p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Insulin effects on apolipoprotein B production by normal, diabetic and treated-diabetic rat liver and cultured rat hepatocytes.

1. The effect of insulin on apolipoprotein (apo B) secretion was studied in 24 h recirculating liver perfusions of isolated normal, diabetic and insulin-treated diabetic rats. In single perfusions from each group apo B accumulated in the media in a linear fashion. 2 In perfusions of normal rat livers, when the medium contained insulin plus cortisol, apo B production was significantly inhibited (by 35.8%), demonstrating a hormone effect on apo B secretion. 3. In perfusions of diabetic-rat livers, apo B production was decreased to 11.8% of normal when the medium contained no hormones, and was not significantly changed by the addition of insulin plus cortisol to the medium, suggesting that the hormone effect on apo B secretion is missing in long-term hypoinsulinaemic states. 4. Treatment of diabetic rats with daily insulin injection restored apo B production and restored the effect of insulin plus cortisol in the medium to inhibit apo B secretion during perfusion. 5. Parallel studies of apo B secretion with insulin alone, cortisol alone and insulin plus cortisol in the medium were performed in primary cultures of hepatocytes to compare results from liver perfusions. 6. Apo B secretion by hepatocytes from normal, diabetic and treated-diabetic rats was inhibited (by 36.8%, 57.1% and 57.9% respectively) when insulin alone was added to the medium. 7. Insulin plus cortisol inhibited apo B secretion by hepatocytes from normal and treated diabetic rats (by 30.2% and 47.2% respectively), but failed to inhibit apo B secretion by hepatocytes from diabetic rats.     

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.     

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.