High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris

Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.     

Effects of vomeronasal organ removal on the sperm motility in male mice

Odors play important roles in the communication of house mice. They release behaviors and prime changes of the physiological conditions of other individuals. In our previous study, we showed that sperm motility was lowered in the subordinate mice comparing with dominant mice. Our hypothesis is that the lowered sperm motility was due to some primer effects by odor substances derived from dominant mice. To test the hypothesis, we destroyed the vomeronasal organ (VNO) of male mice (VNX male) at 5 weeks of age and paired them with intact male mice (Experimental Group). As control group males, intact male mice were kept in pairs (Control Group). At 15 weeks of age, the sperm motility and weights of reproductive organs, and social dominance was analyzed. The subordinate VNX males were found to have high sperm motility comparable to the dominant males. It was suggested that there is male-to-male primer effects, mediated by VNO, that suppress sperm motility of the subordinate mice.     

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.     

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.     

ATP hydrolysis coupled to microtubule sliding in sea-urchin sperm flagella

Using sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1,000 microM) at 0-20 degrees C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes.     

Studies on Nicotinoids as Insecticides

Synthesis of two nicotine isomers, 5′-methylnornicotine and 6-methylnornicotine is described. The latter shows slightly insecticidal action. Myosmine type bases are easily reduced to nornicotine type with sodium borohydride.     

Excitatory and inhibitory actions of norepinephrine on the Ba2+ current through L-type Ca2+ channels of smooth muscle cells of guinea-pig vas deferens

The effect of norepinephrine (NE) was examined on the whole-cell Ba²⁺ current through L-type Ca²⁺ channels of freshly isolated smooth muscle cells of guinea-pig vas deferens. The magnitude of maximum Ba²⁺ current [IBa(max)] varied in different cells, although the capacitance of the cell membrane was similar (∼50 pF). Application of dbcAMP augmented IBa(max) by 37%, which was canceled by Rp-cAMPs, while PMA decreased the current by 32%, which was canceled by staurosporine. NE increased IBa(max) of the cells which originally showed relatively small IBa(max), and decreased the current of the cells which showed larger IBa(max). In the presence of phentolamine, NE increased IBa(max), and this effect was remarkable in cells showed smaller IBa(max). In the presence of propranolol, NE decreased IBa(max). The excitatory β-adrenoceptor activation was canceled by Rp-cAMPs, and the inhibitory α-adrenoceptor effect was canceled by staurosporine. It is suggested that NE shows dual (excitatory and inhibitory) actions on the L-type Ca²⁺ channels of smooth muscle of guinea-pig vas deferens. The excitatory β-adrenoceptor action mediated through cAMP/PKA is predominant in cells with lower density of the Ca²⁺ channels, while inhibitory α-adrenoceptor action mediated through PKC is predominant in cells with higher channel density. © 1996 Wiley-Liss, Inc.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.