Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence

Cofilin is a widely distributed, pH-sensitive, actin-modulating protein with an apparent molecular mass of 21 kDa, which forms intranuclear and/or cytoplasmic actin/cofilin rods in cultured fibroblastic cells under specific conditions. In this study, a cDNA library from porcine brain mRNA was constructed, and full-length brain cofilin cDNA clones were isolated by screening with oligonucleotide probes. The deduced amino acid sequence of cofilin is 166 residues long and contains a sequence of Lys-Lys-Arg-Lys-Lys which is very similar to the nuclear transport signal sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) of SV40 large T antigen. The sequence may act as a signal capable of inducing nuclear accumulation of cofilin in cells exposed to heat shock or dimethyl sulfoxide. The cofilin sequence contains a hexapeptide (Asp-Ala-Ile-Lys-Lys-Lys) identical to the amino-terminal sequence (residues 2-7) of muscle and nonmuscle tropomyosin. Cofilin also has in the carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. Furthermore, the overall amino acid sequence of cofilin shows weak homology with the rod portion of myosin and suggests a high alpha-helical content.     

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

A preliminary note on the intestinal parasites of wild chimpanzees in the Mahale Mountains,Tanzania

Feces of wild chimpanzees in the Mahale Mountains, Tanzania, were inspected for intestinal parasites under a compound microscope. Eggs or larvae ofOesophagostomum, Strongyloides, Trichuris, Prosthenorchis, andBertiella were found. Intestinal nematodes significantly increased in the mid-rainy season. This finding supports (or, at least, is not in conflict with) the hypothesis thatAspilia leaves which are occasionally swallowed by chimpanzees may function as a vermicide, since ingestion of such leaves also increases significantly in the mid-rainy season.     

Changes in mouse brain monoamine oxidase activity in the first, second and fourth generations after manganese administration

The present study was conducted to explore whether or not manganese effect on brain monoamine oxidase (EC 1.4.3.4) is subject to hereditary genetic amplification. Mice of both sexes were given manganese through four generations, and the enzyme activity was measured in the cerebral cortex, cerebellum, hypothalamus and hippocampus of each of the generations except for the third, whose activity we were not in a position to measure. Intrinsic enzyme activity was highest in the cerebellum, and was followed by those in the cerebral cortex and hypothalamus. The activity in the hippocampus was the lowest. Manganese administration greatly stimulated the activity in the cerebellum. However, as generation succeeded, the level of susceptibility to manganese gradually declined. Manganese concentration in pooled suborgan fractions proved to be, in every case, higher in the cerebral cortex, cerebellum and hippocampus and lower in the hypothalamus. No indication was found that the manganese effect is genetically inherited.     

Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.     

Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization

It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.     

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.