Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae

By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S. (1982) J. Bacteriol. 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library. The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively. The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor. Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter. Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions. Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily. Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters. The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose. Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA. The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.     

Both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells

Differentiation-stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL-6 are reported to be cytokines having multifaced functions including the induction of differentiation in mouse myeloid leukemia M1 cells. We here report that both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells induced by retinoic acid alone or combined with dibutyryl cAMP. From the microscopic observation as well as Northern blot analysis using cDNA probes encoding several marker proteins for differentiation of F9 cells, we concluded that D factor/LIF and IL-6 are functionally closely related in the induction of differentiation in M1 cells and in the inhibition of F9 differentiation.     

Partially folded state of the disulfide-reduced N-terminal half-molecule of ovotransferrin as a renaturation intermediate

A previous report (Hirose, M., Akuta, T., and Takahashi, N. (1989) J. Biol. Chem. 264, 16867-16872) has shown that for the efficient oxidative refolding of disulfide-reduced ovotransferrin, a preincubation under reduced conditions at a low temperature is essential. To study the renaturation pathway, the disulfide-reduced N-terminal half-molecule of ovotransferrin was analyzed by CD spectrum. The reduced protein was found to take, at low temperatures, a partially folded conformation that can be distinguished from both the native and denatured states. The folded protein was in a metastable state with delta GD value of 2.2-2.8 kcal/mol at 6 degrees C. The conformation was variable depending on temperature conditions; its stability was decreased at a lower temperature (1.0-1.2 kcal/mol at 0 degrees C). Subsequent reoxidation at 6 degrees C by oxidized glutathione led efficiently the reduced protein to the correctly renatured form having the iron-binding capacity, indicating that the partially folded state is the immediate precursor to subsequent oxidative refolding.     

Cellular and subcellular localization of progastricsin in calf fundic mucosa: colocalization with pepsinogen and prochymosin

The presence of gastricsin in bovine abomasal juice has been reported previously, but its exact site of origin has not yet been established. Specific polyclonal antibodies were used in the peroxidase-antiperoxidase method or the protein A/gold technique to label cells producing progastricsin. This immunocytolocalization was correlated with that of pepsinogen and prochymosin using specific polyclonal antibodies against those zymogens. The present study clearly established that progastricsin was located exclusively in chief, mucous neck, transitional mucous neck/chief, foveolar epithelial and surface epithelial cells of the calf fundic mucosa. Furthermore, progastricsin was found to be colocalized with pepsinogen and prochymosin in the same secretory granules of these cells. Progastricsin was not observed in parietal, gastric endocrine and undifferentiated neck cells.     

Studies on ecology and behavior of Japanese black swallowtail butterflies. 6. Nectar feeding ofPapilio helenus nicconicolens Butler andP. protenor demetrius Cramer as main pollinators of glory bower,Clerodendron trichotomum,Thunb

The butterfliesPapilio helenus andP. protenor were shown to feed mainly on the nectar of the glory bower,Clerodendron trichotomum, which was the most abundant nectar plant in summer in the study area. Both the species were found to have a proboscis longer than 24 mm corresponding to the length of the corolla tube ofC. trichotomum. Visits to the flowers by these butterflies were observed more frequently than visits by sphingid moths which had previously been believed to be the major pollinators ofC. trichotomum. The male butterflies visited trees ofC. trichotomum frequently, while visits by the females were less frequent. However, once females had visited the tree ofC. trichotomum, they remained there longer than the males. Since the flower ofC. trichotomum has long protruding sexual organs, its pollen grains were found to adhere efficiently to the bodies of butterflies, mainly the thorax, during nectar feeding. Most of the butterflies became loaded withC. trichotomum pollen, and the mean number of pollen grains per butterfly was 1,776 forP. helenus and 2,817 forP. protenor. The flowers opened at any time of day but more frequently in the morning. The nectar was secreted throughout the day. In the maturation of the protandrous flower ofC. trichotomum, the duration of the pistillate phase was about twice as long as the staminate phase. The long flowering period and the short duration of the staminate phase resulted in asynchrony of the flowering stages even within a single cyme on a tree. Such asynchrony and the abundance of attractive flowers on a tree facilitates efficient pollination by the butterflies.     

Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells

We analyzed inward Ca2+ currents in single bovine adrenal glomerulosa cell using whole-cell patch clamp techniques. Two types of voltage-gated Ca2+ channel currents were identified. One was a transient (T) type which decayed within 100 ms, characterized by a low threshold voltage (about -70 mv) similar to that seen in rat adrenal glomerulosa cells (Matsunaga, H. et al. (1987) Pflügers Arch. 408, 351-355.) Another was a long-lasting (L) type which shows a more positive threshold potential. The present results suggest that while T type Ca2+ channels may explain initial calcium influx in response to an elevation in extracellular K+, L type Ca2+ channels may allow sustained calcium influx which is necessary for sustained aldosterone secretion.     

Reduced sensitivity to catecholamine in Werner's syndrome fibroblasts

The beta-adrenergic receptor-coupled adenylate cyclase system has been investigated in normal and Werner's syndrome fibroblasts. The basal levels of cAMP in Werner and normal control cells were similar, whereas the isoproterenol-induced increase in cAMP levels was far less for Werner cells than for control cells. In the broken cell preparations isoproterenol stimulated the adenylate cyclase of only control cells, not of Werner cells, although NaF or prostaglandin E1 stimulated the enzyme of both cells to the same extent. The beta-adrenergic receptor concentrations analyzed with hydrophilic radioligand were nearly equal in Werner and in control cells. A reduction of functional activity of the beta-adrenergic receptor in Werner cells is thus suggested.     

Localization of neutrophil adherence-inhibiting factor in guinea pig platelets

The effect of constituents of guinea pig platelets on neutrophil adherence was examined. The platelet sonicate supernatant contained adherence-inhibiting activity which strongly inhibited neutrophil adherence to glass. When the platelet sonicate supernatant was treated with neuraminidase or trypsin, the adherence-inhibiting activity was significantly inhibited, suggesting that the adherence-inhibiting factor (AIF) is a glycoprotein. The subcellular fractionation experiments indicated that the AIF activity was present at about 40% in both the cytosol and granule fractions. From the Sephadex G-200 gel filtration analysis, AIF of cytosol fraction and granule fraction proved to be different molecules, with molecular masses of about 230 and 12 kDa, respectively. When platelets were stimulated with thrombin, about 20% of total AIF was released extracellularly without the release of the cytoplasmic enzyme lactate dehydrogenase. These results suggest the possibility that a biologically active substance, AIF, is released from platelets in response to stimuli and regulates neutrophil functions through interference with neutrophil adherence.     

Inhibition of cellular phosphatidylinosito, turnover by psi-tectorigenin

Psi-tectorigenin, an isoflavonoid, was isolated from a culture filtrate of actinomycetes as an inhibitor of epidermal growth factor-induced phosphatidylinositol turnover in cultured A431 cells. It inhibited phosphatidylinositol turnover with an IC₅₀ of about 1 μg/ml; thus, its inhibitory activity was 6-times stronger than that of genistein or orobol. When added to cultured A431 cells psi-tectorigenin inhibited phosphatidylinositol turnover without inhibiting epidermal growth factor receptor tyrosine protein kinase. Thus, psi-tectorigenin is a specific inhibitor of phosphatidylinositol turnover and may be a useful tool for the functional analysis of phosphatidylinositol turnover.