Vitamin K-dependent carboxylase: the carboxylation of exogenous substrates in different systems

Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of warfarin-treated cows. Their enzymatic activities were compared with substrate-free carboxylase in microsomes from normal cows and substrate-bound carboxylase in microsomes from warfarin-treated cows. A number of exogenous substrates for carboxylase have been purified and tested. We found that large substrates, such as descarboxyprothrombin, are carboxylated only by substrate-free carboxylase and not by the substrate-bound enzyme. No differences in apparent Km values between solid-phase carboxylases II and X were observed.     

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.     

Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria.

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.     

Molecular aspects of the bilayer stabilization induced by poly(l-lysines) of varying size in cardiolipin liposomes

The interaction between poly(l-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and ³¹P- and ¹³C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca²⁺ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca²⁺, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca²⁺ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca²⁺, the lipid was organized in the hexagonal H_II phase in the presence of Ca²⁺. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

The orientation of the distal part of the quadriceps femoris muscle as a function of the knee flexion-extension angle

Lateral view radiographs of ten autopsy knees were used to determine the orientation of the patellar ligament, patella and quadriceps tendon relative to tibia and femur at different flexion-extension angles (0-120 degrees) of the knee. The results show a linear relationship between the angle of flexion and the movement of the patellar ligament relative to the tibia and of the movement of the patella relative to tibia and femur. There is a non-linear relationship between angle of flexion and the movement of the quadriceps tendon relative to the patellar ligament, patella and femur. The angular changes between patella and patellar ligament are negligible. The complicated movements of the distal part of the quadriceps femoris muscle may significantly influence biomechanical parameters such as the forces acting at the patella and tibial tuberosity.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Modulation of beta-adrenergic response in rat brain astrocytes by serum and hormones

Purified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta-adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum-free medium plus hydrocortisone, putrescine, prostaglandin F2 alpha, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum-supplemented medium, astrocytes grown in the chemically defined medium were nonresponsive to isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta-adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta-adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum-supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta-adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta-adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta-adrenergic response.     

Transplacental antibodies. Part II: Maternal antibodies against the toxins of C. diphtheriae and C. tetani

Examinations of 297 sera for diphtheria antitoxin and 160 sera for tetanus antitoxin were carried out in 1981. All sera were obtained from the cord blood of mothers between 15 and 34 years of age. The mothers were divided into four age groups each of which was further subdivided into the primipara and multipara subgroups. The aim was to assess the age-specific variations in response to active immunization against diphtheria and tetanus. The protective level of diphtheria antitoxin (at least 0.01 I.U./ml) was recorded in the serum of 96.3% of examinees and the rates of seropositivity were found to fall with increasing age. The protective level of tetanus antitoxin (at least 0.1 I.U./ml) was found in the serum of 95.2% of mothers. The serologic response encountered in groups of older mothers was a clear-cut demonstration that the country-wide mass immunization against tetanus carried out between 1974 and 1975 was highly effective and fully justified. The variations in the diphtheria and tetanus antitoxin levels found in the primipara and multipara subgroups were not statistically significant.     

Production and response to interleukin 2 in vitro and in vivo after bone marrow transplantation in mice

Lymphoid cells from bone marrow radiation chimeras do not produce normal levels of IL 2 but are capable of responding to IL 2 in mitogenic and cytotoxic assays in vitro. The administration of recombinant human IL 2 into host mice that have received allogeneic, H-2-compatible marrow did not enhance mortality.