Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide

The polysaccharides of cranical cartilge were isolated by ethanol precipitation after papain digestion and β-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39200 on weight basis $\text{(}\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{)}$ and 31400 on number basis $\text{(}\text{M}{}_{\mathrm{<mtext>n</mtext>}}\text{)}$.     

Increased synthesis of a low molecular weight protein in vincristine-resistant cells

A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ($\text{DC-3F}\text{VCRd-5}$) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of $\text{DC-3F}\text{VCRd-5}$, and only trace amounts were detected in control DC-3F cells. A similar protein (M_r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in $\text{DC-3F}\text{VCRd-5}$ cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.     

Structure of the capsular polysaccharide (K6-antigen) from Escherichia coli LP 1092

The linkage pattern of the K6-antigen was investigated using material from the urinary pathogen, $\text{Escherichia coli}$ LP 1092. The polysaccharide consists of ribose and 3-deoxy-$\text{D}\text{-}\text{manno}$-2-octulosonate (KDO) in a ratio of 2:1. Colorimetric procedures, Smith degradation, methylation analysis, and nuclear magnetic resonance spectroscopy were applied to the whole polysaccharide and to a trisaccharide “repeating unit” obtained by mild-acid catalyzed hydrolysis. Together, the data are compatible only with a branched chain structure …$\text{3Rib}\text{f}\text{β1→7KDO}\text{p}\text{β2→3Rib}\text{f}\text{β}$…

     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Occurrence of cis-7-tetradecenoic acid in the envelope phospholipids of Escherichia coli K12

We have isolated a tetradecenoic acid from $\text{E}\text{.}\text{coli}$ and have identified this new acid as $\text{cis}$-7-tetradecenoic by its ¹³C nuclear magnetic resonance spectrum. This identification was confirmed by conventional structural studies. The acid is a component of the phospholipids of $\text{E}\text{.}\text{coli}$ and comprises about 15% of the total phospholipid unsaturated fatty acid.     

Xylans from the tropical grass Panicum maximum

Two xylans have been isolated from the mature tissues of the tropical grass Panicum maximum—an arabino(4-O-methylglucurono)xylan and an acidic galactoarabinoxylan. Both consist of a main chain of β(1 → 4) linked d-xylopyranosyl residues. The former has average of ca 46 such residues to which are attached ca 7 l-arabinofuranosyl and (ca 2 4-O-methyl-d-glucopyranuronosyl residues at C3 and C2 positions respectively. The acidic galactoarabinoxylan has a $\text{DP}$_n of ca 90 and contains arabinose, galactose, xylose and uronic acid residues in the molar ratio 10:5:22:4. Methylation analysis and periodate oxidation indicated the highly branched nature of this polysaccharide.     

Purification of penicillin-insensitive DD-endopeptidase, a new cell wall peptidoglycan-hydrolyzing enzyme in Escherichia coli, and its inhibition by deoxyribonucleic acids.

Activity of a penicillin-insensitive $\text{DD}$-endopeptidase that splits the $\text{D}$-alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of $\text{Escherichia coli}$. The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments.     

Biochimica et Biophysica Acta: Vol. 307 (1973)

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ($\text{12-Me-14:0}$) or $\text{cis-Δ}{}^9\text{-}\text{octadecenoate}$ ($\text{cis-δ}{}^9\text{-18:1}$). When this strain is grown for many generations on $\text{12-Me-14:0}$, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of $\text{12:0}$ (8%) and $\text{14:0}$ (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.     

DNA strand scission in E.coli by electronically excited state molecules generated by enzymatic systems

$\text{E.}\text{coli}$ containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D₂ system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in $\text{E.}\text{coli}$ cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O₂ system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

Interaction of the lectin limulin with capsular polysaccharides from Neisseria meningitidis and Escherichia coli

The lectin limulin from the serum of the horseshoe crab $\text{Limulus polyphemus}$ binds to $\text{N}$-acetylneuraminic acid and 2-keto-3-deoxyoctonate residues. These interactions were examined using capsular polysaccharides from strains of $\text{Neisseria meningitidis}$ and $\text{Escherichia coli}$. Our findings indicate that limulin has greatest reactivity with homopolymers of $\text{N}$-acetylneuraminic acid as compared with heteropolymers of either sugar. Polysaccharides with α(2→9) ketosidic linkages were most efficient in precipitating this lectin. Finally, $\text{O}$-acetylated homopolymers of $\text{N}$-acetylneuraminic acid were more reactive than their $\text{O}$-acetyl-negative counterparts.     

Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpiusrotundacauda

The sialic acid binding loctin carcinoscorpin agglutinates $\text{Escharichia}\text{coli}\text{K12}\text{and}\text{Salmonella}\text{minnesots}$ R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. $\text{Vibrio}\text{cholerae}$, a Grum negative bectarium devoid of 2-keto-3-deoxyoctonate and $\text{Staphylococcus}\text{sureus}$ a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoa crab.     

Drug-induced changes in lipid composition of E. coli and of mammalian cells in culture: ethanol, pentobarbital, and chlorpromazine.

Both Chinese hamster ovary cells in culture and $\text{E.}$$\text{coli}$ cells change their lipid composition when grown in the presence of ethanol, pentobarbital, and chlorpromazine. The effects of ethanol and the cross-tolerant drug, pentobarbital, are similar. Both cause a shift from 18:0 fatty acid to 16:0 fatty acids in CHO cells and a decrease in the proportion of saturated fatty acids in $\text{E.}$$\text{coli}$. Chlorpromazine, a non-cross-tolerant drug, causes the opposite effect in $\text{E.}$$\text{coli}$, a decrease in the proportion of unsaturated fatty acids. Chlorpromazine has little effect on the fatty acid composition of CHO cells. These changes in lipid composition are proposed as an adaptive response and a part of the mechanism for the development of drug tolerance.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

Release of chromatin template restriction in rabbit spermatozoa

The addition of the acidic polymers heparin or polyxanthylic acid to rabbit spermatozoa or sperm heads previously exposed to disulfide reducing agents released sperm DNA template restriction and stimulated high levels of incorporation of DNA precursor into DNA, as assayed with exogenous DNA polymerase. Incorporation did not occur in the presence of DNAase, or in the absence of magnesium ion, any of the four deoxyribonucleotides, or $\text{E. coli}$ DNA polymerase. This represents the first report that spermatozoa can synthesize DNA $\text{in vitro}$.     

Glycogen accumulation by pleomorphic cells of Streptococcus sanguis

Cells of $\text{Streptococcus sanguis}$ undergo gross morphological changes when cultured in the presence of oxygen. These cells accumulate large amounts of an intracellular polysaccharide. The chemical nature and location of this polysaccharide were investigated and we showed that it is an intracellular polymer composed primarily of glucose in a linkage of the glycogen-amylopectin type. Glycogen accumulation appeared to coincide temporally with the onset of pleomorphism while its disappearance coincided temporally with cell death.     

Steroids from starfish

Starfish $\text{Linckia multifora, Protoreaster nodosus, Protoreaster lincki, Culcita schmideliana, Nardoa variolata}$ and $\text{Acanthaster planci}$ were examined for sterols and sapogenins. All asteroids contained cholestanol in addition to C₂₇ to C₃₀ mono and diunsaturated sterols. A planci contained largest amounts of 5α-cholesta-9(11), 20(22)-diene-3β, 6α-diol-23-one and 5α-pregn-9(11)-ene-3β,6α-diol-20-one whereas $\text{P. nodosus, P. lincki and C. schmideliana}$ contained only small amounts. Neither pregnane nor cholestane genins were detected in $\text{L. Multifora}$ and $\text{N. variolata}$.