A rapid method for the detection of potentially viable Mycobacterium leprae in human biopsies: a novel application of PCR

Abstract A simple procedure based on the polymerase chain reaction has been developed to detect Mycobacterium leprae , rapidly and unambiguously, in biological samples. Its application to small numbers of M. leprae cells (∼ 10²) isolated from armadillo liver, mouse footpads or human biopsies is discussed.     

Epidermal growth factor receptor binding is not a simple one-step process

The binding kinetics of 125I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture were measured at 4 degrees C. Initial binding rates as a function of hormone concentration allowed estimation of simple two-state on-off rate constants of 1.2 x 10(6) M-1 s-1 and 4.9 x 10(-3) s-1, respectively. These two-state parameters gave inadequate computer fits to long term kinetic and equilibrium-binding data, suggesting that an additional process(es) was occurring. Nonlinear equilibrium Scatchard plots and transient "pseudo-Scatchard" plots taken at pre-equilibrium times support the idea that at least one other process is occurring during receptor binding. 125I-EGF-receptor dissociation kinetic plots were biphasic, yielding rate constants of 1.5 x 10(-2) s-1 and 5.6 x 10(-5) s-1 with the ratio of the two components changing with the time of initial incubation with 125I-EGF. Application of a ternary complex model which assumed complexation of the bound receptor with a cell surface interaction molecule gave satisfactory fits to all data.     

Variation among Species in the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K_m) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25°C, the reappearance of variable fluorescence during a dark incubation was determined at 5°C intervals from 15°C to 45°C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20°C in wheat, 30°C in cotton, 20°C to 25°C in tomato, 30 to 35°C in bell pepper and 25°C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K_m of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K_m values (viz. the thermal kinetic window).     

Thermal Dependence of the Apparent K(m) of Glutathione Reductases from Three Plant Species

The thermal dependencies of the apparent K_m of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent K_m of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent K_m in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent K_m of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber.     

Genetic variability for esterase enzyme in Onobrychis species

Summary Understanding polymorphism at the enzyme level is basic to its use in population and genetic studies. However, no such information is available on the variability among different sainfoin (Onobrychis) species. Therefore, our objective was to study the existence of genetic polymorphism for esterase in 17 Onobrychis species and three cultivars of O. viciifolia Scop. Three regions of banding were observed in all the materials tested, with the number of bands varying from 0 to 3, 3 to 14, and 1 to 2 bands in each of these zones, which have been designated EST1, EST2, and EST3 respectively. All the materials studied had unique banding patterns, the only common feature being that all of them, except one species, had isozyme 1. Identification was possible only for four species (O. iberica, O. kachetica, O. transcaucasica, and O. bieberstenii) and one cultivar (Nova) based on the banding patterns. Large diversity was evident from the wide range of percent similarity values (0%–79%). Subsequent studies should be directed in using these isozyme banding patterns as markers to the desirable agronomic and quality traits of different germplasm lines.This work was supported by USDA Specific Cooperative Agreement No. 58-7MN1-8-143 from the Plant Stress and Water Conservation Unit, USDA-ARS, Lubbock, Texas. Joint contribution of the Texas Tech University, Lubbock, Texas and the USDA-ARS. TTU Journal no. T-4-302     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Variability of entrainment of cohesive sediments in freshwater

Estimates of sediment entrainment are required for models of particle transport in lakes and estuaries but are difficult to make because of the multiplicity of factors affecting cohesiveness of surficial sediments. We present results of sediment resuspension studies performed in an annular flume calibrated with laser-Doppler velocimetry. In our experiments, using sediments collected from two sites in the R. Raisin which flows into L. Erie and from one site in the western basin of L. Erie near the mouth of the R. Raisin, we applied shear stresses at the sediment-water interface in steps from 2 to 12 dyne/cm². Percent water content at the surface of the sediments was either 77 or 74%, and trials were run with and without oxygenating the water overlying the sediments. Entrainment rates as a function of shear stress at the sediment-water interface were best described by a power-law relationship. All but 14% of the variability in the power law expression was due to shear stress and percent water content; the variability not accounted for was due to differences in particle size distributions, chemical properties, and biological activity in the sediments.     

Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand.

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.     

Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant.

We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.     

Making sense of the combined effect of interleukin-2 and interleukin-4 on lymphocytes using a mathematical model

The cytokines are the information superhighway of the immune system. They are an important component of the integrated behavior of the system. In order to be able to have a good understanding of the immune system, we must be able to model the effect of cytokines and their combined effect. This work is a step in that direction. We study the combined effect of two cytokines: interleukin-2 (IL-2) and interleukin-4 (IL-4) on some cells of the immune system. Interleukin-2 and interleukin-4 are important growth and differentiation factors for B and T cells. Interleukin-4 antagonizes the effect of interleukin-2 on B cells and some T cells while it synergizes with interleukin-2 on other T cells. We build a mathematical model of the interaction of both cytokines on T and B cells as a building block toward a model of the Th1/Th2 cross-regulation. The response of a given cell to the combination of interleukin-2 and interleukin-4 is shown to involve competing dynamical effects which can lead to either antagnostic or synergistic combined effect. Author to whom correspondence should be addressed at Department of Engineering and Public Policy. Work supported by NIH grant nv: Ai31427.