Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

Structure-activity relationships in human interleukin-1 alpha: identification of key residues for expression of biological activities.

To identify the sites important for the different biological activities of human interleukin-1 alpha (hIL-1 alpha), 56 single-amino acid-substituted mutants of hIL-1 alpha were produced in Escherichia coli using site-directed mutagenesis, and were examined for their biological activities such as mouse lymphocyte activating factor activity (LAF activity), cytostatic activity against human melanoma cells A-375 (A375 activity) and prostaglandin E2 (PGE2) inducing activity in human osteosarcoma cells MG-63 (PEI activity). Two amino acid residues, Asp26 and Asp151, were found to be important for these activities. The replacement of Asp26 by Val caused a decrease in LAF and PEI activities by one or two orders of magnitude and a slight decrease in A375 activity. The Tyr or Phe substitution for Asp151 caused decreases in LAF and A375 activities by one or two orders of magnitude and complete loss of PEI activity. The change from Asp151 to Lys or Arg resulted in marked decrease in LAF activity and complete loss of A375 and PEI activities. Since Asp26 and Asp151 are close to each other in the three-dimensional structure, the region involving these amino acids seems to be important for the biological activities of hIL-1 alpha.     

Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.     

Host discrimination modulates brood guarding behaviour and the adaptive superparasitism in the parasitoid wasp Trissolcus semistriatus (Hymenoptera: Scelionidae)

Because hosts utilized by parasitoids are vulnerable to further oviposition by conspecifics, host guarding benefits female wasps. The present study aims to test whether female adults regulate brood guarding behaviour by host discrimination in a solitary parasitoid Trissolcus semistriatus by presenting an intact or parasitized host egg mass to a female adult. Virgin females without oviposition experience have host discrimination ability, which enables them to adjust the number of eggs laid in the hosts. Mating experience increases superparasitism by female adults, whereas mated females achieve a higher discrimination ability as a result of oviposition experience and show a lower superparasitism rate. As expected, females exhibit brood guard after parasitizing an intact host egg mass, whereas those females visiting a previously parasitized host egg mass, do not. Because the survival of eggs in superparasitized hosts is relatively low, regulating brood guarding behaviour by host discrimination is adaptive for female wasps.     

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.     

Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Movement of the actin filament bundle in Mytilus sperm: a new mechanism is proposed

An actin filament bundle approximately 2-5 microns in length is present in the sperm of the blue mussel, Mytilus. In unfired sperm this bundle extends from the midpiece through a canal in the center of the nucleus to terminate on the membrane limiting the inside of the cone-shaped acrosomal vacuole. The bundle is composed of 45-65 actin filaments which are hexagonally packed and regularly cross-bridged together to form an actin paracrystal so well ordered that it has six nearly equal faces. Upon induction of the acrosomal reaction, a needle-like process is formed in a few seconds. Within this process is the actin filament bundle which appears unchanged in filament number and packing as determined by optical diffraction methods. Using fluorescein-conjugated phalloidin we were able to establish that the bundle does not change length but instead is projected anteriorly out of the midpiece and nuclear canal like an arrow. Existing mechanisms to explain this extension cannot apply. Specifically, the bundle does not increase in length (no polymerization), does not change its organization (no change in actin twist), does not change filament number (no filament sliding), and cannot move by myosin (wrong polarity). Thus we are forced to look elsewhere for a mechanism and have postulated that at least a component of this movement, or cell elongation, is the interaction of the actin filament bundle with the plasma membrane.     

Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.