Sex differences in vasopressin neurons in the bed nucleus of the stria terminalis by in situ hybridization

To determine whether a sex difference exists in the biosynthetic capacity of vasopressingergic (AVP) neurons in the bed nucleus of the stria terminalis (BNST), we have used in situ hybridization and quantitative autoradiography to measure propressophysin messenger RNA levels in these cells from adult male and female rats. We have found that significantly more (p less than 0.01) neurons are labeled in male rats than in female rats and that these labeled cells averaged more grains/cell (p less than 0.05) in males than in females. Therefore, the sexual dimorphism of AVP pathways in the BNST and lateral septum recently shown by immunohistochemistry results from a sex difference in the biosynthetic capacity of these AVP neurons.     

Electric field-induced lateral mobility of photosystem I in the photosynthetic membrane: A study by electrophotoluminescence

Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 · 10^-5 cm²V^-1s^-1. The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of ~5 · 10^-9 cm²s^-1 was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism

Lactoferrin has been previously shown to be bactericidal for Legionella pneumophila. The current study showed that CaCl2, Mg(NO3)2, and MgCl2, but not NaCl, blocked killing. Activity was pH dependent with the greatest activity at 5.0. Sensitivity of the organism was dramatically affected by the growth conditions. Log phase 12 h, broth-grown cells were most sensitive, with older cultures becoming more resistant. Plate-grown cells were completely resistant. Lactoferrin binding, as detected by immunofluorescence microscopy, was temperature dependent (no binding at 4 degrees C), but was independent of killing.     

Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.     

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.     

Amino acid sequence of a mouse mucosal mast cell protease

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.     

Protease peptide mapping of affinity-labeled rat pancreatic cholecystokinin-binding proteins

Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.     

Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel

This paper demonstrates that local electric fields originating from negatively charged groups on a K+-specific ion channel modify its behavior. Single high-conductance, Ca2+-activated K+ channels were studied in neutral phospholipid bilayers. The channel protein surface charges were manipulated experimentally by carboxyl group esterification using trimethyloxonium (TMO) or by electrolyte screening. Three channel properties--ion conduction, ion blockade, and voltage-dependent gating--are affected by surface electrostatics. Negative charges increase the affinity of cationic pore blockers by establishing a local negative potential at the pore entrance; these charges modify channel gating by establishing a potential gradient across the ion channel; finally, both effects influence ion permeation through the pore.