"Haemostasis time", a modified bleeding time test and its comparison with the Duke and Ivy/template bleeding times. I. Normal values, application in thrombocytopenic patients and evaluation of heparin and aspirin effects

The occlusion time ("haemostasis time" - HT) of a thin, short cannula inserted into the cubital vein, was compared with the skin bleeding times of the Duke and Ivy/template techniques. 25 male and 25 female volunteers without a history of bleeding were divided into 5 equally large age groups ranging from 10 to over 50 years of age. They exhibited a range of 46 s-6 min 38 s (95% tolerance interval), while the Duke and Ivy/template bleeding times, which were simultaneously determined, corresponded to values given by other authors. HT is different from the skin bleeding times in that endothelium is replaced by a standard foreign surface which allows better standardization of the method. Similar results were obtained with HT compared to the skin bleeding times. These and a similar, non-significant heparin response with all three techniques suggest that HT is not more influenced by clotting factors than the Duke and Ivy/template bleeding times and, indeed, may be regarded as a bleeding time modification. HT, like both of the skin bleeding times, reflected lowered platelet counts and is even more sensitive in this respect. As tested in a group of 20 male and 20 female volunteers, HT showed a significant prolongation two hours after ingestion of 1 g aspirin. While male individuals exhibited longer bleeding times than females with the Ivy/template technique (sex-related difference p = 0.01), no male to female differences were found both with HT and the Duke bleeding time. HT is easy to perform, inexpensive, leaves no scars and is safe even for the patient with severe bleeding. Moreover, compared to the skin bleeding times, it permits a differential evaluation of vessel wall and tissue effects.     

Microscopic and submicroscopic anatomy of the parabronchi, air sacs, and respiratory space of the budgerigar (Melopsittacus undulatus)

The normal microscopic and submicroscopic structure of the lower respiratory tract of the budgerigar (Melopsittacus undulatus) is described and compared with other birds and mammals. Granular (type II) pneumocytes are confined to linings of air sacs, parabronchi, and their atria; however, their secretions (surfactant) cover the surfaces of the infundibula and respiratory space. Infundibula extend from the atria and give rise to the air capillaries, which branch and anastomose freely with those of adjacent infundibula and other parabronchi (interparabronchial septa are not found). Infundibula and the respiratory labyrinth are lined by a continuous epithelium of squamous pneumocytes, whose perikarya are concentrated in the infundibula and whose peripheral cytoplasm is markedly attenuated. The squamous pneumocytes of the respiratory labyrinth share a basal lamina with the blood capillaries that they envelop.     

Lack of correlation between extensive accumulation of bisnucleoside polyphosphates and the heat-shock response in eukaryotic cells

The accumulation in large amounts of bisnucleoside polyphosphates (Ap4X) after heat shock in Xenopus laevis oocytes or cultured hepatoma cells (HTC cells) is observed after exposure to temperatures of 45 degrees C or higher. The accumulation is a transient phenomenon, with the collapse in cellular ATP concentration severely affecting the rate of synthesis of Ap4X, allowing degrading activities to empty the pool of these compounds under prolonged heat shock. This accumulation of Ap4X to high levels, compared to the basic content, is only observed under conditions leading to irreversible damage, ultimately resulting in the death of the cell. It is shown that the increase in Ap4X after hyperthermia is due to the partial or almost complete inhibition of their degradation pathways, rather than to a stimulation of their rate of synthesis. Finally, the synthesis of heat-shock proteins could be observed under conditions which do not lead to important accumulation of Ap4X, therefore ruling out the possibility that these adenylylated nucleotides would behave as chemical signals ("alarmones") triggering the synthesis of heat-shock proteins. Nevertheless, on the basis of our earlier results (Guédon, G., Sovia, D., Ebel, J. P., Befort, D., and Remy, P. (1985) Embo J. 4, 3743-3749), it cannot be excluded that Ap4X might play a role in the regulation of the heat-shock response; this would, however, rely on variations in Ap4X concentrations which do not exceed a factor of 2.     

Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography

The metabolism of mephenytoin to its two major metabolites, 4-OH-mephenytoin (4-OH-M) and 5-phenyl-5-ethylhydantoin (nirvanol) was studied in human liver microsomes by a reversed phase HPLC assay. Because of preferential hydroxylation of S-mephenytoin in vivo, microsomes (5-300 micrograms protein) were incubated separately with S- and R-mephenytoin. After addition of phenobarbital as internal standard, the incubation mixture was extracted with dichloromethane. The residue remaining after evaporation was dissolved in water and injected on a 60 X 4.6-mm reversed-phase column (5 mu-C-18). Elution with acetonitrile/methanol/sodium perchlorate (20 mM, pH 2.5) led to almost baseline separation of mephenytoin, metabolites, and phenobarbital. Quantitation was performed by uv-absorption at 204 nm by the internal standard method. Propylene glycol was found to be the best solvent for mephenytoin, but inhibited the reaction noncompetitively. 4-OH-M and nirvanol could be detected at concentrations in the incubation mixture as low as 40 and 80 nM, respectively. The rates of metabolite formation were linear with time and protein concentration. The reaction was found to be substrate stereoselective. At substrate concentrations below 0.5 mM S-mephenytoin was preferentially hydroxylated to 4-OH-M, while R-mephenytoin was preferentially demethylated to nirvanol at all substrate concentrations tested (25-1600 microM). These data provide a mechanistic explanation for the stereospecific pharmacokinetics in vivo. The dependence of both metabolic relations on NADPH and the inhibition by CO suggest that they are mediated by cytochrome P-450-type monooxygenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.     

Covalent modification of phenylalanyl-tRNA synthetase with phenylalanine during the amino acid activation reaction catalyzed by the enzyme

Yeast phenylalanyl-tRNA synthetase (PRS) is shown to undergo autoaminoacylation with phenylalanine under in vitro amino acid activation conditions. Phenylalanyl adenylate enzyme complex yields a covalent phenylalanyl isopeptide exclusively with the beta subunit of the alpha 2 beta 2 enzyme. Contrary to previously reported cases of autoaminoacylation of aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, the autoaminoacylation of PRS occurs under a specific set of conditions and results in the identification of only one labeled tryptic peptide on two types of high pressure liquid chromatography columns. The ability of PRS to undergo this covalent modification directly correlates with its ability to catalyze the synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate from enzyme-bound phenylalanyl adenylate. Both reactions require the presence of low levels of zinc or cadmium and are inhibited by tRNAPhe or by low levels of low molecular weight thiols. Since diadenosine 5',5"'-P1,P4-tetraphosphate synthesis is known to be catalyzed in vivo in response to oxidation stress, it is also likely that the autoaminoacylation of phenylalanyl-tRNA synthetase may occur in vivo under a similar set of conditions. These reactions are thus not simply the result of accumulation of phenylalanyl adenylate and probably reflect conformational changes in the protein which are brought about by its interaction with zinc or cadmium.     

Effects of Undernutrition and Thyroid State on the Ontogenetic Changes of D1, D2, and D3 Brain-Specific Proteins in Rat Cerebellum

Abstract: Disturbances in metabolic balance brought about by alterations in thyroid state and undernutrition during early life had a marked effect on the concentrations of the brain-specific proteins, D1, D2, and D3 in the developing rat cerebellum. In normal rats, the concentrations of D1 and D3 increased and that of D2 decreased during the first 3 weeks after birth. In the hyperthyroid state a small but consistent advancement was observed in the developmental curves of these proteins. The hypothyroid state caused a marked retardation in the maturational pattern of D1 and D2 but not of D3. In undernutrition, at 6 days the concentrations of D1 and D3 proteins were higher than in controls, but thereafter the developmental increase was markedly delayed for D1 only. The concentration of D2 was normal at 6 days, but after the first week a marked retardation was observed in the maturational pattern of this protein in undernourished rats. In addition, the "anodic-immature"form of D2 predominated in 6-day-old controls, but this was gradually replaced by a "cathodic-mature"form which progressively became the dominant form of D2 in 35-day-old rat cerebellum. The developmental switch in terms of the two forms was also advanced in hyperthyroidism and retarded in thyroid deficiency and undernutrition. Furthermore, daily treatment of hypothyroid rats with physiological doses of thyroxine from birth restored the concentrations of D1 and D2 to normal, but that of D3 was increased above control levels, indicating differences between the proteins in their sensitivity to mechanisms of control by thyroid hormone. Also, the overall effects of undernutrition were markedly different from those of hypothyroidism.     

Queuosine modification of the wobble base in tRNAHis influences ''in vivo'' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.     

Astrocytes support incomplete differentiation of an oligodendrocyte precursor cell.

Glial fibrillary acidic protein-positive astrocytes, but not neurons or fibroblasts, support the differentiation of an oligodendroglial precursor cell expressing O4 antigen and vimentin into an O4 antigen-positive, but vimentin-negative oligodendrocyte. Further maturation into galactocerebroside (O1)-positive oligodendrocytes is, however, not achieved under the culture conditions used, neither in the presence of astrocytes nor neurons.     

Antigenic variation is associated with DNA rearrangements in a relapsing fever Borrelia

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation in its host. Surface-exposed proteins with differing primary structures determine the serotype of each organism. Using amino acid sequence data from two of these variable proteins, we synthesized two mixed-sequence oligonucleotides and then used the oligonucleotides to probe mRNA and DNA of three isogenic serotypes of B. hermsii. In Northern blots the probes were specific for the mRNA of the homologous serotype. Southern blots revealed two classes of hybridizing fragments: those common to the three serotypes and those specific for a particular serotype. A serotype-specific DNA fragment, which had hybridized to both oligonucleotide probes, was cloned. Subsequent use of the cloned fragment as a probe provided further evidence that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.