Biomedical subjects
Publications and source records attributed to R Knowles.
A case of S. intercalatum infection in a young girl from Sao Tomé who had never previously left the island is described. The expansion of this species and its possible hybridization are discussed.
Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony-forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.
In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.
Nitrapyrin inhibited growth, CH(4) oxidation, and NH(4) oxidation, but not the oxidation of CH(3)OH, HCHO, or HCOONa, by Methylosinus trichosporium OB3b, suggesting that nitrapyrin acts against the methane monooxygenase enzyme system. The inhibition of CH(4) oxidation could be reversed by repeated washing of nitrapyrin-inhibited cells, indicating that its effect is bacteriostatic. The addition of Cu did not release the inhibition. Methane oxidation was also inhibited by 6-chloro-2-picoline. These data suggest that the mode of action of nitrapyrin on M. trichosporium is different from that on chemoautotrophic NH(4) oxidizers or methanogens.
zospirillum brasilense Sp7 was grown anaerobically with N2O as the terminal electron acceptor and NH4Cl as the nitrogen source. Hydrogen uptake activity (O2-dependent H3H oxidation) was expressed in the presence and absence of 5% H2; it reached its maximum in late logarithmic phase as the malate became limiting. This activity was very stable in stationary phase, even in the absence of exogenous H2, compared with microaerobically grown cultures; this supports the hypothesis that the exclusion of O2 is critical for maintaining the integrity of the H2 uptake system in this organism. Oxygen, as well as methylene blue and N2O, supported H2 uptake, indicating the presence of electron transport components leading to O2 in anaerobically grown A. brasilense. Nitrite (0.5 mM) inhibited H2 uptake. In cultures grown with NO3- as the terminal electron acceptor and NH4Cl as the nitrogen source, in the presence and absence of exogenous H2, only low H2 uptake activity was observed. Methylene blue, O2, N2O, NO3-, and NO2- were all capable of acting as the electron acceptor for H2 oxidation. Nitrite (0.5 mM) did not inhibit H2 uptake in NO3--grown cells, as it did in N2O-grown cells. A. brasilense appears to be one of the few organisms capable of expressing the H2 uptake system under denitrifying conditions in the absence of molecular H2.
Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction.
Two monoclonal antibodies were obtained from a fusion following immunization with human thymic cells. BL6 reacted with an antigen present on 80% of thymocytes found in the cortex, but absent from all other circulating or non circulating T or B lymphocytes. It immunoprecipitated an antigen of 49 Kd heavy chain with a small 12 Kd subunit. BL6 also reacted with dendritic cells of the epidermis identified as Langerhans cells carrying histocompatibility class II antigens and possessing Birbeck granules. The second antibody, BL7, did not stain thymocytes, but reacted with a cytoplasmic antigen of certain thymic epithelial cells. In skin sections, BL7 detected a cytoplasmic antigen of the keratinocytes of the basal cell layer. These cross reactivities between skin and thymic cells are discussed in view of a possible functional similarity between the two tissues.
The oxidation of NH(4) by Nitrosomonas europaea was insensitive to 10 mM NaClO(3) (sodium chlorate) but was strongly inhibited by NaClO(2) (sodium chlorite; K(i), 2 muM). The oxidation of NO(2) by Nitrobacter winogradskyi was inhibited by both ClO(3) and ClO(2) (K(i) for ClO(2), 100 muM). N. winogradskyi reduced ClO(3) to ClO(2) under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO(2) was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH(4) and NO(2) was inhibited in the presence of 10 mM ClO(3) after a 2-h lag period, despite the fact that, under these conditions, ClO(2) was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH(4) oxidation is inhibited by ClO(2) produced by reduction of ClO(3) by the NO(2) oxidizer. The use of ClO(3) inhibition of NO(2) oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH(4) is not affected.
Nitrogen fixation (C(2)H(2) reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C(2)H(4) production occurring in the presence of 8 mumol of S per ml of stream water. Sodium sulfide at concentrations of 16 mumol of S per ml or greater inhibited nitrogen fixation, with 64 mumol of S per ml being completely inhibitory. Sulfide at levels of 16 mumol/ml or above inhibited CO(2) production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO(2) production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 mumol of S per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H(2), H(2)S, and other sulfur species at various pH values, it appeared that H(2)S was the toxic entity and that HS was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS from H(2)S.
Production of H2 by Azospirillum brasilense under N2-fixing conditions was studied in continuous and batch cultures. Net H2 production was consistently observed only when the gas phase contained CO. Nitrogenase activity (C2H2 reduction) and H2 evolution (in the presence of 5% CO) showed a similar response to O2 and were highest at 0.75% dissolved O2. Uptake hydrogenase activity, ranging from 0.3 to 2.5 mumol H2/mg protein per hour was observed in batch cultures under N2. Such rates were more than sufficient to recycle nitrogenase-produced H2. Tritium-exchange assay showed that H2 uptake was higher under Ar than under N2. Uptake hydrogenase was strongly inhibited by CO and C2H2. Cyclic GMP inhibited both nitrogenase and uptake hydrogenase activities.
An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N mh, respectively. Acetylene reduction at an average rate of 0.07 mmol of C(2)H(4) formed mh was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N mh in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.
Samples of sediment from Lake St. George, Ontario, Canada, were incubated in the laboratory under an initially aerobic gas phase and under anaerobic conditions. In the absence of added nitrate (NO(3)) there was O(2)-dependent production of nitrous oxide (N(2)O), which was inhibited by acetylene (C(2)H(2)) and by nitrapyrin, suggesting that coupled nitrification-denitrification was responsible. Denitrification of added NO(3) was almost as rapid under an aerobic gas phase as under anaerobic conditions. The N(2)O that accumulated persisted in the presence of 0.4 atm of C(2)H(2), but was gradually reduced by some sediment samples at lower C(2)H(2) concentrations. Low rates of C(2)H(2) reduction were observed in the dark, were maximal at 0.2 atm of C(2)H(2), and were decreased in the presence of O(2), NO(3), or both. High rates of light-dependent C(2)H(2) reduction occurred under anaerobic conditions. Predictably, methane (CH(4)) production, which occurred only under anaerobiosis, was delayed by added NO(3) and inhibited by C(2)H(2). Consumption of added CH(4) occurred only under aerobic conditions and was inhibited by C(2)H(2).
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The diagnosis of pyogenic arthritis may be difficult to confirm since culture results are sometimes negative. This study attempted to evaluate the utility of the limulus lysate assay for the early detection of pyogenic arthritis due to gram-negative organisms. Seven-one specimens of synovial fluid from 46 patients were evaluated for reactivity in the limulus test, pyrogenic responses in rabbits, total white blood cell count, total neutrophil count, total red blood cell count, and protein and glucose concentrations. All patients with culture-proven septic arthritis or presumptive septic arthritis had joint fluid specimens that yielded a positive result in the limulus assay (12 patients). However, 52.9% of patients (18 of 34) who had a nonseptic cause for their joint effusion also had a joint fluid specimen that was positive in the limulus test. A positive limulus test result showed a significant correlation with an elevated total white blood cell count (P less than 0.0005), an elevated absolute neutrophil count (P less than 0.0005), and a decreased concentration of glucose (P less than 0.005) in synovial fluid, and the production of fever in rabbits after injection of synovial fluid (P less than 0.05). Thus, this study suggests that a positive result in the limulus test on joint fluid is nonspecific for a septic process, but a negative result would be evidence against it.
Azospirillum brasilense was grown continuously at various levels of dissolved oxygen (O2) in a nitrogen-free medium containing malates as the carbon source. Steady-state cultures were established only at O2 concentrations less than 0.0150 atm (1 atm = 101.325 Pa) and rates of acetylene reduction (N2 fixation) and efficiencies of N2 fixation were maximal between 0.0050-0.0075 atm dissolved O2. These cultures appeared to be O2- or N2-limited. There was no evidence of a respiratory protective mechanism in this organism. Anaerobic denitrifying steady-state cultures were established with nitrate (NO3-) as the sole nitrogen source with no detectable N2 fixation. N2 fixation, but no denitrification, was observed when NO3- was decreased to 10 microgram N per millilitre at 0.003 atm dissolved O2. In samples removed from the culture vessel, either activity could be induced with a lag of approximately 120 min by incubation under appropriate conditions.
Potassium homeostasis was evaluated in 13 patients with diabetes mellitus. In eight, plasma renin activity was low; plasma aldosterone concentration was decreased in all; seven had a history of spontaneous hyperkalemia. After administration of glucose orally, there were paradoxical increases in serum potassium levels in seven patients. After potassium loading, maximal values and increments of serum potassium were higher and fractional potassium excretion was lower in the diabetic than in the control subjects, although the differences were not statistically significant. Abnormalities of potassium homeostasis in diabetes are probably related to insulin and mineralocorticoid deficiency. Diabetic patients with hypoaldosteronism have the potential for severe hyperkalemia should renal or extrarenal mechanisms for potassium homeostasis be challenged by severe acidosis be challenged by severe acidosis, diminished renal function, marked hyperglycemia, or administration of potassium salts or potassium-sparing diuretics.
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