Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.     

Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of Cryptosporidium Oocysts and Giardia Cysts in Water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

Circular dichroism of the parallel β helical proteins pectate lyase C and E

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel β-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel β-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel β-sheets these proteins provide a unique opportunity to study the effect of parallel β-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca²⁺, derive the parallel β-helical components of the spectra, and compare these results with previous CD studies of parallel β-sheet structure. The shape and intensity of the parallel β-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel β-helical folding motif. © 1995 Wiley-Liss, Inc.     

The Late Quaternary history of Rhamnus frangula in Norway

Finds of pollen and macrofossils of Rhamnus frangula L. from Eem and Holocene are discussed and compared with the present pollen production and dispersal of the species, and its present distribution. It is presumed that there was little difference between the potential distribution area of R. frangula and its actual geographical range because of its rapid spread during Preboreal and Boreal in South Norway. A small, temporary expansion of R. frangula occurred around 5 500 BP in a mountain valley in W Norway. A simultaneous local expansion of the species has been registered in Vestvågøy, Nordland county, N Norway. In these two areas, which are outside its present distribution, the maximum of R. frangula is dated to between 5 000 and 4 800 BP. The maxima of R. frangula in profiles from other Norwegian areas are discussed. Factors such as changes in climatic condition, in–filling stages of local successions in the sedimentation basins, or human activity may explain the differences found.     

Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.     

Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.

The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.