Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

PHOTOACCLIMATION IN THE TROPICAL CORALLINE ALGA HYDROLITHON ONKODES (RHODOPHYTA, CORALLINACEAE) FROM A FRENCH POLYNESIAN REEF

Pigment concentration, in vivo absorption, and photosynthetic parameters of the coralline alga Hydrolithon onkodes (Heydrich) Penrose and Woelkerling were compared among samples from a lagoon and from a reef crest of Tahiti Island. Four groups of specimens were considered, differing in their natural exposure to PAR. For specimens collected from the lagoon, the tissues from low-light samples had significantly higher pigment concentration, particularly chl a and phycobilins, compared with the high-light exposed plants that contained more total carotenoids. The in vivo absorption spectra normalized to chl a (called a* values) also revealed differences. The low-light samples had a reduced absorption capacity and a well-marked phycobilin absorption signature, whereas sunlit samples showed a greater absorption at wavelengths absorbed mainly by chl a and carotenoids. The decrease of a* when pigment concentration increased is interpreted as a consequence of the pigment packaging. Significantly lower α (chlorophyll basis) and higher E_k values were found in the shaded plants. The values of P _max for the four groups of specimens were not significantly different. The samples showed various degrees of photoinhibition depending on the light exposure during growth, and this effect was more pronounced in the shaded plants. The specimens from the reef crest deviate from the general model presented for the lagoon samples and show a mix of sun- and shade-exposed characteristics. We have shown that the coralline alga H. onkodes responds to its light environment, probably by acclimation rather than ecotypic genetic variation, by adjusting its physiology, but some morphological differences are also involved. Photoacclimation can explain partly the wide distribution of this species over the reef ecosystem and its major contribution to the building of the reef.     

Target size analysis of neurotensin receptors

The neurotensin receptors from rat brain synaptosomal membranes differed in subunit structure from those in rat gastric fundus smooth muscle plasma membranes when studied following photoaffinity labelling or after exposure to cross-linking reagents. However, these two receptors were similar in their recognition properties. In this study we compared the target size of the two receptors by radiation inactivation and observed that the receptors in both tissues had similar target sizes (mean values 103,000 and 108,000 daltons). This suggests that the differences in size observed in biochemical studies may reflect changes occurring during the isolation procedures, or, on the other hand, there might be inherent difference in the subunit structure of these receptors.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Specific Antisera Against the Catecholamines: L-3,4-Dihydroxyphenylalanine, Dopamine, Noradrenaline, and Octopamine Tested by an Enzyme-Linked Immunosorbent Assay

Antisera were raised against L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catecholamine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the nonconjugated compound was poorly recognized. The nonreduced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.     

Cortical activity in vertebrate eggs. I: The activation waves

We present a physical model for the propagation of chemical and mechanical waves on the surface of vertebrate eggs. As a first step we analyzed the propagation of the calcium wave observed to sweep over the surface of the Medaka egg (Gilkey et al., 1978). It has been assumed that this wave is driven by a mechanism of calcium-stimulated-calcium-release. By formulating this hypothesis mathematically we can use the observed wavefront data to obtain a map of cortical reactivity. This map indicates a gradient of reactivity along the egg: highest in the animal hemisphere and tapering off towards the vegetal hemisphere. The cortex of Xenopus eggs is also capable of propagating a calcium wave (Busa & Nuccitelli, 1985). At about the same time a wave of expansion followed by a wave of contraction sweeps across the egg surface (Takeichi et al., 1984). We have proposed a mechanism for this wave pair based on the physical chemistry of actomyosin gels. The calcium wave activates solation factors which sever some of the actin chains which leads to an osmotic swelling of the gel. Calcium also activates the contractile machinery of the actomyosin system which causes the gel to contract. The contraction lags the swelling because of the nature of the kinetics: solation and swelling is a more rapid process than contraction. By writing the equations for gel expansion and contraction we can mimic the mechanical and chemical wave propagation by a computer simulation. If the model is correct this provides a method for using the waves as a diagnostic of the mechanochemical properties of the egg cortex.     

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

Summary A library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.     

Cloning of the cysB gene of Erwinia carotovora subsp. carotovora, and the identification of its product

Summary The suicide vector pJB4JI was used to generate a range of Tn5-induced mutants of Erwinia carotovora subsp. carotovora (Ecc). One mutant, HC500, was a cysteine auxotroph which had a non-pectolytic, non-cellulolytic, non-proteolytic phenotype when grown under sulphate-limitation. The cysteine lesion of HC500 was shown to be analogous to the cysB mutation of Escherichia coli. The Ecc-cysB ⁺ gene product was identified as a protein of M_r 36000.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.