Protein kinase from Mucor rouxii

Summary Cyclic AMP binding to Mucor rouxii protein kinase holoenzyme and free regulatory subunit was measured by the classical membrane filtration technique and by equilibrium dialysis. The results obtained demonstrate that the filtration method can be used without loss of any cyclic AMP binding site. Both methods unambiguously demonstrate that the number of molecules of cyclic AMP bound to the holoenzyme are half of those bound to the regulatory subunit. This result suggests that unshielding of new cyclic AMP binding sites occurs upon dissociation of the ternary complex holoenzyme-cyclic AMP.     

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.     

Structure and function of the mitochondrial bc1 complex. A mutational analysis of the yeast Rieske iron-sulfur protein

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to a single complementation group (G12) have been determined to have lesions in the iron-sulfur protein (Rieske protein) of ubiquinol: cytochrome c reductase. Mutants capable of expressing the protein were chosen for further studies. The genes from 13 independent isolates were cloned and their mutations sequenced. Twelve mutations were ascertained to cause single amino acid substitutions in the carboxyl-terminal regions of the protein between residues 127 and 173. This region is proposed to be part of the catalytic domain with the ligands responsible for co-ordinating the two irons of the 2Fe-2S cluster. Based on the catalytic properties of the ubiquinol: cytochrome c reductase complex and the electron paramagnetic resonance (e.p.r.) signals of the iron-sulfur protein, the mutants describe two different phenotypes. A subset of mutants have no detectable iron-sulfur cluster and are completely deficient in ubiquinol: cytochrome c reductase activity. These strains identify mutations in residues considered to be essential for binding of the iron or for maintaining a proper tertiary structure of the catalytic domain. A second group of mutants have reduced levels of enzymatic activity and exhibit e.p.r. spectra characteristic of the Rieske iron-sulfur cluster. The mutations in the latter strains have been ascribed to residues that influence the redox properties of the cluster by distorting the iron-binding pocket. A secondary and tertiary structure model is presented of the carboxyl-terminal 65 residues constituting the catalytic domain of the iron-sulfur protein. It is postulated that the two irons of the cluster are co-ordinated by three cysteine and a single histidine residue located in a loop structure. The catalytic domain also contains two short alpha-helices and three beta-strands that form a partial beta-barrel. Most of the hydrophilic amino acids are present in turns that map to one pole of the domain. When viewed in the context of the model, mutations that abolish the iron-sulfur cluster are mostly in residues defining the boundaries of the alpha-helices and beta-strands. The notable exception is a cysteine residue that has been assigned to the loop with the iron ligands. This cysteine residue is proposed to co-ordinate one iron of the cluster. Mutations that reduce ubiquinol: cytochrome c reductase activity and alter the redox potential of the cluster occur in residues located in the loop that contains the ligands of the cluster.     

Fine structure of the excretory system of the deep posterior (Ebner's) salivary glands of the human tongue

Human deep posterior lingual glands (von Ebner's glands) are located beneath the circumvallate papillae. They are formed by tubuloalveolar adenomeres, intercalated ducts and excretory ducts coming together in the main excretory duct. The tubuloalveolar cells, pyramid-shaped, show large and dense secretory granules (clear cored) throughout the cytoplasm, rare basal folds and packed cisternae of rough endoplasmic reticulum (RER) at the basal pole. The columnar cells of the intercalated ducts are arranged in a monolayer. They are characterized by dense, clear-core secretory granules (mostly in the apical cytoplasm), a basal nucleus, well-developed RER and Golgi apparatus, and thin filaments distributed in supra- and perinuclear cytoplasm. Striated ducts are absent. Excretory ducts, coming together in the main duct, are lined by a bistratified epithelium. The inner layer consists of columnar cells showing bundles of tonofilaments with scarce secretory activity. The outer layer is composed of basal cells lying on the basal lamina. The main excretory duct, which opens at the bottom of the vallum, shows a stratified epithelium. The outer side is composed of 2-3 layers of malpighian cells lying on the basal lamina. The inner side consists of a single layer of cuboidal-columnar cells with dense apical granules and well-developed organelles synthesizing and condensing secretions. These cells interpolate with goblet cells, rare mitochondria-rich cells, ciliated cells and numerous small globous cells showing a clear matrix and lacking secretory granules. The cilia show a 9 + 2 microtubular structure with basal bodies provided with striated rootlets. Myoepithelial cells surround with their processes the basal portions of the secretory cells and the intercalated ducts. The conclusions concern some comparative aspects and some hypothesis on the functional role of goblet cells, ciliated cells and epithelial cells lining the different ducts, also in relation to the final secretory product.     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β₄subunit is present in association with α₆chain on both normal and transformed epithelial cells. Recently α₆β₄heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β₄subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α₆β₄has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β₁(TGF-β₁) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α₆β₄expression is modulated by TGF-β₁, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β₁cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β₄subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β₁.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.