A sulfhydryl group of the canine cardiac beta-adrenergic receptor observed in the absence of hormone

Canine cardiac beta-adrenergic receptors contain a free sulfhydryl group in the adrenergic ligand binding site. [125 I]-Iodohydroxybenzylpindolol [( 125 I]-IHYP) binding to cardiac beta-receptors was inhibited 80% by treatment with 1 mM p-chloromercuribenzoic acid (pCMB). Occupation of the beta-receptors by an antagonist prior to treatment with pCMB prevented this effect suggesting that a sulfhydryl group is present in or near the ligand binding site of the cardiac beta-receptor. In the presence of agonists, the sensitivity of cardiac beta-receptors to pCMB was increased. Incubation of isoproterenol-occupied cardiac beta-receptors, resulted in a 57% inhibition of [125 I]-IHYP binding measured after extensive washing to remove bound agonist. The ability of isoproterenol to increase the reactivity of cardiac beta-adrenergic receptors supports the hypothesis that agonists produce a conformational change upon binding.     

Structural analysis of purified beta-adrenergic receptors

We have characterized the structure of purified beta-adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)-tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta-adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]-iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta-receptors were subjected to complete trypsin digestion and subsequent reverse-phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta-receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod-shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta-receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta-adrenergic receptor and its quaternary structure.     

Localization of penicillin-binding proteins to the splitting system of Staphylococcus aureus septa by using a mercury-penicillin V derivative.

Precise localization of penicillin-binding protein (PBP)-antibiotic complexes in a methicillin-sensitive Staphylococcus aureus strain (BB255), its isogenic heterogeneous methicillin-resistant transductant (BB270), and a homogeneous methicillin-resistant strain (Col) was investigated by high-resolution electron microscopy. A mercury-penicillin V (Hg-pen V) derivative was used as a heavy metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the presence of an abnormally large (17 to 24 nm in width) splitting system within the thick cross walls or septa of Hg-pen V-treated bacteria of all strains. Untreated control cells possessed a thin, condensed splitting system, 7 to 9 nm in width. A thick splitting system was also distinguishable in unstained thin sections, thereby confirming that the electron contrast of this structure was not attributed to binding of bulky heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membranes as well as sodium dodecyl sulfate-treated cell walls and that two or more PBPs in each strain bound to this antibiotic. In contrast, the splitting system in penicillin V-treated bacteria was rarely visible after 30 min in the presence of antibiotic. These findings suggest that while most PBPs were associated with the plasma membrane, a proportion of PBPs were located within the fabric of the cell wall, in particular, in the splitting system. Inhibition of one or more high-M(r) PBPs by beta-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesis and architectural design of these structures in S. aureus.     

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

The availability of the complete 1.83-megabase-pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.     

Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation

The gene encoding a human alpha 2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated alpha 2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)     

No evidence of kin bias in dispersion of young‐of‐the‐year Atlantic salmon Salmo salar L. in a natural stream

Ninety‐one young‐of‐the‐year Atlantic salmon Salmo salar were captured using a non‐invasive snorkelling technique in a 38 m section of Catamaran Brook, New Brunswick, Canada, to test whether related fish settle closer to one another than unrelated fish. A maximum likelihood estimate of parentage relationships assessed by genotyping eight microsatellite loci revealed five half‐sibling families in the sample of fish. Related juvenile S. salar were not found closer to one another than unrelated fish in three analyses at two spatial scales: a comparison of the relatedness of focal fish to their nearest neighbour and to their four nearest neighbours, and a correlation of the pair‐wise relatedness and distance matrices for all fish in the sample. The lack of a kin‐biased dispersion pattern may be related to the lower density of fish or the scarcity of full‐siblings at the study site compared to laboratory conditions.