Hepatic transformation of prostaglandin D2 to a new prostanoid, 9 alpha,11 beta-prostaglandin F2, that inhibits platelet aggregation and constricts blood vessels

The metabolic transformation of tritium-labeled prostaglandin D2 ([3H]PGD2) was investigated in the isolated Tyrode's-perfused rabbit liver. One major product was isolated and identified in the perfusate as a new prostanoid. The structure of this metabolite was further confirmed by gas chromatography-mass spectrometry and chemical methods to be 9 alpha,11 beta,15-L-trihydroxyprosta-5-cis, 13-trans-dienoic acid, namely (9 alpha,11 beta-PGF2). This new prostanoid was found to be an inhibitor of platelet aggregation and to cause constriction of canine coronary artery strips. These results suggested that on passage through the hepatic circulation exogenous PGD2 is converted to 9 alpha,11 beta-PGF2, the latter having a biological profile which differs from that of PGD2 and PGF2 alpha.     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

Abortion due to infection with Chlamydia psittaci in a sheep farmer's wife.

A farmer''s wife who had helped with lambing aborted spontaneously in March after a short febrile illness in the 28th week of her pregnancy. She developed disseminated intravascular coagulation post partum with acute renal failure and pulmonary oedema. Recovery was complete after two weeks of hospital care. A strain of Chlamydia psittaci, probably of ovine origin, was isolated from the placenta and fetus. The patient''s serum showed rising titres of antibody against chlamydia group antigen; the placental and fetal isolates; and a known ovine abortion, but not a known avian, strain of C psittaci. IgG against both ovine abortion and enteric strains of C psittaci was detected, but IgM against only an abortion strain was detected. Histological examination showed pronounced intervillus placentitis with chlamydial inclusions in the trophoblast but no evidence of fetal infection or amnionitis. Laboratory evidence of chlamydial infection was found in an aborting ewe on the farm in January and in remaining sheep and lambs in July. Doctors should recognise the possible risk to pregnant women in rural areas where chlamydial infections in farm animals are widespread.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

The respiratory burst of human neutrophils treated with various stimulators in vitro is dampened by exogenous unsaturated fatty acids

Cis-unsaturated free fatty acids (FFA) at concentrations between 10 and 30 microM suppressed the superoxide respiratory burst induced in human neutrophils by the chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP). Corresponding trans-isomers had a reduced efficacy while saturated FFA were inert. The effects of unsaturated FFA were maximally achieved after several min of preincubation with cells and reversed upon washing. Increased concentrations of Ca2+ in the medium also relieved the inhibition. Unsaturated FFA were equally effective in dampening the respiratory burst induced by fluoride ions but less so with bursts elicited by 9 nM phorbol myristate acetate (PMA). Moreover reactions triggered by higher concentrations (e.g., 100 nM) of PMA were resistant to the effects of FFA. Radioimmunoassays showed that unsaturated FFA directly elevated intracellular cyclic adenosine monophosphate (cAMP) by severalfold above basal levels. It is suggested that inhibition is brought about by unsaturated FFA perturbation of the neutrophil membrane structure, perhaps with an independent contribution from a cAMP-dependent mechanism.     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Fluorescent inhibitors of a cell surface protease used to locate leukaemia cells in kidney sections

Guanidinobenzoatase is a trypsin-like protease on the surface of cells capable of migration, for example leukaemia cells. We have used a number of fluorescent probes that are competitive inhibitors of guanidinobenzoatase to locate leukaemia cells in resin sections of kidney tissue obtained from leukaemic rats. We have demonstrated how this competitive inhibition system can be used to direct desired molecules (such as cytotoxic drugs) to these cells and to monitor the arrival of such compounds at the active site of guanidinobenzoatase. The principles developed in this study could equally well be applied to other enzymes on other cells provided suitable competitive inhibitors were designed. The presence of an enzyme on the surface of a cell can be used to direct molecules to that cell provided that these molecules contain a functional group that acts as an inhibitor for the chosen enzyme.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.