Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development

Summary The digestive glands of many marine molluscs are rich sources of arylsufatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone, Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43 000 Da each, as measured by gel filtration. The affinities for p-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes have K _m values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A₂) has a K _m of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45°C, respectively. Three of the four enzymes have-t_1/2(50°C) values of 3.5 min; the enzyme A₄ has a t_1/2 has a t_1/2(50°C) of 8.5 min. A monoclonal antibody directed against form A_1b does not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes.Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation. The secoad phase of enzyme expression is dependent upon the induction of settlement and metamorphosis of the competent veliger larvae, strongly suggesting that the expression of arylsulfatase synthesis (and the maturation of the digestive gland, the hepatopancreas) is controlled by genetic events which occur as a result of metamorphosis. Competent veliger larvae express only two arylsulfatase forms, which share many physicochemical and kinetic characteristics with the adult hepatopancreatic enzymes. However, neither of the larval arylsulfatases is recognized by the monoclonal antibody to form A_1b from adult hepatopancreas. Endogenous enzyme inhibitor levels in larvae remain constant throughout the period of arylsulfatase induction, and therefore do not contribute to the control of arylsulfatase activity levels during development.These results are the first documentation of the developmental induction of a specific protein(s) in abalone as a result of metamorphosis. The significance of the timing of arylsulfatase expression is discussed in relation to potential physiological substrates and the dietary switching which occurs at metamorphosis. Possible genetic events which are consistent with the observed patterns of expression of these enzymes also are considered.Abbreviations BSA bovine serum albumin - C centigrade - Da daltons - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - FPLC fast protein and polynucleotide liquid chromatography - GABA -aminobutyric acid - HAT hypoxanthine, aminopterin, thymidine - Hepes N-(2-Hydroxythyl)piperazine-N'-(2-ethanesulfonic acid) - HPLC high pressure liquid chromatography - KBS Kantor's balanced salt solution - K _m Michaelis constant - PBS phosphate buffered saline - R _m relative mobility - RPMI Roswell Park Memorial Institute - SDS sodium dodecyl sulfate - T time - TBS TRIS buffered saline - V _max maximal velocity     

Morphological, Cultural, Biochemical, and Serological Comparison of Japanese Strains of Vibrio parahemolyticus with Related Cultures Isolated in the United States

Morphological, cultural, biochemical, and serological characteristics of 79 strains of Vibrio parahemolyticus isolated from patients suffering from gastroenteric disease in Japan were compared with 17 suspected V. parahemolyticus cultures isolated from wound infections and 14 nonpathogenic vibrios isolated from an estuarine environment in the United States. These groups were differentiated on the basis of several key reactions which included: the range of growth temperature and salt tolerance; the production of catalase and acetoin; the hydrolysis of starch; the fermentation and utilization as single carbon source of sucrose, cellobiose, and arabinose; and the ability to swarm on 1% agar. The separation of the groups on the basis of cultural and biochemical analyses was confirmed by means of slide agglutinations with specific anti-K antisera. The results of this study strongly suggest that the wound infection isolates are V. parahemolyticus species which are easily distinguished from the nonpathogenic estuarine vibrios.     

The microbial plankton of Lake Fryxell,southern Victoria Land,Antarctica during the summers of 1992 and 1994

 Samples collected from Lake Fryxell, southern Victoria Land, Antarctica in January 1992 and 1994 were analysed for the abundance of bacterioplankton and the diversity and abundance of protistan plankton. At the times of sampling, 14 ciliate species and 10 species of autotrophic flagellate were recorded. The samples contained two species of rotifer (Philodina spp.), which formed the first record of planktonic metazoans in the Dry Valley lakes of this region of Antarctica. Bacterial concentrations ranged between 1.0 and 3.8×10⁸ l^-1 in the upper oxic waters increasing to 20×10⁸ l^-1 in the anoxic waters. Heterotrophic flagellates decreased in abundance down the oxygenated water column, disappearing completely at 9 m, and ranged between 0.28 and 7.39×10⁵ l^-1 in abundance. Autotrophic flagellates were much more abundant exhibiting a number of distinct peaks down the water column (1.89–25.3×10⁸ l^-1). The ciliated protozoa were very abundant (up to 7720 l^-1) in relation to flagellate and bacterial numbers, typical of oligotrophic lakes world-wide. The distribution of the protistan plankton showed marked zonation, probably in response to the differing salinity and temperature gradients in the water column. Possible trophic interactions are discussed and comparisons with other continental Antarctic lakes made. Received: 29 November 1995/Accepted: 18 February 1996     

HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation.

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.     

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2α,3α,22α, 23α-tetrahydroxy-24α-methyl-B-homo-7-oxa-5α-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22β, 23β-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydrocampesterol (24α-methyl) and brassicasterol (24β-methyl) from oysters. The method of synthesis favored the formation of the 22β, 23β-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain $\text{cis}$-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 μg level. The least active was the 22β,23β-isomer of brassinolide. The isomers with the 22α, 23α and 24β, and the 22β, 23β and 24β configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22β, 23β and 24β configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.     

Cholesterol, 7-ketocholesterol and 25-hydroxycholesterol uptake studies and effect on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in human fibroblasts.

In human fibroblasts two oxidized derivatives of cholesterol, 7-ketocholesterol and 25-hydroxycholesterol, but not cholesterol itself, are potent inhibitors of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (mevalonate: NADP+ oxidoreductase (Co-enzyme A acylating), (EC 1.1.1.34), the rate-limiting enzyme in sterol biosynthesis. In addition, these derivatives of cholesterol are effective regulators in cells from homozygous familial hypercholesterolemic individuals. The differences in the inhibitory potencies of the sterols cannot be explained in terms of the amount of uptake into the cell.     

Prolonged Vitamin D Intoxication in a Patient with Hypoparathyroidism

Pitfalls in the management of hypoparathyroidism are illustrated by the case of a patient who developed hypervitaminosis D while receiving doses of calciferol and of calcium in amounts commonly recommended for treatment. Either the patient was very slow to obtain maximum vitamin D effect or else her sensitivity to vitamin D increased, because she did not become hypercalcemic until two years after treatment was started. The dose of vitamin D was halved to 50,000 units per day and the dose of calcium was lowered to 0.26 g. daily. She failed to remain under medical supervision for the next four years and presented with hypercalcemia and evidence of renal impairment. After vitamin D was discontinued she remained hypercalcemic for nine months.These findings are discussed in the light of current knowledge concerning the actions of parathyroid hormone and vitamin D. The influence of adrenocortical hormones on calcium metabolism is considered. The need to follow up hypoparathyroid patients closely, and to check the level of calcium in the serum, is emphasized.     

Dangers in the Use of Some Potent Drugs

The chief dangers reported with some common drugs are reviewed. Hazards of antibiotic therapy include: the increasing incidence of sensitization to penicillin with occasional anaphylactic reactions; aplastic anemia with chloramphenicol, and the poor tolerance of infants for chloramphenicol; staphylococcal enterocolitis; unnecessary “prophylactic” use of antibiotics. Thiazide diuretics may precipitate potassium depletion, skin reactions, pancreatitis, blood dyscrasias, gout, diabetes mellitus and hepatic coma. Reserpine can increase gastric acidity, induce mental depression, and when used with digitalis lead to ventricular premature beats. Hydralazine may aggravate angina pectoris, cause tachycardia, and bring about a syndrome resembling disseminated lupus erythematosus. Guanethidine may result in loose stools, impotence, and postural hypotension. Hazards of phenothiazines include jaundice, parkinsonian states and tremors, convulsions, hypotension, and blood dyscrasias. The butanediols have numerous side effects including gastrointestinal, cutaneous and hypotensive reactions. Prolonged corticosteroid therapy introduces a new danger in surgical treatment. The progesterone-like drugs may induce masculinization of the female fetus.