The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes.

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the 'cobblestone' or the 'sprouting' phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a 'cobblestone' monolayer. In contrast, cells displaying the 'sprouting' morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by 'cobblestone' endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels ('sprouting' cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a 'cobblestone' or 'sprouting' morphology.     

The chicken liver cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for insulin-like growth factor II

The chicken liver cation-independent mannose 6-phosphate receptor has been purified to apparent homogeneity by affinity chromatography on pentamannose phosphate-Sepharose and tested for its ability to bind iodinated human IGF-I, human IGF-II, and chicken IGF-II. In contrast to the bovine, rat, and human cation-independent mannose 6-phosphate receptors, which bind human IGF-II and IGF-I with nanomolar and micromolar affinities, respectively, the chicken receptor failed to bind either radioligand at receptor concentrations as high as 1 microM. The bovine receptor binds chicken IGF-II with high affinity while the chicken receptor binds this ligand with only low affinity, which we estimate to be in the micromolar range. These data demonstrate that the chicken cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for IGF-II. These results provide an explanation for the failure of previous investigators to identify the type II IGF receptor by IGF-II cross-linking to chicken cells and indicate that the mitogenic activity of IGF-II in chick embryo fibroblasts is most likely mediated via the type I IGF receptor.     

Use of an alternative method for monitoring total nitrogen concentrations in Florida lakes

We studied a recently described method for the determination of total nitrogen in natural waters involving sample oxidation with persulfate and subsequent determination of nitrate-nitrogen with second derivative spectroscopy and compared it to the USEPA approved method involving the sum of nitrate-nitrogen and Kjeldahl-nitrogen as measured with an automated analyzer. The overall objective was to determine if the two methods gave the same estimates of total nitrogen and if the detection limits, precision and accuracy of the new method were as good as those of the USEPA method. The new method was used to make measurements on replicated blanks, standards and lake water samples covering a range of concentrations. We also collaborated with certified laboratories to make comparative measurements on 5 standards and 21 lake water samples that were run by us with the new method and by them with the USEPA method. The new method had an instrument detection limit of 0.07 mg 1^–1, and the standard deviation of 20 sets of lake water samples averaged 0.03 mg 1^–1. The new method gave concentrations equivalent to those found with the USEPA method, was more sensitive, and had a higher degree of precision. We concluded that the new method is suitable as a substitute for the USEPA method. We also found that the addition of acid to lake water samples stored under refrigeration was not necessary to preserve them for later determinations of nitrate-nitrogen and total nitrogen and that freezing was an effective means of sample preservation for 90 days.     

Fate of elemental sulfur in an intertidal sediment

Abstract: Sediment from a tidal flat at Wedderwarden, near the mouth of the Weser estuary, northern Germany, was amended with elemental sulfur, and concentrations of metabolic end products were monitored. The production of both sulfate and sulfide was consistent with disproportionation as the most important fate of the added elemental sulfur. A population of bacteria conducting active elemental sulfur disproportionation was also enriched from the sediment. In the enrichments, containing both elemental sulfur and Fe oxides as a sulfide 'scrub', sulfide and sulfate were produced in a ratio of     , somewhat lower than the predicted ratio of     . The mismatch between predicted and observed production ratios is explained by the channelling of electrons into autotrophic or mixotrophic CO₂ fixation rather than sulfide formation. The production of organic carbon, in the correct amount to explain the observed sulfide to sulfate production ratio, was verified by organic carbon analysis. Finally, rates of sulfate reduction were identical in the elemental sulfur amended sediment, and in control sediment with no added sulfur. Hence, the heterotrophic bacterial community was completely unaffected by an active metabolism conducting elemental sulfur disproportionation.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.     

Growth of Octopine-Catabolizing Pseudomonas spp. under Octopine Limitation in Chemostats and Their Potential To Compete with Agrobacterium tumefaciens

The growth characteristics of five octopine-catabolizing pseudomonads have been determined in batch and continuous cultures. All five strains belonged to rRNA homology group I and showed a more psychrotrophic growth pattern than did Agrobacterium tumefaciens B6 and ATCC 15955. In chemostats limited by octopine, either as the source of carbon and nitrogen or the sole source of nitrogen, maximum specific growth rates and substrate affinities were lower than those in chemostats limited by glutamate. These growth dynamics were similar to those observed for Agrobacterium strains B6 and ATCC 15955 even though the catabolic genes and pathways are believed to be different in the two genera. An analysis of the yields in octopine-limited chemostats indicated that the use of octopine as the sole source of carbon and nitrogen was grossly inefficient. Octopine and presumably lysopine and octopinic acid provided a better source of nitrogen than of carbon. One of the Pseudomonas fluorescens strains, E175D, was able to produce its highest yield on octopine as a nitrogen source. Competition models formulated on pure culture parameters indicated that two of the Pseudomonas spp. would dominate A. tumefaciens B6 and ATCC 15955 when in simple competition for octopine as a limiting substrate.     

Partial amino acid sequence of the preprotein form of the alpha subunit of human choriogonadotropin and identification of the site of subsequent proteolytic cleavage

Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH₂-terminal extension of 24 amino acids was found and its partial sequence is:

The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH₂-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit.