Characterization of Schistosoma mansoni antigen-reactive T cell clones that form granulomas in vitro

Granuloma formation and modulation in schistosomiasis are a consequence of discrete subpopulations of T lymphocytes and the mediators they produce. In the present study, T cell clones reactive to soluble egg antigen (SEA) were developed to analyze the roles of T cells in Schistosoma mansoni egg-induced granuloma formation. In an in vitro granuloma assay, 1 X 10(5) T cells specifically augmented the response of 2 X 10(6) normal spleen cells to SEA-coupled but not purified protein derivative-coupled polyacrylamide beads. In vitro granulomatous responses by individual clones were correlated with their capacity to mediate local delayed-type hypersensitivity reactions in footpad swelling assays. Phenotypic analysis of the seven clones characterized in the present study demonstrated that they were L3T4+, Ly-2.2-. An analysis of supernatants of T cells pulsed with concanavalin A or SEA + antigen-presenting cells was also undertaken in an attempt to correlate in vitro granuloma formation with lymphokine production. Stimulated T cells (but not unstimulated T cells) produced interleukin 2, macrophage activating factor, migration inhibitory factor, and eosinophil stimulation promoter in response to both mitogenic and antigenic stimuli. The results suggest that individual clones of T cells are capable of producing a variety of mediators that influence their ability to activate and to recruit cells into granuloma formation. The model may be useful in the analysis of specific antigens and regulatory interactions and their contribution to granuloma formation.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

Efficient separation of subfossil Chironomidae from lake sediments

Electron transport system (ETS) activity, CO₂ evolution, O₂ consumption, N₂-fixation (C₂H₂ reduction) and methanogenesis were appropriately measured in aerobic and anaerobically incubated sediment at 4, 10 and 20 ° C to better characterize these activities under different incubation conditions. ETS activity was always higher in the aerobically incubated sediment at all three incubation temperatures, whereas (C₂H₂ reduction was always greater in the anaerobic sediment. Carbon dioxide evolution was detected only in the aerobic sediment at 10 and 20 ° C but not at 4 ° C. Methane evolution in anaerobic sediment increased gradually with an increase in the incubation temperature.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.     

Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus.

The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. Relatively few differences in polypeptide composition exist when microaerobic cells are compared with anaerobic cells, but these changes correlate with a change from the mycelial to the yeast morphology. We hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

Organogenesis in pepper tissue cultures

Knowledge concerning in vitro growth and developmental responses of bell and chile peppers (Capsicum annuum L.) has been limited. Shoot and root organogenesis in cultures of seedling explants was restricted to primary cultures or those less than three months old under 12-and 16-h photoperiod at 25°C. Shoot organogenesis was extended to 5 months under continuous light at 25°C, and to 8 months under continuous light at 28.5°C. Murashige and Skoog basal media containing 0.05mg/l each of IAA and BA promoted shoot elongation and rooting of some explant sources, while 0.05-4 mg/l IAA with 10–50 mg/l BA promoted adventitious shoot bud formation. Glucose was superior to sucrose as the carbon source. Leaf discs collected from greenhouse-grown plants regenerated shoots for at least 2 months. Incubation environment, carbon source, explant source, growth regulator treatment and passage number were not independent variables as demonstrated by statistical analysis. The plant regeneration techniques described here have useful but limited applications, not extending to unorganized callus or cell suspension cultures.Journal article no. 1151 of the New Mexico Agricultural Experiment Station.     

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.