A culture system for the maintenance and proliferation of shark and sting ray immunocytes

A method designed to support the in vitro maintenance and proliferation of elasmobranch peripheral blood leucocytes (PBLs) has been developed. This method includes protocols for leucocyte isolation, the preparation of media, and culturing conditions. Leucocytes were isolated from whole blood using a Ficoll-hypaque density gradient followed by nylon wool filtration. Recovered cells were washed and incubated at a density of 0.054 × 10⁶ cells per well of a microtitre plate using a novel medium designed for shark cells. Incubation was carried out at 17 and 37° C. These media represent a modification of RPMI₁₆₄₀ or DMEM to account for isotonic and isoosmotic differences between mammalian and elasmobranch blood via the addition of trimethylamine- N -oxide, urea and sodium chloride to the RPMI or DMEM.     

Cyclopropane fatty acid synthase of Escherichia coli: deduced amino acid sequence, purification, and studies of the enzyme active site.

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.     

Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol.

Rabbit polyclonal antibodies were raised against rat liver bile salt-independent neutral cholesteryl ester hydrolase (CEH) and used for subcellular localization and immunological comparison with isoforms from other tissues. Antibodies exhibited a high degree of specificity for the liver CEH through all stages of purification and neutralized 70-80% of the activity of liver cytosolic CEH. They exhibited various levels of cross-reactivity with cytosolic proteins from other tissues, but reacted weakly with pancreatic and intestinal proteins and did not inhibit pancreatic CEH. Cytosol contained 78% of total cellular CEH activity and 75% of CEH immunoreactive protein. Washed microsomes contained 3% of CEH activity and 5% of CEH protein.     

Iron binding by phosvitins: variable mechanism of iron release by phosvitins of diverse species characterized by different degrees of phosphorylation

The rate of reductive iron release from Fe(III) complexes of phosvitins of diverse fish species, at varied initial degrees of saturation with iron, was studied with particular attention to the effect of the degree of phosvitin phosphorylation on the kinetics of iron release. The reaction was followed colorimetrically as phosphorprotein-bound iron was transferred to an excess of o-phenanthroline, in the presence of hydroquinone as a reducing agent. The principal finding was the variability of the kinetic order or iron release by phosvitins, depending on their degree of saturation with iron and the extent to which their serine residues were phosphorylated. Highly phosphorylated proteins, especially at high initial degrees of iron saturation, obey first-order kinetics. Partially phosphorylated proteins, especially at low initial degrees of iron saturation, release their iron in a zero-order fashion. First-order rates imply that the iron binding sites are kinetically independent of each other. Zero-order behavior appears to reflect iron release from hypothetical iron-binding clusters serving as kinetically effective reactive centers of unchanging concentration for most of the time course of the reaction. Variations of the initial degree of iron saturation of given phosvitins produced variations in their kinetic behavior. The results are considered in terms of a dynamic model of phosvitin iron binding sites which may constitute themselves diversely, in response to the amount of iron that is to be accommodated, or may reconstitute themselves as their molecular environment becomes altered.     

Isolate B12, which harbours a virus-like element,represents a new species of the archaebacterial genus Sulfolobus,Sulfolobus shibatae,sp. nov.

The Sulfolobus isolate B12 and its endogenous virus-like element SSV1 have provided a fruitful system for detailed analysis of certain aspects of archaebacterial molecular biology, especially those concerning gene expression. In the course of clarifying this isolate's taxonomic position, we determined DNA base composition, ability to grow autotrophically, nucleotide sequence of 16S ribosomal RNA, and level of total genomic homology to other Sulfolobus strains. Although the results generally demonstrate a similarity to S. solfataricus, DNA-DNA hybridisation and 16S rRNA sequence data indicate that isolate B12 in fact represents a distinct species.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1 B, D-3300 Braunschweig, FRG - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis