Amylase activity, glycoprotein and electrolyte concentration in rat's submaxillary salivary gland during heat acclimation.

1. Amylase activity, glycoproteins, Na and K concentrations were measured in submaxillary salivary gland of the rat during heat acclimation (34 degrees C). 2. Acclimation resulted in a decrease in glycoprotein concentration and amylase activity, whereas Na and K concentrations and the Na/K ratio increased. 3. It is suggested that heat acclimation results in an increase in glandular activity leading to increased water secretion and depletion of the glycoprotein store. The decrease in amylase activity is probably due to liver atrophy which occurs during prolonged heat exposure.     

Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

G alpha 11 and G alpha q guanine nucleotide regulatory proteins differentially modulate the response to thyrotropin-releasing hormone in Xenopus oocytes.

Xenopus oocytes that express mouse thyrotropin-releasing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R cDNA respond to THR by a depolarizing current. This response is transduced by activation of phosphoinositide-specific phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotide-binding regulatory (G) protein(s). The alpha subunit of G11 and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differences between these proteins, we have co-expressed the TRH-R with either alpha 11 or alpha q. alpha 11 potentiated the response to TRH (by 61 +/- 16%), while alpha q inhibited the response (by 37 +/- 9%). The changes in amplitudes were accompanied by inverse changes in response latencies. These data show that alpha 11 and alpha q differentially modulate signal transduction in Xenopus oocytes.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

Insulin action in intact mouse diaphragm I.

Summary The incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2–10 mm) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0-methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.Abbreviations GS Glycogen synthase - GS-I Glycogen synthase activity independent of G6P - GS-D Glycogen synthase activity dependent on G6P - G6P Glucose-6-phosphate - ATP Adenosine triphosphate - EDTA Ethylene diamine tetracetic acid - Mops Morpholinopropane sulfonic acid - 2DG 2-Deoxy glucose - 3-0-MG 3-0-Methyl glucose - tricine N-tris(Hydroxymethyl)methyl glycine Enzymes: Glycogen Synthase — UDPGlucose — Glycogen Glucosyl — Transferase (EC 2.4.1.11) J. Larner is an established investigator of the American Diabetes Association.     

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes

Summary Commercially available concanavalin A binds Ca²⁺ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca²⁺ uptake (defined as an increased association of ⁴⁵Ca²⁺ with cells) in rat splenocytes and Ca²⁺ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in ⁴⁵Ca²⁺ uptake over control. The concanavalin A stimulated uptake of ⁴⁵Ca²⁺ occurred within minutes, and required concentrations of concanavalin A which promoted [³H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca²⁺ uptake than concanavalin A. Sodium periodate inhibited Ca²⁺ uptake at concentrations which promoted ³H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca²⁺ uptake which is not due to binding of ⁴⁵Ca²⁺ to concanavalin A. Although the concanavalin A-promoted Ca²⁺ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by ³H-thymidine incorporation, the role of Ca²⁺ in this event remains unclear.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.