Chemistry of the collagen cross-links. Origin and partial characterization of a putative mature cross-link of collagen.

The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen.     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

Electroantennogram responses of the oriental fruit fly, Dacus dorsalis, to a spectrum of alcohol and aldehyde plant volatiles

Electroantennograms (EAGs) were recorded from unmated, laboratory-reared, male and female oriental fruit flies, Dacus dorsalis, in response to a range of between C₁ and C₁₂ carbon chain-length saturated and unaturated aliphatic alcohols and aldehydes, most all of which are known host-plant volatiles. Only two of the 35 compounds tested elicited significantly larger EAGs from female than male antennae. For the two functional-group series tested, aldehydes elicited responses greater than or equal to the responses to the alcohols. In general, the unsaturated alcohols did not elicit responses significantly different from the saturated alcohols. However, the unsaturated aldehydes, (E)-2-hexenal and 10-undecenal, elicited larger amplitude EAGs than their saturated analogs. EAGs were significantly greater for a particular carbon chain-length, with responsiveness to primary alcohols peaking at C₆ and aldehydes peaking at C₇. The (E)-2- monoenic alcohols peaked at C₆, while the (E)-3-alcohols plateaued between C₅ and C₈. The greatest EAG responses of all compounds tested were elicited by the saturated and unsaturated C₆ alcohols and aldehydes which are constitutents of the general green-leaf volatile complex that emanates from most plants. The potential adapative benefit of selective sensitivity to green-leaf volatiles is discussed in regards to foraging behaviors of oriental fruit flies.

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Résumé Des électroantennogrammes (EAG) ont enregistré les réponses, en élevages de femelles et mâles vierges de Dacus dorsalis, à une gamme de chaînes de carbones de C₁ à C₁₂ saturés et non-saturés d'alcools aliphatiques et d'aldéhydes, dont beaucoup sont connus comme substances volatiles des végétaux. Seulement 2 des 35 composés examinés ont provoqué des EAG significativement plus importants chez les femelles que chez les mâles. Pour les séries des deux groupes fonctionnels examinés, les aldéhydes ont provoqué des réponses supérieures ou égales aux alcools. En général, les réponses aux alcools nonsaturés n'étaient pas significativement différentes des réponses aux alcools saturés. Cependant, les aldéhydes non-saturés, (E)-2-hexénal et 10-undécénal, ont induit des EAG de plus grande ampleur que leurs analogues saturés. Les EAG étaient significativement les plus importants pour une chaîne de longueur particulière, la réponse aux alcools primaires culminant en C₆ et les aldéhydes en C₇. Les alcools monoéniques (E)-2- culminaient en C₆, tandis que les alcools (E)-3- étaient étales entre C₅ et C₈. Les EAG les plus importants ont été obtenus pour tous les composés examinés avec les alcools et aldéhydes en C₆ qui appartiennent à l'odeur verte complexe émise par beaucoup de plantes. Le bénéfice adaptatif potentiel de la sensibilité sélective à l'odeur verte des feuilles est examinée en fonction du comportement de prospection de D. dorsalis.

     

Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid R1

Initiation of replication of the resistance plasmid R1 is carefully regulated by the two negatively acting factors, CopA and CopB. It is shown here that the temperature-dependent runaway-replication phenotype of an R1 plasmid mutant is caused by two point mutations in each of the promoters for the genes of these control factors. Expression of the two genes is affected in the following way: (1) one C-to-T transition in the putative -35 box of the copB-repA operon creates a two- to three-fold stronger promoter from which expression is temperature-dependent; (2) another C-to-T transition in a G + C-rich area immediately downstream from the -10 box of the copA promoter reduces expression of the copA gene three-fold. The phenotypic consequences of the two mutations are discussed in the light of the current model for R1 replication control.     

Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata to identified volatile constituents from calling males

Fifty-six compounds from the odor of calling, sexually mature, laboratory reared males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) were isolated by headspace trapping on Tenax columns and identified using GC/MS techniques (69 total compounds were detected). Electroantennogram responses (EAGs) to 54 of the 56 identified compounds as well as 5 analogs were tested on both sexes. Significant differences between the sexes in their responsiveness were found in 9 of the 54 identified compounds tested. There was no correlation between the amplitude of the EAG response and the relative abundance of compound identified from headspace analysis. Of the five major identified components, three elicited relatively small EAG responses, while two elicited large EAGs compared to the hexan-1-ol standard. The relative ranking of EAG responses were: methyl and ethyl hexenoates and hexanoates > C₄–C₆ esters and/or acetates > ethyl and methyl octenoates > monoterpenes > sesquiterpenes > C₂–C₅ acetates, alcohols and ketones. Behavioral bioassays on each of the five major identified components as well as a blend of six of the compounds showed some degree of attractancy to virgin females which in some cases approached the response to a pheromonal standard (male odors absorbed onto filter paper). These results are discussed in relationship to the insect's antennal sensitivity to putative pheromone components and/or allomonal components and to other reported C. capitata pheromone studies.

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Résumé Cinquante-six composés de l'odeur de mâles de C. capitata Weidemann, élevés en laboratoire, sexuellement mûrs et en appel, ont été isolés par piégeage sur colonnes tenax et identifiés par la technique GC/MS (69 composés avaient été détectés en tout). Les électroantennogrammes (EAGs) ont été examinés chez les deux sexes pour 54 des 56 composés identifiés et 5 de leurs analogues. Des différences significatives entre les sexes ont été observées pour 9 des 54 composés identifiés. Il n'y avait pas de corrélation entre l'ampleur de l'EAG et l'abondance relative du composé lors de son isolement. Pour les 5 principaux composés identifiés, 3 ont induit des EAGs relativement faibles, tandis que 2 étaient importants, par comparaison avec l'Hexane-1-ol utilisé comme témoin. Le classement relatif des EAG a été: hexénoates et hexanoates d'éthyl et de méthyl C₄–C₆ esters et/ou acétates octénoates d'éthyl ou de méthyl monoterpènes sesquiterpènes C₂–C₅ acétates, alcools et kétones. Les expériences de comportement avec chacun des 5 composés principaux identifiés, comme avec des mélanges de 6 composés ont mis en évidence une attraction des femelles vierges qui dans quelques cas avoisine la réponse à la phéromone témoin (odeur du mâle absorbée sur papier filtre). Ces résultats sont discutés en fonction de la sensibilité de l'antenne d'insexte aux composés supposés de la phéromone et aux composés allomonaux, et en fonction des autres études connues sur les phéromones de C. capitata.

     

Natural taxonomy of collagen based on amino acid composition

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.     

Effect of alveolar hypoxia on pulmonary fluid filtration in in situ dog lungs

We have studied the effect of alveolar hypoxia on fluid filtration characteristics of the pulmonary microcirculation in an in situ left upper lobe preparation with near static flow conditions (20 ml/min). In six dogs (group 1), rate of edema formation (delta W/delta t, where W is weight and t is time) was assessed over a wide range of vascular pressures under two inspired O2 fraction (FIO2) conditions (0.95 and 0.0 with 5% CO2-balance N2 in both cases). delta W/delta t was plotted against vascular pressure, and the best-fit linear regression was obtained. There was no significant difference (paired t test) in either threshold pressure for edema formation [18.3 +/- 1.8 and 17.1 +/- 1.2 (SE) mmHg, respectively] or the slopes (0.067 +/- 0.008 and 0.073 +/- 0.017 g.min-1. mmHg-1.100g-1, respectively). In another seven dogs (group 2), delta W/delta t was obtained at a constant vascular pressure of 40 mmHg under four FIO2 conditions (0.95, 0.21, 0.05, and 0.0, with 5% CO2-balance N2). Delta W/delta t for the four conditions averaged 0.60 +/- 0.11, 0.61 +/- 0.11, 0.61 +/- 0.10, and 0.61 +/- 0.10 (SE) g.min-1.mmHg-1.100g-1, respectively. No significant differences (ANOVA for repeated measures) were noted. We conclude that alveolar hypoxia does not alter the threshold for edema formation or delta W/delta t at a given microvascular pressure.     

Evidence for glucose-mediated covalent cross-linking of collagen after glycosylation in vitro.

Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics. The tendons treated with glucose contained up to three times as much hexosyl-lysine and hexosylhydroxylysine as did control tendons as judged by assay of NaB3H4-reduced samples. Measurement of the force generated on thermal contraction showed significant increases in glycosylated tendons compared with controls, indicating the formation of new covalent stabilizing bonds. This conclusion was supported by the decreased solubility of intact tendons and re-formed fibres glycosylated in vitro, and by the evidence from peptide maps of CNBr-digested glucose-incubated tendons. The latter, when compared with peptide maps of control tendons, revealed the presence of additional high-Mr peptide material. These peptides appear to be cross-linked by a new type of covalent bond stable to mild thermal and chemical treatment. This system in vitro provides a readily controlled model for the study of the chemistry of changes brought about in collagen by non-enzymic glycosylation in diabetes.