Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution

A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.     

Effect of the external donors on the polymerization of 4-methyl-1-pentene with high activity MgCl2/TiCl4 catalytic system

In order to find out correlations between the structure of an external donor and the obtained polymer, the effects of different external donors on the activity and stereospecificity of the MgCl₂/TiCl₄ catalytic system in the bulk polymerization of 4-methyl-1-pentene (4MP) were carefully studied. Different silane compounds of the structure R_nSi(OR')_4-n (where: n = 1–3, R = alkyl/phenyl, R = alkyl) and Al(i-Bu)₃ (TIBA) were used as external donors and cocatalyst, respectively. The effect of the donor/TIBA mole ratio on the activity and stereospecificity of the catalytic system was studied. Some major effects were observed for the three different external donors, namely, Me₃Si(OMe), Me₂Si(OMe)₂, and Me₃Si(OBu)₃, in the 4MP polymerization process. It was observed that the effect of the external silane donor on the polymerization strongly depends upon the size of the alkoxy and hydrocarbon (alkyl/phenyl) groups which are attached to the silicon atom. A selective deactivation of the non-stereospecific centers, as well as a transformation of the non-stereospecific into isospecific centers, is assumed to occur. On the basis of the obtained results and literature data available for the propene polymerization, the concept of structural conformity between the ligand-surrounded active center and the monomer molecule was carried forward.     

Detection by PCR and isolation assays of the anaerobic intestinal spirochete Brachyspira aalborgi from the feces of captive nonhuman primates

The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the feces of captive nonhuman primates (n = 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the B. aalborgi PCR. B. aalborgi was not detected by PCR in any of the other animal or bird species tested, and B. pilosicoli was not detected in the primates or any of the other animals or birds. B. aalborgi was isolated from both PCR-positive vervet monkeys. This is the first time that B. aalborgi has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species.     

The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia

Tissue factor (TF) is an integral membrane protein that binds factor VIIa and initiates coagulation. The extracellular domain of TF is responsible for its hemostatic function and by implication in the dysregulation of coagulation, which contributes to death in endotoxemia. The role of the cytoplasmic domain of tissue factor in endotoxemia was studied in mice, which lack the cytoplasmic domain of TF (TF(deltaCT/deltaCT)). These mice develop normally and have normal coagulant function. Following i.p injection with 0.5 mg of lipopolysaccharide (LPS), TF(deltaCT/deltaCT) mice showed significantly greater survival at 24 hours compared to the wt mice (TF(+/+)). The serum levels of TNF-alpha and IL-1beta were significantly lower at 1 hour after LPS injection and IL-6 levels were significantly lower at 24 hours in TF(deltaCT/deltaCT) mice compared to TF(+/+)mice. Neutrophil recruitment into the lung was also significantly reduced in TF(deltaCT/deltaCT) mice. Nuclear extracts from tissues of endotoxemic TF(deltaCT/deltaCT) mice also showed reduced NFkappaB activation. LPS induced leukocyte rolling, adhesion, and transmigration in post-capillary venules assessed by intravital microscopy was also significantly reduced in TF(deltaCT/deltaCT) mice. These results indicate that deletion of the cytoplasmic domain of TF impairs the recruitment and activation of leukocytes and increases survival following endotoxin challenge.     

Parvovirus B19 replication in human umbilical cord blood cells.

The human parvovirus B19 is now known to be one of the causative agents of nonimmune hydrops fetalis and spontaneous abortions in pregnant women. The presence of the viral proteins and antibodies in fetuses of B19-infected women suggests that the virus can cross the placental barrier. In order to gain an insight into the mechanism of intrauterine fetal infection and the virus-induced hydrops fetalis, we examined whether human umbilical cord blood cells were permissive for B19 replication. Cord blood cells were infected with B19 in vitro, and Southern blot analyses of low M(r) DNA isolated from these cells revealed the presence of the characteristic replicative intermediates of B19 DNA. In addition, B19 genome expression in cord blood cells was detected by Northern blot analysis. Quantitative DNA dot blot analysis of culture supernatants documented complete assembly and release of B19 progeny virions in these cells. The progeny virions were biologically active in secondary infections of normal human bone marrow cells. The human umbilical cord blood cells may be a useful alternative to bone marrow and fetal liver culture systems for further studies on B19 since the need for bone marrow donors is obviated and, unlike fetal tissues, there are no ethical questions associated with the experimental use of cord blood because it is normally discarded. These studies also suggest that the umbilical cord blood may be a site for active replication of parvovirus B19 in vivo and may thus provide a means for transmission of the virus during intrauterine fetal infections.     

The effect of midazolam on median nerve somatosensory evoked potentials

Objective. To quantify the effect of an induction dose of midazolam on median nerve somatosensory evoked potentials.Methods. We studied 10 patients undergoing lumbar spine surgery. After an induction dose of intravenous midazolam was given, MNEPs were collected for ten minutes. After ten minutes the patients were intubated and their anesthetic was supplemented with 0.5% isoflurane, narcotic, and N₂O.Results. We found a clinically significant decrease in amplitude and an insignificant delay in latency.Conclusion. When midazolam is used as an anesthetic induction agent, a decrease in amplitude can be expected.