The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.     

Kinetic evidence for cytoplasmic calcium as an inhibitory messenger in parathyroid hormone release

A sudden change of extracellular Ca2+ from 0.5 to 3.0 mM resulted in a transient rise of the cytoplasmic Ca2+ concentration (Ca2+i) followed by a sustained increase in parathyroid cells loaded with the Ca2+-indicator fura-2. The initial transient could be eliminated by increasing the Ca2+ buffering capacity of the cytoplasm. Under such conditions the rise of Ca2+i exhibited kinetics reminiscent of those for 45Ca uptake and cell depolarization. Addition of 0.5 mM Mn2+ mimicked the effect of raising the extracellular Ca2+ concentration, since there was an initial Ca2+i transient followed by a slower entry of Mn2+ into the cells. This reaction pattern was different from that of pancreatic alpha 2-cells in which there was no substantial influx of Mn2+ before depolarization with arginine. When measuring the kinetics of parathyroid hormone (PTH) release it was apparent that Ca2+ inhibition of secretion followed Ca2+i and thus became substantially delayed after eliminating the initial transient. The results support the concept of a depolarizing Ca2+ permeability in the parathyroid cell membrane which can be activated by external Ca2+, and indicate that Ca2+i is an inhibitory messenger of importance for the physiological regulation of PTH release.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.     

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

Lipid phase transitions in membranes involving intrinsic periodic curvature

A conformation of the lipid bilayer of membranes is proposed, with periodic curvature corresponding to the minimal surface structure of cubic lipid phases. Evidence is given indicating that activities of lipid synthesis/modification enzymes embedded in the membrane are controlled by the lateral "packing-pressure", so that the lipid bilayer is close to a transition from the lamellar (L alpha) type of conformation to this periodically curved conformation. Such a phase transition mechanism is assumed to be involved in numerous cooperative membrane functions.     

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.