Mechanisms of voltage transients during current clamp inNecturus gallbladder

Summary Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (V _ms ) observed during transmural current clamps in the isolatedNecturus gallbladder. The results indicate that: a) part of V _ms is due to a transepithelial resistance change (R _t ), and part to a tissue emf change. b) R _t is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the R _t -independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (E _b ), with smaller changes at the luminal membrane (E _a ) and the paracellular (shunt) pathway (E _s ). It is shown that two-thirds of the transient are caused by E _s , and one-third by (E _b –E _a ). E _s can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. E _a and E _b are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.     

Changes in protein synthesis in rats in response to endurance training

Experiments were conducted to investigate the influence of endurance exercise training on protein synthesis in skeletal muscle, heart, and liver. Training decreased incorporation of [¹⁴C]-leucine into proteins of the stromal fraction of muscle but there was no change in amino acid incorporation into proteins of the sarcoplasmic and myofibrillar fractions. Incorporation of [¹⁴C]-leucine into the protein of heart, liver, and plasma was depressed in trained rats compared to untrained rats. The specific radioactivity of [¹⁴C]-leucine was similar in tissues of trained and untrained rats and thus the depressed amino acid incorporation represents a decrease in the rate of protein synthesis. These observations demonstrate that the adaptation of muscle protein metabolism to endurance training is quite different than the alterations during work-induced hypertrophy of muscle. The difference in adaptation probably relates to the functional differences between the types of exercise. However depression of protein synthesis in trained rats is a general effect in several tissues and not an effect localized in muscle tissue.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light     

The presence of lysophosphatidylcholine in chromaffin granules

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N₂ and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N₂ but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [¹⁴C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.     

Oxidative demethylation of t-butyl alcohol by rat liver microsomes

Tertiary butyl alcohol has often been used experimentally as a “non-metabolizable” alcohol. In this report, evidence is presented that t-butanol serves as a substrate for rat liver microsomes and that it is oxidatively demethylated to yield formaldehyde. The apparent K_m for t-butanol is 30 mM while V_max is about 5.5 nmol per min per mg microsomal protein. Formaldehyde production is stimulated by azide, which prevents destruction of H₂O₂ by catalase. Hydroxyl radical scavenging agents, such as benzoate, mannitol, and 2-keto-4-thiomethylbutyrate, suppress formaldehyde production. Therefore, the microsomal reaction pathway appears to involve the interaction of t-butanol with hydroxyl radicals generated from H₂O₂ by the microsomes. Formaldehyde is also produced when t-butanol is incubated with model hydroxyl radical-generating systems such as the iron-EDTA-stimulated oxidation of xanthine by xanthine oxidase or the iron-EDTA-catalyzed autoxidation of ascorbate. These results indicate that t-butanol cannot be used to distinguish metabolically-linked from non-metabolically-linked actions of ethanol.     

Purification of cytoplasmic antigens from the mycelial phase of Candida albicans: Possible advantages of its use in Candida serology

The serology of candidiasis is complicated by the use of poorly defined antigens. Total extracts of the yeast phase have been commonly used as cytoplasmic antigen, without regard to the significant amounts of carbohydrate that may contaminate such preparations. This is particularly true in the case of commercially available antigens that have been used as cytoplasmic antigens but actually are richer in carbohydrate than in protein. Affinity chromatography in concanavalin A — Sepharose provides a simple procedure to separate carbohydrates, mainly mannan, from protein antigens in whole Candida extracts. By using mannan-poor antigens, the specificity of serological reactions can be increased considerably, since both the positive reactions seen in asymptomatic donors and the cross-reactions seen in patients infected with other fungi are due to anti-mannan antibodies. In contrast, both anti-mannan and anti-cytoplasmic antigen antibodies can be detected in patients suspected of systemic candidiasis. On the other hand, absolute specificity may never be achieved for systemic candidiasis. We have found antibodies against cytoplasmic antigen in a patient allergic to C. albicans, in whom the microorganism was isolated from fecal material. It appears that, under favorable conditions, mucosal sensitization may also trigger a systemic reaction directed against both mannan and cytoplasmic antigens.Publication no. 341 from The Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.     

The molecular basis of disorders of human hemoglobin synthesis

Summary The structure and organization of the human globin genes at the nucleotide level has been established by restriction endonuclease digestion of cellular DNA, and by the isolation and purification of these. genes in phage vectors. With this approach it has been possible to define alterations at the DNA level resulting in a group of inherited diseases of man known as the thalassemia syndromes, and related disorders. Combined with other known genetic and biochemical data, these studies provide a framework for understanding the pathogenesis of these disorders at the molecular level.     

The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

Immunological characterization of mouse mammary tumor virus p10 and its presence in mammary tumors and sera of tumor-bearing mice.

Mouse mammary tumor virus (MMTV) p10 and gp52 were purified and used as radiolabeled antigens in sensitive radioimmunoassays. These radioimmunoassays were specific for MMTV proteins since detergent-disrupted MMTV from C3H/HeN, RIII, and GR/N mice gave complete competition, whereas C3H/HeNf liver extracts and other lysed retroviruses did not. Both gp52 and p10 are coded by the viral genome, since MMTV grown in a heterologous cell line (feline kidney cells) competed in these assays. Sera from mammary tumor-bearing mice and mammary tumors from C3H/HeN and C3H/HeNf mice competed in both the gp52 and the p10 assays. Although these radioimmunoassays detected predominantly group-specific antigenic determinants in C3H/HeN and C3H/HeNf tumor extracts, type specificity was also found with gp52. Absorption of the anti-MMTV serum with C3H/HeNf tumor extracts removed all antibodies directed against p10 and decreased the anti-gp52 titer approximately 30-fold. When this absorbed antiserum was used at limiting dilution in the gp52 radioimmunoassay, C3H/HeN tumor extracts gave complete competition, whereas no competition was found with C3H/HeNf tumor extracts.