C.S. Russell

Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging

Three dermatan sulfates (DS18, DS28, and DS35) were isolated from womens skin of ages 19 +/- 2.5, 35 +/- 3.5, 47 +/- 1.7, 60 +/- 0.8, and 75 +/- 5 years. They sequentially precipitated with 18, 28, and 35% ethanol. Their sulfate content was: 23.5, 25.3, and 29.0% (w/w) for DS18 at ages 19-35, 47, and 60 years, respectively; 29.0, 24.0, and 18.8% for DS28; and 18.0, 20.0, and 20.6% for DS35 at ages 19-47, 60, and 75 years, respectively. Both DS18 and DS28 decreased, respectively, from 0.030% (of wet-skin weight) to traces at age 75, and from 0.020 to 0.010% at 60 years. At age 75, DS28 apparently increased by 30%. The DS35 values (traces-0.006%) had no age-related trend. Hyaluronic acid (HA) precipitated with 45% ethanol, was 0.030% of skin-weight at ages 19-47, and decreased to 0.015 and 0.007% at 60 and 75 years, respectively. Its electrophoretic mobility was slower at age 47. In the oldest group, i.r. spectra of HA and DS35 displayed no bands at 1650-1600, 1380, and 1320 cm-1, and a new band at 1560 cm-1. Moreover, ninhydrin-positive material of HA and DS35 increased by 75 and 95%, respectively, and the reducing GlcNAc content of HA decreased. These data showed three chemically different dermatan sulfates (two of which were preponderant) and N-deacetylation of HA and DS35 of the oldest group. After age 47, total DS and HA considerably decreased, DS18 and DS35 were oversulfated, and DS28 became undersulfated with aging.

Cloning and structure of the hemA gene of Escherichia coli K-12

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552].

The structure of the Escherichia coli hemB gene

The Escherichia coli hemB gene, which encodes 5-aminolevulinic acid dehydratase, and was cloned into pTZ18U, a multicopy plasmid, was sequenced. The hemB insert was double-digested with restriction enzymes and recloned back into pTZ18U and pTZ19U to allow for sequencing in two directions. In a second procedure, used to fill in gaps and to confirm the sequence derived from the first procedure, the whole insert was cloned into M13 phages. A nested set of deletions was constructed and recloned into M13. Both the double-digested fragments cloned into plasmids pTZ18U and pTZ19U and the overlapping fragments contained in M13 phages were sequenced using the dideoxy procedure with [35S]dATP. Computer software was used to identify coding regions and the correct reading frame. Two promoter regions, two Shine-Dalgarno sequences and two possible start sites were identified. Extensive homologies with yeast (36%), human liver (40%) and rat liver (40%) amino-acid (aa) sequences were observed, especially in the 16-aa Zn-binding region (75%) and the 4 aa surrounding the essential lysine at the active site (100% for rat and human proteins). Computer analysis of promoter strength and two independent analyses of codon usage indicated that the hemB gene is moderately expressed.

Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy

Age-mediated deacetylation of hyaluronic acid and dermatan sulfate, and shift of sulfate ester configuration were indicated by infrared spectroscopy. Hyaluronic acid and the three dermatan sulfates (DS18, DS28 and DS35), sequentially precipitated from adult skin with 18%, 28% and 35% ethanol, were analyzed at varying ages. At age 75 years, loss of infrared bands in the 1,650-1,600 cm-1 region, at 1,380 cm-1 and 1,320 cm-1 and appearance of a band at 1,560 cm-1 were characteristic of hyaluronic acid and DS35; moreover, in DS28 and DS35 the intensities of the bands at 840 cm-1 and 860 cm-1 were, respectively, decreased and increased. A low intensity band in the 805-785 cm-1 region was observed in the spectra of DS18 (19-35 years), DS28 (70-80 years) and DS35 (all ages). It intensified in DS28 of the 80-year-olds. In the 75 +/- 5-year-old group, ninhydrin-positive material of hyaluronic acid and DS35 increased, while reducing GlcNAc of hyaluronic acid decreased. The data demonstrated hyaluronic acid and DS35 deacetylation and suggested a decrease of equatorial sulfates with infrared band at 840 cm-1 and an increase of axial sulfates with band at 860 cm-1 in DS28 and DS35 of the 75 +/- 5-year-old set. Equatorial Equatorial sulfates with band in the 805 +/- 785 cm-1 region apparently decreased in DS18 after 35 years and increased in DS28 of the oldest group.

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.

Expression of glutamyl-tRNA reductase inEscherichia coli

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.

Heating unsaturated fatty acids in air produces hemagglutinins

When mono-unsaturated fatty acids are heated in air, they form hemagglutinins. When the double bond is delta-6,7 or delta-9,10, the titer is higher than for delta-11,12. Stearic acid does not become a hemagglutinin on heating. Hydroxy-monounsaturated fatty acids, ricinoleic (cis-12-OH-delta-9) and ricinelaidic (trans-12-OH-delta-9) are not hemagglutinins unless they are heated. Oleic acid (delta-9-octadecenoic acid, OA) has a very low agglutination titer but lyses red cells at higher concentrations. Rabbit and rat erythrocytes (RBC) give the highest titers but RBCs of other species are also agglutinated. OA was chosen for further study. Its specific titer against rat RBCs increases with time of heating in air. Thin-layer chromatography (TLC) and mass spectroscopy (MS) show that higher molecular weight compounds are formed and that activity is associated with these materials. Synthetic (oxidation of oleic acid with tert-butyl peroxide) and commercial preparations of oleic acid dimers (Emery and Unichema) and a commercial preparation of oleic trimer mixed with polymer (Emery) have high hemagglutination titers against rat erythrocytes. A cyclic, long-chain dicarboxylic acid, 5(6)-carboxy-4-hexyl-2-cyclohexene-1-octanoic acid (Westvaco) gives a very low titer unless heated and no lysis. Sialidase treatment of the red cells increases the titer. Removal of cations does not alter the titer but addition of Ca2+ or Mg2+ lowers the titer. Light microscopy was used to characterize and visualize the agglutination process with rat RBCs. Agglutination without lysis or fusion is observed for low concentrations of heated oleic acid and C-18 dimers and trimer-polymer preparations, and no large vesicles are seen. We conclude that the oligomeric fatty acids with two or more hydrophobic chains of seven or more carbons are agglutinins at physiological pH. Agglutination by dimer may be the result of the its two hydrophobic side chains inserting into adjacent RBC membranes or the result of dimer inserting completely into RBC membranes and altering their properties. The carboxyl groups may also play a role in the process by interacting with polar headgroups in the RBC membrane.