The involucrin genes of the mouse and the rat: study of their shared repeats

The involucrin genes of the mouse (Mus musculus) and the rat (Rattus norvegicus) have been cloned and sequenced. The coding region of each gene contains, at site P, a segment of repeats homologous to that of other nonanthropoid mammals. In contrast to the repeats of species belonging to different mammalian orders, many individual repeats of the mouse and the rat can be matched. Both before and after the divergence of the two species, these repeats have been the site of systematic alterations in nucleotide sequence. One of the alterations is the correction of nucleotides of one repeat by those of another. Corrected nucleotides may be closely linked to flanking nucleotides that are uncorrected; the systematic correction process therefore appears to be due to gene conversion. There is a stretch of 18 reiterated CAGs in the segment of repeats of the Mus gene; most of these reiterations were introduced recently, supporting the idea that the gene was generated originally from poly CAG. An antiserum to a synthetic peptide encoded by the segment of repeats of the Mus gene reveals differentiation- specific expression of the gene in the epidermis.      

Camphor-grown Pseudomonas putida,a multifunctional biocatalyst for undertaking Baeyer-Villiger monooxygenase-dependent biotransformations

Summary Both NADH- and NADPH-dependent Baeyer-Villiger monooxygenase activities with potential uses as biocatalysts for biotransformations are present to different extents throughout the growth of Pseudomonas putida NCIMB 10007 on (+)-camphor. The two activities give a different pattern of stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Evidence that beta-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable beta-d-Glycosidase

A survey of Sulfolobus isolates showed all to contain thermostable enzyme activities hydrolyzing various glycosidic compounds. Of those not previously reported, the beta-glucosidase activity of Sulfolobus solfataricus isolate P2 was chosen for further study and found to have the same kinetics of inactivation, apparent molecular weight, and many (though not all) other biochemical properties of the beta-galactosidase also present in this strain. The two activities copurified approximately 850-fold to apparent homogeneity. The enzyme, whose subunit M(r) was estimated to be 60,000 to 65,000 by gel permeation chromatography of the active enzyme and 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured form, hydrolyzed a variety of low-molecular-weight, beta-linked glycosides and could account for most of the corresponding activities found in crude extract. Kinetic analyses indicated that chromogenic beta-d-galactosides and beta-d-glucosides are hydrolyzed at a common active site and that beta-glucosides and beta-fucosides represent the preferred substrates. The liberation of aglycone from aryl beta-d-glucosides was stimulated by alcohols in a manner suggesting specific interaction between alcohol and enzyme.     

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

The transverse location normal to the bilayer surface of a series of n-(9-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 greater than or equal to 3 greater than 6 greater than or equal to 7 greater than 9 greater than 12 greater than 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.     

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.     

Assessment of the lymphocyte response to silicone

The biocompatibility of silicone is once again the focus of increased interest. Long considered inert, silicone has now been reported to be responsible for macrophage inhibition in rats and to possibly cause adjuvant disease in humans, and the related compound silica has elicited an antibody response in mice. The present study evaluates lymphocytic response to silicone as expressed by the demonstration of immunologic memory, or changes in specific lymphocyte subpopulations. Thirty-six female Lewis rats (250 gm body weight) were used as test animals. Group 1 (n = 12) was injected subcutaneously with 2.5 ml Freund's Complete Adjuvant (FCA) alone. Group 2 (n = 12) was injected with 2.5 ml FCA sonicated with silicone gel. Group 3 (n = 6) was injected with 2.5 ml FCA, and at 4 weeks, gel-filled silicone implants were placed subcutaneously. Group 4 (n = 6) was injected with 2.5 ml FCA sonicated with silicone gel, and gel-filled silicone implants were placed at 4 weeks. An additional group of six rats (group 5) served as control for the experimental animals, and a group of four rats (group 6) served as naive control. Groups 1 and 2 were sacrificed at 4 weeks, and splenic lymphocytes were obtained for lymphocyte transformation assays performed against silicone. Assays also were run with the addition of the known mitogens Con A, PHA, LPS, and pokeweed. Cytofluorographic analysis of pan-T, T-helper, T-suppressor, and B-cell populations was performed. Groups 3, 4, 5, and 6 were harvested at 8 months, and splenic lymphocytes were subjected to lymphocyte transformation assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorus nuclear magnetic resonance of diverse phosvitin species

1. High resolution 31P nuclear magnetic resonance (NMR) spectra, with and without proton decoupling, of the principal egg phosphoproteins--phosvitins--of a bird (Gallus gallus), an amphibian (Xenopus laevis) and a fish (Salmo gairdneri) were obtained. 2. The spectra were evaluated with special reference to available amino acid sequences and the major NMR resonance in all three spectra was assigned to phosphoserine clusters. 3. The resolution of numerous additional phosphorus resonances provides the basis for further investigation of the particular molecular environments of phosvitin-bound phosphoryl groups and their involvement in the diverse binding modes for metal complex formation by phosvitins.     

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.     

Interaction of respiration and luminescence in a common marine bacterium

Investigators have proposed for some time that bacterial luciferase forms a shunt around the pathway of respiratory electron transport. Certain physiologic evidence for coupling between luminescence and respiration has supported such a view. In this study, Vibrio harveyi cells were monitored for luminescent responses to artificial manipulation of respiratory electron flow. The effects of cyanide under aerobic and anaerobic conditions confirmed that luminescence and respiration compete for oxygen. The effects of an uncoupler of oxidative phosphorylation indicated that luminescence and respiration compete for a common reductant. Treatment with uncoupler also induced aldehyde deficiency in vivo.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Tris tris(hydroxymethyl) aminomethane     

Electronic sorting of granulocyte precursor cells from stimulated bone marrow.

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.