Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.     

Studies on the structures of the normal and abnormal goat thyroglobulin genes

We have cloned the thyroglobulin (Tg) gene of normal goats and goitrous goats which have a Tg synthesis defect. At the 5'-end of the gene, we studied cosmid clones covering a region from 20 kilobases (kb) upstream from the Tg gene to 42 kb into it. Electron microscopy and restriction mapping show that this part of the gene contains 20 exons of 90-1190 bp, in total 4.9 kb of exonic information (56% of the mRNA) split by 19 introns of 150-9100 bp. The exons comprise 12% of the 5' sequences cloned. At the 3'-end, 55 kb were cloned, containing 10 kb of the gene which comprises only 3 exons of 550 bp in total. Sequence analysis of the 3'-end of the normal and abnormal Tg genes has revealed one transition mutation 3' to the reading frame in a stem-loop structure region of the last exon near the poly(A) addition site. Analysis of the promoter site and the first 5 exons has revealed only one difference between the normal and goitrous Tg genes: a Ser----Leu transition in exon 5. We also found an insertion in the fifth intron of the abnormal gene.     

Pathogenicity of some chrysosporium species isolated in France

In order to appreciate the pathogenicity of several geophilic Chrysosporium species (including Anixiopsis stercoraria, Chrysosporium keratinophilum, C. tropicum, C. pannorum, C. state of Arthroderma curreyi, C. state of A. multifidum, and C. state of A. tuberculatum), the authors have realized two series of experimental infestations. Inoculation of these fungi on the back of guinea pigs produced rare erythematous scaling lesions which spontaneously disappeared 3–5 weeks later. No real hair invasion was observed. In white mice, eight weeks after intraperitoneal inoculation, granulomas with necrotic center were observed in the peritoneal tissue with C. keratinophilum, C. tropicum, C. state of A. curreyi and C. state of A. tuberculatum. Conidia were often intact in necrotic centers and retrocultures were positive. With C. state of A. curreyi, spherical spores associated with rare budding cells were noted. The pathogenic role of these keratinophilic fungi is uncertain. However, their ability to remain viable for several weeks in skin and peritoneal tissue indicates that they could become pathogen in certain circumstances.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

The effect of the H2 partial pressure on the metabolite pattern ofLactobacillus casei,Escherichia coli andClostridium butyricum

Summary The metabolite pattern of batch cultures ofLactobacillus casei LMG 6400,Clostridium butyricum LMG 1213t1 andEscherichia coli LMG 2093 was effected only for the latter organism when the H₂ partial pressure was below 1 atmosphere: high hydrogen partial pressures increased the formate formation, low pressures gave rise to increased acetate production and higher cell yields.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

Larval and adult morphology ofCamallanus anabantis Pearse, 1933 andC. kulasirii (Yeh, 1960) (Nematoda: Camallanidae) from freshwater fishes,with notes on the validity of some related forms

Larvae and adults ofCamallanus anabantis andC. kulasirii, recovered from the West Bengali freshwater fishes,Anabas testudineus andOphicephalus punctatus, respectively, are described on the basis of detailed morphological studies under the light microscope. Larval forms collected fromA. testudineus are deemed to be of the third and fourth stages when compared with those from experimental studies of the life cycle ofC. anabantis. Moreover, the present fourth stage female larvae are similar to the females ofC. pearsei, both morphologically and metrically.C. pearsei is, therefore, believed to represent the fourth stage female larvae ofC. anabantis. Similarly, adult males and females recovered fromO. punctatus closely resembleC. kulasirii andC. fernandoi, respectively. The larval forms from this host are fourth stage and can be distinguished as males and females, but both possess a buccal capsule bearing beaded longitudinal ridges similar to that of adult males. The late fourth stage (just prior to the final moult) female larva is, however, found to possess a buccal capsule transitional between that of the adult male and female and also betweenC. kulasirii andC. fernandoi. C. fernandoi is, therefore, presumed to represent the females ofC. kulasirii. However,C. pearsei andC. fernandoi are regarded, for the present, asspecies inquirendae.     

Mitochondrial genome in animal cells. Structure, organization, and evolution

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.     

VIP and related peptides induce rapid homologous desensitization in the human lymphoma SUP T1 cell line

Incubation of human SUP T1 lymphoblasts with VIP, helodermin and related peptides induced homologous desensitization within 5 min as indicated by: 1) a secondary decrease in cellular cyclic AMP levels, even in the presence of phosphodiesterase inhibitors, 2) a reduced capacity of cells to bind [125I]helodermin, 3) decreased helodermin stimulation of adenylate cyclase activity in membranes, and 4) unaffected NaF- and Gpp[NH]p-stimulated adenylate cyclase activities. The desensitizing ability of all peptides correlated with their efficacy to occupy cell receptors, except for [D-Phe2]VIP, a partial VIP agonist with low intrinsic activity, that did not desensitize.     

Induction of NADP-dependent malic dehydrogenase activity in brain of singi fish, heteropneustes fossilis (bloch), by 3,5,3′-triiodothyronine

For elucidation of thyroid hormone-induced responsiveness of fish brain, various doses (0.012, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 and 4 μg/g) of triiodothyronine (T₃) were injected in Singi fish, Heteropneustes fossilis (Bloch), for 3 consecutive days and the changes in cytosolic NADP-dependent malic enzyme (ME, EC 1.1.1.40) activity in whole brain tissue were determined. Compared to the control, the ME activity increased with lower doses (0.012, 0.025 and 0.05 μg/g) and decreased with higher doses (1, 2 and 4 μg/g) of T₃, showing a biphasic nature of thyroid hormone action. The enzyme activity remained unaltered with 0.1, 0.25 and 0.5 μg of T₃/g in comparison to the control. Immersion of the fishes in cycloheximide-containing medium (0.5 mg/l) inhibited the T₃ (0.025 μg/g)-induced rise in ME activity. On the other hand, the NAD-dependent cytosolic malate dehydrogenase (EC 1.1.1.37) activity and the total protein content of brain cytosol remained unaltered with all doses of T₃ used. The thyroid hormone specificity of cytosolic NADP-dependent malic enzyme in fish brain is thus documented.     

Gene replacement in Dictyostelium: generation of myosin null mutants.

The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5' coding sequence and 0.9 kb of 3' coding sequence or 1.5 kb of 5' flanking sequence and 1.1 kb of 3' flanking sequence. Myosin null mutants (mhcA- cells) were obtained after transformation with either of these plasmids. The mhcA- cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.