E. Kodicek

The uptake of (35S) sulphate by mucopolysaccharides of granulation tissue

In order to gain some insight into the formation and fate of the sulphated polysaccharides of granulation tissue, the (35S) sulphate uptake of regenerating tendons of guinea-pigs was determined in vivo. Hexosamine and hydroxyproline contents were estimated at various stages of healing. The maximum 35S uptake occurred after the seventh day and was maintained for at least 8 more days, returning to low values on the 23rd day after operation. The relationship between sulphate uptake, hexosamine and hydroxyproline contents was determined and discussed. In scurvy, the uptake of 35S was lower than in controls, while hexosamine values were unchanged. This was presumed to indicate that though the total amount of polysaccharide in scorbutic granulation tissue was undiminished, it was not sulphated in the normal way. A polysaccharide component, which was highly radioactive and had a mobility of approximately the same order as chondroitin sulphate, was separated from normal granulation tissue by extraction and ionophoresis on paper. It was thus shown that sulphation of a mucopolysaccharide was responsible for the uptake of 35S. The mechanism of (35S) sulphate uptake was studied in vitro by incubating slices of granulation tissue in a medium containing radioactive sulphate. Disruption of cells by homogenization or freezing-and-thawing greatly reduced the incorporation of sulphate. Little or no uptake of 35S occurred in boiled tissue, in the cold, or in the absence of oxygen. 2:4-Dinitrophenol, azide, cyanide and certain sulphydryl-binding substances were found to inhibit the reaction. Salicylate caused an inhibition, while cortisone had no such effect. Scorbutic granulation tissue showed a decreased fixation of sulphate. It was concluded that sulphate uptake by granulation tissue was dependent on an enzymatic reaction, possibly involving oxidative phosphorylation.

The metabolism of acetate and mevalonic acid by lactobacilli IV. Analysis of the fatty acids by gas-liquid chromatography

Abstract The fatty acids of Lactobacillus casei, Lactobacillus arabinosus and Lactobacillus acidophilus were analysed by gas-liquid chromatography using columns with polyethylene glycol adipate or Apiezon L grease as stationary phase. Two groups of acids were analysed, those prepared by saponification and those prepared by acid hydrolysis. Acid hydrolysis was necessary to obtain the total fatty acids but it resulted in the destruction of lactobacillic acid. In addition to the whole cells, the fatty acids from the bacterial membranes of L. casei , prepared by differential centrifugation, were examined and compared with those from the membranes of Micrococcus lysodeikticus and Bacillus megaterium .

The influence of dietary calcium deficiency and parathyroidectomy on bone collagen structure

Abstract Bone collagen synthesized in chicks fed a calcium-deficient diet had the same chain composition as that normally synthesized, but differed in that in both α1- and α2-chains there was an increased conversion of lysine to hydroxylysine, of about 50–70%, equivalent approximately to an extra five residues of hydroxylysine in the α1- and an extra six to seven residues in the α2-chain. This effect was not due to the hyperparathyroidism accompanying the calcium deficiency since increased hydroxylation was also observed when calcium levels were reduced following parathyroidectomy. It is suggested the effect may be directly attributable to reduced calcium levels and that the increased hydroxylation of bone collagen lysine occurring in rickets may also be attributable to the reduced level of calcium existing in vitamin D deficiency. Examination of skin collagen indicated a slight increase in hydroxylation, equivalent only to an extra one to two residues of hydroxylysine, in the α1-chains. A similar slight increase in the α2-chains was not unequivocably demonstrated. The results suggest lysine hydroxylation in bone collagen to be much the more sensitive to changes in calcium level and the possible significance of this, particularly in relation to mineralization, is considered.

Separation of binding proteins for cholecalciferol and 25-hydroxycholecalciferol from chick serum

Abstract The serum transport proteins in the chick of vitamin D and 25-hydroxyvitamin D have been studied by means of analytical polyacrylamide disc gel electrophoresis, gel filtration and column chromatography on DEAE-Sephadex. It has been possible to show the presence of, and subsequently separate, two carrier proteins from chick serum. One was found to bind predominantly 25-hydroxycholecalciferol while the other protein binds predominantly cholecalciferol.

The metabolism of acetate and mevalonic acid by lactobacilli I. The effect of acetate and mevalonic acid on growth

Abstract The requirement of three species of lactobacillus for acetate and its replacement by mevalonic acid was investigated. It was found that, in the presence of 1% glucose, Lactobacillus acidophilus had a requirement for sodium acetate which could be replaced by 1/8000 times its concentration of mevalonic acid, but when the glucose concentration was 2%, acetate was no longer required. Lactobacillus arabinosus also required acetate, but mevalonic acid replaced only partially the acetate requirement. Acetate was not required, under the present experimental conditions for Lactobacillus casei . The acetate requirement of L. acidophilus could also be replaced by β-hydroxy-β-methylglutaric acid and by 2,2′-dimethylacrylic acid, but not by farnesol or geraniol.

Excretion of hydroxyproline and other amino acids in scorbutic guinea-pigs

Abstract 1. 1. A fall in the urinary excretion of total ( i.e , peptide-bound plus free) amino acids, including hydroxyproline, occuring in scorbutic guinea-pigs and commencing around the 15th day of vitamin deficiency, was also observed in pair-fed controls and is thought, therefore, to be due to inanition. The significance of this finding in relation to the known impairment of collagen synthesis in scurvy is discussed. 2. 2. Although, with the exception of glycine and histidine, the excretion of free amino acids by scorbutic guinea-pigs was comparable to that in normal controls, the excretion in pair-fed controls was reduced. It is suggested, therefore, that scurvy is associated with some degree of general aminoaciduria which offsets the decreased excretion of free amino acids to be exprected as a consequence of the inanition which accompanies the vitamin deficiency in guinea-pigs. 3. 3. An increased excretion of free glycine and histidine observed in scorbutic guinea-pigs is thought to be attributable in part to the general aminoaciduria associated with scurvy and in part to the anomalous excretory response of these two amino acids to inanition. 4. 4. Ammonia excretion was increased 4–10 fold in scorbutic guinea-pigs, and since values in pair-fed controls remained normal, it is considered that the increased excretion in the scorbutic animals is directly attributable to the deficiency of ascorbic acid per se .

The metabolism of acetate and mevalonic acid by lactobacilli II. The incorporation of [14C]acetate and [14C]mevalonic acid into the bacterial lipids

Abstract Using [ 14 C]acetate and [ 14 C]mevalonic acid, it was shown that Lactobacillus casei, Lactibacillus arabinosus and Lactobacillus acidophilus incorporate from 20–35 times as much sodium acetate as mevalonic acid, the cellular concentration of mevalonic acid being from 0.5–0.9 μg/mg dry weight. Most of the radioactive material was found in the lipid fraction, probably bound to protein. Acetate was shown to be a precursor of both fatty acids and unsaponifiable lipids, mevalonic acid gave only unsaponifiable material. Part of the fatty acids could only be liberated from the whole lipids by acid hydrolysis and was not hydrolysed under alkaline conditions. Analysis of the unsaponifiable material by reversed-phase paper chromatography showed the presence of at least six components. The chemical nature of these substances was studied. Since mevalonic acid did not yield fatty acids, [ 4 C]glucose in absence of acetate was shown to be a precursor of the bacterial fatty acids. Biotin was found to be necessary for fatty acid synthesis from acetate but not for the synthesis of the unsaponifiable lipids from mevalonic acid.

The metabolism of acetate and mevalonic acid by lactobacilli III. Studies on the unsaponifiable lipids derived from mevalonic acid

Abstract In an investigation of the synthesis of the unsaponifiable lipids of Lactobacillus casei from mevalonic acid, it was shown that although no preferential order of synthesis of the components could be detected there was a turnover of the principal one with a simultaneous increase in the others. The presence of glucose and of all the amino acids was found to be necessary for the incorporation of mevalonic acid by L. casei and L. arabinosus . Since, in addition, the incorporation of mevalonic acid was inhibited by chloramphenicol it appears that protein synthesis is necessary for the uptake of mevalonic acid. This requirement for protein synthesis could not be explained by the necessity for inducing an enzyme before mevalonic acid could be utilised by the bacteria. Of other inhibitors tested, only p -chloromercuribenzoate completely inhibited the incorporation of mevalonic acid. Analysis of the cell fractions, prepared by differential centrifugation, suggested that the lipid from mevalonic acid is located in the soluble fraction of the cell. Only a trace of this lipid was found in the protoplast membrane of Micrococcus lysodeikticus . The unsaponifiable lipids could not replace mevalonic acid or acetate in the growth of L. acidophilus and they were not incorporated into the cell lipids by L. casei, L. arabinosus or L. acidophilus . The effect of mevalonic acid deficiency on the incorporation of various radioactive biochemicals was investigated and glucose uptake was found to be significantly reduced. A smaller reduction was found in the rate of acid production.

Captain Cook and scurvy

‘We were all hearty seamen no cold did we fear And we have from all sickness entirely kept clear Thanks be to the Captain he has proved so good Amongst all the Islands to give us fresh food.’ (Song by T. Perry, from H.M.S. Resolution) (23). The first encounter with scurvy at sea for the young James Cook, then 28 years old, might have been in July 1756, when he rejoined H.M.S. Eagle at Plymouth. The ship was being refitted and Captain Palliser had reported to the Admiralty the terrible effects of scurvy on maintaining his ships at sea (1): Put ashore to the hospital 130 sick men, most of which extremely ill; buried in the last month 22. The surgeon and four men died yesterday, and the surgeon’s two mates are extremely ill; . . . so that we are now in a very weak condition. A year later Cook’s ship, H.M.S. Pembroke, with others lying at Halifax in Canada, had so many sick on board that it took no part in the military action of Wolfe against the French. Cook remained with the Pembroke till 1762, for the most part in the basin of Quebec, and then for a further five years surveying Newfoundland. He probably had not heard of Jacques Cartier, the first European to sail up the St Lawrence River, who wintered in 1535-36 at the site of the present city of Quebec, at Stadacona with a crew ill and dying from a strange and fatal disease, ‘la grosse maladie’. Nor would he have heard of their miraculous cure, on the advice of a friendly Indian, with a decoction of the leaves and bark of the tree Annedda, the ‘arbor vitae’ of the natives.