3 as determined using a Coulter counter. When NH4Cl is supplied as nitrogen source, the mean cell volume fluctuates from 4.6 to 7.4 microns3. Parallel experiments using flow cytometric measurements show similar characteristic fluctuations in the narrow forward angle light scattering signal and also in cellular protein content as determined using fluorescein isothiocyanate (FITC) fluorescence. Fluctuations in the perpendicular light scatter signal during batch growth are similar for both sets of growth conditions. Changes in cell morphology and ultrastructure are also similar for both sets of growth conditions, as demonstrated by electron microscopic examination. We conclude that narrow forward angle light scatter is a close correlate of cell size, whereas right angle scatter is an indicator of morphological variations other than size.

Ammonium Chloride↗

PubMed1990

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage.

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.

Biopsy↗

PubMed1989

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

Amino Acid Sequence↗

PubMed1988

Limitations of flow cytometry for the specific detection of bacteria in mixed populations.

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.

Antibodies, Bacterial↗

PubMed1988

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests.

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B. anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.

Antibodies, Bacterial↗

PubMed1987

Direct and indirect immunofluorescence analysis of bacterial populations by flow cytometry.

Bacillus anthracis spores and Escherichia coli were stained with fluorescein-conjugated antibody using direct and indirect methods, then analyzed by means of a commercial flow cytometer. To reduce the cytometer's fluorescence component resulting from unreacted conjugate, reaction mixtures were either diluted or were centrifuged through a sucrose solution using a moving zone technique. Evidence is produced that the fluorescence statistics for centrifuged samples closely represent the fluorescence distribution of stained single bacteria in the reaction mixture at the end of incubation; in particular, centrifugation did not cause aggregation of bacteria. Centrifugation is proposed as more effective than mere dilution for use with a wide range of bacterial concentrations, and the moving zone technique is to be preferred to conventional centrifugation in which bacteria tend to aggregate in the pellet. In indirect assays, it was shown that the washing step after reaction with antibacterial antibody may be omitted. The performance of direct and indirect staining methods was compared, including the use of either Staphylococcus aureus protein A or polyclonal sheep anti-rabbit antibody as the indirect reagent. When the bacterial concentration in reaction mixtures was increased the median fluorescence intensity fell, indicating that specific antibody had become limiting at low concentrations of the polyclonal antibody preparations. The implications of this for the design of flow cytometry assays of bacteria are discussed.

Antibodies, Bacterial↗

PubMed1985

Serum stimulation and repression of flow immunofluorescence staining of bacteria.

A flow cytometer was used to measure the fluorescence intensity of Bacillus anthracis spores, B. subtilis spores and Escherichia coli stained in suspension with specific rabbit fluorescein-conjugated antibody. The effect of normal sera and a number of other additives on the binding of conjugate to the surface of the homologous bacteria was assessed by measuring the median fluorescence intensity of the bacterial population in the reaction mixture. Non-ionic detergent depressed binding of one conjugate (anti-E. coli) by up to 22%. Bovine serum albumin, gelatin, foetal calf serum and normal rabbit serum did not affect the median fluorescence value for these 3 bacterial species by more than 14%. Normal serum from 5 goats reduced the specific staining of B. anthracis by up to two-thirds. Anti-B. anthracis antibodies were detected in goat serum by indirect immunofluorescence microscopy, and it is inferred that these goat antibodies were in competition with fluorescein conjugate for the bacterial antigens. Normal goat and sheep serum stimulated the specific staining of B. subtilis and E. coli measured by the cytometer; in the case of goat serum previous heating of the serum to 56 degrees C resulted in repression of staining of E. coli. Since anti-E. coli antibody was detected in this normal sera by indirect immunofluorescence assays, it is proposed that repression was caused by anti-bacterial antibodies and stimulation by a separate factor, heat-labile in the case of goat serum. The stimulatory factor was also apparently inactivated by increasing the NaCl concentration, suggesting that stimulation depends heavily on charge interactions. Preliminary evidence is presented that the stimulatory factor may be anti-antibody, possibly of the IgA or IgG class.

Animals↗

PubMed1985

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores.

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.

Bacillus↗

PubMed1985

Hepatic membrane antibodies: studies of prevalence and specificity.

Using a newly developed assay for hepatic membrane-associated antibodies (HMA), we studied the prevalence and specificity of HMA in a variety of acute and chronic liver diseases to evaluate the usefulness of such an assay system. Sera were examined by indirect immunofluorescence on rat liver sections previously fixed in Bouin's fluid. As compared with a prevalence of 4% in 45 healthy controls, HMA were detected in 73% of 45 patients with chronic active hepatitis (CAH) (P less than 0.001) and in all 11 patients with primary biliary cirrhosis (PBC) (P less than 0.001). HMA were also present in 38% of 40 patients with acute viral hepatitis, 47% of 27 with alcoholic liver disease and 33% of 24 with other liver diseases not thought to be immunologically mediated. In 53 patients studied with other liver, gastrointestinal or autoimmune diseases, the prevalence of HMA was not increased over controls. Absorption studies showed that seven of 14 HMA positive sera contained specificities for polymerized human serum albumin (pHSA) or liver-specific protein (LSP) or both antigens. HMA positive samples reacted occasionally with rat kidney tubular epithelial membranes but did not appear to react with other tissues. These results demonstrate that HMA are present at higher prevalence in the autoimmune liver diseases than many previously described autoantibodies and are reactive in part to specificities on pHSA and LSP.

Antibody Specificity↗

PubMed1984

The choice of methods for immunoglobulin IgG purification: yield and purity of antibody activity.

Six methods for the purification of immunoglobulin G (IgG) from serum were compared, using rabbit antiserum to Bacillus anthracis spores as a model. Antibody activity was monitored by a solid-phase immunoradiometric assay (IRMA). Salt precipitation/ion exchange chromatography and ethanol precipitation both resulted in IgG of high purity but there was considerable inactivation of antibody. Salt precipitation/affinity chromatography gave poor yields of antibody. PEG precipitation and gel filtration of Sephacryl S-300 gave moderate yields and purity of IgG, with little evidence of antibody inactivation. Salt precipitation was marginally more destructive than the last 2 methods, but is recommended for routine use on grounds of its simplicity. Should IgG prepared by salt precipitation prove inadequate for particular applications, gel filtration is recommended since it allows the balance of yield and purity to be altered at will.

Animals↗

PubMed1984

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores.

Staphylococcus aureus Protein A (SpA) labelled with [125I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [3H] by propionylation also performed well but would be expensive to use. SpA labelled with [3H] fluorodinitrobenzene, or labelled with [125I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a microfluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[125I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR:SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fluorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.

Antibodies, Anti-Idiotypic↗

PubMed1983

Comparison of immunoradiometric assays of Bacillus anthracis spores immobilised on multispot slides and on microtitre plates.

Indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores are described in which the spores were heat fixed either on multispot microscope slides or on polyvinyl or polystyrene microtitre plates. Assays on plastic plates sometimes suffered from poor intra-experiment reproducibility. Signals were higher in assays in flat-bottomed microtitre wells than in assays in slides, but assay noise, due to non-specific absorption of antibody, was higher also, giving an overall disadvantage in signal-to-noise ratio. Elution of antibody with acid gave up to a doubling in signal-to-noise ratio. When small volumes of antibody reagents were used in assays in round-bottomed plastic wells, signal and noise characteristics were similar to those of the slide assay. While the assays on plastic wells tended to be less sensitive in terms of anti-spore antibody concentration or the number of spores detectable, the larger volume of antigen suspension that could be used in plastic wells gave them an advantage in terms of the minimum antigen concentration detectable.

Antibodies, Bacterial↗

PubMed1983

Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry.

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.

Antigens, Bacterial↗

PubMed1983

Comparison of direct and indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores immobilised on multispot microscope slides.

A solid phase immunoradiometric assay (IRMA) is described in which Bacillus anthracis spores were heat fixed to the wells of glass multispot microscope slides. Assays for spores of B. anthracis Vollum and Sterne strains with 3H labels were evaluated in the direct and indirect versions. Neither single nor signal-to-noise characteristics of indirect assays were greatly improved by the use of immunopurified antibody (IPAB) or IgG anti-bacterial reagents rather than antiserum. However, the specificity of the direct and indirect assays for B. anthracis strains and B. cereus NCTC 8035 was altered by immunopurification of the anti-bacterial reagent. Although the signal-to-noise ratio was sometimes higher in indirect than in direct assays, signal values were usually no better. Evidence was produced that the overall ratio of the indirect : direct antibody molecules bound by preparations of B. anthracis spores rarely exceeded two but the antibody-molecular ratio for antigens on extracellular material in spore preparations was much higher than the ratio for antigens on the spores themselves.

Antibodies, Bacterial↗

PubMed1983

Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.

Bacillus anthracis↗