Studies of the characterisation of prostanoid receptors: A proposed classification

Comparison of rank orders of agonist potency of the anturally occurring prostanoids. PGD₂, PGE₂, PGF₂α and PGI₂ as well as the stable TxA₂ mimetic, U-46619, on a range of smooth muscle preparations provides evidences evidence for the existence of distinct receptors for PGE₂. PGF₂α and TxA₂. Since others have provided evidence for the existence of distinct receptors for PGD₂ and PGI₂, we suggest that receptors exist for each of these naturally occurring 2-series prostanoids. Results obtained with two specific prostanoid receptor blocking drugs, SC-19220 and AH 19437, supported and extend these conclusions. SC-19220 selectively block some but not all PGE-sensitive receptors. While AH 19437 selectively blocks all U-46619/TxA₂-sensitive receptors. A nomenclature for prostanoid receptors is proposed,; in which each receptor is designated the letter P preceded by a letter signifying the most potent natural prostanoid agonist at than receptor, such that receptors sensitivity to PGs D₂, E₂, F₂α, I₂ and TxA₂ become DP-, EP-, FP- and TP- receptors respectively. Where some sub-division is required within a receptor group, e.g. EP-receptors (SC-19220-sensitive and SC-19220-insensitive), subcript numbers may be used such that these are EP₁ and EP₂ subtypes. The resulting scheme is a working hypothesis and its confirmation requires the development of potent selective prostanoid receptor blocking drugs for each postulated type.     

H2Kk can influence whether cytotoxic T lymphocytes recognize trinitrophenyl in association with H2Dk unique or H-2Kk and H-2Dk shared self determinants

The present study investigates the effect of trinitrophenyl- (TNP) modified H-2Kk (TNP-Kk) antigens on the generation of anti-TNP-Dk restricted cytotoxic T lymphocyte (CTL) responses. C3H.OH mice were primed to TNP-self by skin-painting with trinitrochlorobenzene, and spleen cells from these primed mice were subsequently stimulated in vitro with TNP-self. The effector cells generated exhibited appreciable lysis of TNP-modified C3H.OH blast target cells. Cold target inhibition studies demonstrated the generation of two effector cell populations: one that recognizes TNP in association with unique Dk self determinants, and one that recognizes TNP in association with self determinants shared between TNP-Kk and TNP-Dk. This was in contrast to primed C3H/He spleen cells, which did not generate CTL that recognized TNP in association with unique Dk self determinants. When spleen cells from (C3H/He x C3H.OH)F1 mice primed to TNP were stimulated in vitro with TNP-C3H.OH cells, unique Dk self determinants were recognized in association with TNP. However, in vitro stimulation of the same F1 responding cells with TNP-C3H/He or TNP-F1 cells failed to elicit CTL that utilized these Dk-unique self determinants. The findings of this study demonstrate that unique or shared H-2Dk determinants can be differentially utilized by CTL populations, depending on the H-2 alleles expressed by the stimulator cells.     

Prostacyclin-induced contraction of isolated aortic strips from normal and spontaneously hypertensive rats (SHR)

Prostacyclin (PGl₂) (500-5,000 ng/ml) produced a concentration-dependent increase in contractile tension of isolated thoracic aortic strips (AS) from normotensive (WKY) and spontaneously hypertensive rats (SHR). No significant differences were noted between this response to PGl₂ in these two groups. Lower concentrations of PGl₂ (10 pg/ml — 100 ng/ml) caused neither contraction nor relaxation of agonist-contracted tissue. PGl₂ (500-5,000 ng/ml) did not relax KCl or methoxamine contracted AS. In concentrations above 100 ng/ml, PGl₂ caused a further increase in tension in KCl-depolarized preparations. The constrictor effect of PGl₂ on AS was attenuated by verapamil pretreatment or removal of extracellular Ca⁺⁺ from the physiological buffer. This inhibitory effect of Ca⁺⁺ deficiency on the PGl₂ response was significantly greater in AS from SHR compared to WKY tissue. The stable metabolite of PGl₂, 6-keto PGF_1a, caused a weak constrictor effect (40% of KCl reference contraction) over the concentration range 1,000–5,000 ng/ml. Contraction induced by PGl₂ was not prevented by pretreatment with antagonists of adrenergic, histamine, serotonin or cholinergic receptors. The contraction response of the rat AS to PGl₂ is similar to that reported for porcine coronary artery and rabbit aortic tissues in vitro.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

Evidence for an essential lysine residue on the phosphoribosyl transferase subunit of the anthranilate synthetase-anthranilate 5-phosphoribosyl-pyrophosphate phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Pyridoxal 5′-phosphate reacts with the anthranilate synthetase-phosphoribosyltransferase enzyme complex of $\text{Salmonella typhimurium}$ to inhibit PR transferase activity. Glutamine-dependent anthranilate synthetase is not affected. Spectral and kinetic data suggest that the inactivation results from the modification of an essential lysine residue which interacts with 5-phosphoribosyl 1-pyrophosphate.     

Atherocalcin, a gamma-carboxyglutamic acid containing protein from atherosclerotic plaque.

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.     

Characterization of immunologically active peptides from the cell wall of T.mentagrophytes

A low molecular weight fraction from chitinase digested cell walls ofT. mentagrophytes containing both polysaccharide and peptide moieties was found to have immunological reactivity at both the cellular and humoral level. This fraction (UM₂(a)) was further degraded by treatment with either a combination of pronase and carboxypeptidase A or with trypsin. Peptides were separated from the carbohydrate-rich fraction by ultrafiltration. The carbohydrate-rich fraction retained the ability to induce both immediate and delayed skin reactions in sensitized guinea pigs and to stimulate the proliferation of sensitized lymphocytesin vitro. The peptide moieties retained reactivity in that they caused delayed reactions and lymphocyte proliferation but were unable to induce immediate or Arthus reactions in sensitized animals. Tryptic peptides from UM₂(a) were purified by ion exchange chromatography. A high proportion of these peptides demonstrated immunological activity at both the cellular and humoral level since they were capable of inducing delayed reactions and/or lymphocyte transformation, as well as being capable of blocking the complement fixation reaction between UM₂ (a) and specific antiserum.This work was supported by grant number 6411 from the Medical Research Council of Canada.A. Kh. Al-Rammahy was supported by a scholarship from the Ministry of High Education, Iraq.     

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine

Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.     

Human lymphocyte subpopulations: the effect of pregnancy.

Peripheral blood lymphocytes from pregnant and nonpregnant females were studied for the presence of the following surface receptors: (i) Receptor for heat-aggregated human IgG (AggIgG); (ii) Receptor(s) for complement components; (iii) Receptor for sheep red blood cells (SRBC). Absolute numbers of lymphocytes with receptors for complement and those with receptors for SRBC were present in equal numbers in both groups. However, in the pregnant females there was a significantly lower number of lymphocytes with receptors for AggIgG. It is hypothesized that some of the immunological changes which occur during pregnancy may be mediated partly through changes in the total numbers of certain lymphocyte subpopulations.     

Estrogen-dependent trypsin-like activity in the rat uterus. Localization of activity in the 12,000g pellet and nucleus.

Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treatment, indicating organ specificity. The enzyme (or enzymes) from the 12,000g pellet was solubilized and some characteristics were determined. The apparent K_m is about 1.0 × 10^?6m, the temperature optimum is about 44 °C and maximum velocity is achieved in the alkaline range (pH ~ 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin.