Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin.

The ability of many viruses to replicate in host cells depends on cleavage of certain viral glycoproteins, including hemagglutinin (HA). By generating site-specific mutant HAs of two highly virulent influenza viruses, we established that the relationship between carbohydrate in the stalk and the length of the connecting peptide is a critical determinant of cleavability. HAs that lacked an oligosaccharide side chain in the stalk were cleaved regardless of the number of basic amino acids at the cleavage site, whereas those with the oligosaccharide side chain resisted cleavage unless additional basic amino acids were inserted. This finding suggests that the oligosaccharide side chain interferes with HA cleavage if the number of basic amino acids at the cleavage site is not adequate to nullify this effect. Similar interplay could influence cleavage of other viral glycoproteins, such as those of human and simian immunodeficiency viruses and paramyxoviruses.     

Protein Kinases Are Involved in Prolonged Acetylcholine Release from Rat Hippocampus Induced by Thyrotropin-Releasing Hormone Analogue NS-3

Abstract: The effects of various protein kinase inhibitors on acetylcholine release from the rat hippocampus induced by the local application of NS-3 (montirelin hydrate, CG-3703), a thyrotropin-releasing hormone analogue, into the medial septum-diagonal band were examined using in vivo microdialysis. Perfusion of NS-3 (1 µ M ) into the medial septum-diagonal band for 20 min produced a pronounced and prolonged increase in the hippocampal acetylcholine efflux. Pretreatment of the medial septum-diagonal band with either K-252a, a nonselective protein kinase inhibitor, or selective protein kinase A inhibitor H-89 almost completely blocked the acetylcholine efflux evoked by NS-3, and selective protein kinase C inhibitor calphostin C inhibited the action of NS-3. On the other hand, NS-3 (0.1–10 µ M ) or TRH (1–100 µ M ) increased the cyclic AMP efflux from the medial septum-diagonal band in a concentration-dependent manner, as measured by microdialysis. These findings suggest that protein kinases A and C in the neurons of the medial septum-diagonal band are involved in the mechanism of the prolonged stimulation of acetylcholine release from the hippocampus induced by thyrotropin-releasing hormone and its analogue, NS-3.     

Interannual fluctuations in recruitment and growth of the sardine,Sardinops melanostictus,in the sea of Japan and adjacent waters

Recruitment and growth of the sardineSardinops melanostictus fluctuated markedly in the Sea of Japan and adjacent waters between 1978 and 1993. Stock size was calculated using Virtual Population Analysis and average body length in each age class was determined by the number of annual rings on the scales. There is an inverse correlation between average water temperature at a depth of 50 m in the coastal area of the mainland of Japan in winter (January to March) and recruitmentR defined as the number of individuals at 1 year old. There is also an inverse correlation between spawning stock sizeE and reproductive success in (R/E). A multiple regression model using spawning stock size and water temperature in winter as independent variables can explain 73% of variance in reproductive success. It suggests that both density-dependent and density-independent factors perform important roles determining reproductive success. There is an inverse correlation between body length and stock size and this suggests that there is a density-dependent effect on the growth of the sardine.     

A morphological study and hybridization analysis of Caloglossa leprieurii (Ceramiales, Rhodophyta) from Japan, Singapore and Australia

Morphological and hybridization experiments were performed on Caloglossa leprieurii (Montagne) J. Agardh collected from Japan, Singapore and Australia in order to evaluate taxonomic characters of this species. Within C. leprieurii at least four mating groups were recognized from the Indo-Pacific region. These mating groups could be characterized by the blade width at the inter-node and the cell-row numbers on the opposite side derived from the first axial cell at the main axis, though these properties showed a certain variability even in the same plant under both field and culture conditions. The phylogenetic relationship and geographic distribution of the four mating groups are discussed.     

Alteration of Substrate Specificity and Optimum pH of Sarcosine Oxidase by Random and Site-Directed Mutagenesis

The sarcosine oxidase gene was randomly mutated, and F103V, which showed altered specificity, was screened with the plate assay. Five different mutants at position 103 were also constructed. Each mutant exhibited a rather striking change in substrate specificity, except for F103W. The optimum pH was also shifted, depending on the substrate.     

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.