Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K_m and V_max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

Photodegradative surfactants: photolyses of p-alkylbenzyltrimethylammonium and alkylbenzyldimethylammonium halides in aqueous solution.

A series of benzyl-containing ammonium salts, p-alkylbenzyltrimethylammonium halides (C(n)BA: 1a-c) and alkylbenzyldimethylammonium halides (C(n)AB: 2a-d), have been prepared and their photodegradabilities in aqueous solutions have been compared. The photolytic decomposition proceeded by heterolytic and homolytic cleavages of the benzyl-nitrogen bond. The conversion yields were almost the same for all surfactants, whereas the product yields were slightly dependent on the alkyl-chain length. After irradiation, C(8)BA (1b) and C(12)BA (1c) were converted to non-surfactants, whereas C(12)AB (2c) and C(16)AB (2d) still remained surface-active. Their solution properties were concomitantly changed.     

Imperfect preference for singly parasitized hosts over doubly parasitized hosts in the semisolitary parasitoid Echthrodelphax fairchildii: implications for profitable self-superparasitism

Self‐superparasitism can be profitable (i.e., a fitness gain) when conspecific female adult parasitoids prefer singly parasitized hosts over doubly parasitized hosts. This preference is expected to evolve when the value (i.e., the fitness gain from oviposition) of doubly parasitized hosts is lower than that of singly parasitized hosts. We examined whether such a preference is found in the small brown planthopper, Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), and its semisolitary infanticidal parasitoid Echthrodelphax fairchildii Perkins (Hymenoptera: Dryinidae). We compared the preferences and host values between each of four pairs of double and triple parasitism, each of which had the same time interval between the first and last oviposition bouts. Ovipositions on doubly and singly parasitized hosts occurred with similar frequencies in each of the four pairs, even though the doubly parasitized hosts were of lower value than the singly parasitized hosts. However, the value of doubly parasitized hosts with the first and second parasitoid offspring on the same side of the host was higher than that of hosts with the two offspring on different sides, and the value of the former did not differ significantly from that of singly parasitized hosts. The preferences between singly and doubly parasitized hosts with the two offspring on the same or different sides were as expected from differences in their values, except for one pair of double and triple parasitisms. This exception is considered attributable to an imperfect ability to evaluate hosts.     

Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6′-sialyl(N-acetyllactosamine). These properties correlated with the substitution 186S→I in HA1. The major inhibitory activity in rabbit serum was found to be a β inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6′-sialyl(N-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine α₂-macroglobulin because of the mutation 145N→K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine α₂-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.Influenza A viruses possess two envelope glycoproteins:hemagglutinin (HA) and neuraminidase (NA). HA binds to cell surface sialylglycoconjugates and mediates virus attachment to target cells (19, 30). NA cleaves the α-glycosidic linkage between sialic acid and an adjacent sugar residue, facilitating elution of virus progeny from infected cells and preventing self-aggregation of the virus (1, 13). Natural sialylglycoconjugates are structurally diverse (37, 40), and the preferential recognition of distinct sialyloligosaccharides by HA and NA correlates with the host species from which the viruses are isolated (reviewed in references 19, 30, and 38; see also references 4, 6, 7, 11, and 28).The receptor-binding activity of influenza viruses can be inhibited by certain molecules present in the sera and fluid secretions of animals (see references 14 and 21 for reviews). These inhibitors are classified as α, β, and γ types based on their thermal stability, virus-neutralizing activity, and sensitivity to inactivation by NA and periodate treatments. The β inhibitors are thermolabile mannose-binding lectins that interact with the oligosaccharide moieties on viral glycoproteins. They neutralize virus by steric hindrance of HA and by activation of the complement-dependent pathway (2, 3). By contrast, the α and γ inhibitors are heat-stable sialylated glycoproteins that mimic the structure of the cellular receptors of influenza viruses and competitively block the receptor-binding sites of HA. Influenza viruses are neutralized by γ inhibitors but not by α inhibitors, which are considered to be sensitive to viral NA. However, the distinction between α and γ inhibitors is strain dependent and rather arbitrary, as described by Gottschalk et al. (14). Although inhibitors in serum or other body fluids are believed to influence the selection of influenza virus receptor variants in natural hosts, no direct experimental support for this hypothesis has been presented.A potent γ inhibitor of H2 and H3 human influenza viruses, equine α₂-macroglobulin (EM), contains a Neu4,5Ac₂2-6Gal moiety that is insensitive to viral NA and thus resists inactivation by this enzyme (16, 24, 31). Cultivation of human H3 influenza viruses in the presence of horse serum results in the selection of variants that have a decreased affinity for the Neu5Ac2-6Gal-specific receptors due to a single amino acid substitution (226L→Q) in their HA (32, 33). One of these mutants (X31/HS strain) does not bind the Neu4,5Ac₂ (4-O-acetylated sialic acid) species (25). Therefore, there are at least two mechanisms by which a virus can become resistant to the horse serum inhibitor: a change in the recognition of the type of Sia-Gal linkage, and a change in the recognition of the 4-O-acetylated sialic acid. The relative contributions of these mechanisms to the resistant phenotype are yet to be defined.We have previously shown that horse, pig, and rabbit sera all contain distinct heat-resistant inhibitors of the H3N2 human influenza virus A/Los Angeles/2/87 (LA/87), because variants resistant to these sera possess unique mutations in their HA receptor-binding regions (34). The major inhibitor in pig serum was later identified as α₂-macroglobulin that contains predominantly 2-6 linkage between sialic acid and galactose (35). Gimsa et al. (12) recently showed that pig serum-resistant human and swine strains exhibit decreased affinity for human erythrocytes that had been modified to contain terminal Neu5Ac2-6Gal residues. However, the nature of the rabbit serum inhibitor and the mechanisms of influenza virus resistance to each serum inhibitor remain unknown.To understand the molecular mechanisms by which influenza viruses become resistant to horse, pig, and rabbit serum inhibitors, we compared the receptor-binding characteristics of LA/87 and its serum-resistant variants and analyzed these data in relation to the known amino acid substitutions in the HA of the mutants. We then analyzed the receptor-binding properties of viruses isolated during an equine influenza outbreak that was caused by an avian virus, in order to evaluate the influence of natural inhibitors on the evolution of virus in a new host.     

Transparent-Scaled Variant of the Rosy Bitterling, Rhodeus ocellatus ocellatus (Teleostei: Cyprinidae)

Transparent-scaled variant (TSV) of the rosy bitterling Rhodeus ocellatus ocellatus (Kner) was observed on both morphology and heredity. Compared with the normal-scaled type (NST), TSV is characterized by the blackish coloration in both eyes and peritoneum, and the luminescent one over the whole body. Histologically, the density of guanophores containing reflecting platelets was conspicuously low, especially in scale, iris, choroid and peritoneum, while the increase in the number of goblet cells (mucous cells) was recognized all over the dermal/epidermal regions. The heredity of TSV was recessive and supposed to be controlled by a single pair of genes unrelated to sex, judging from the result of crossbreeding experiments between NST and TSV. In growth and reproduction, no difference was seen between these two phenotypes. Transparent-scaled variant of the rosy bitterling can be competent for a genetic marker in experimental and developmental biology.     

Specific tandem GG to TT base substitutions induced by acetaldehyde are due to intra-strand crosslinks between adjacent guanine bases.

Acetaldehyde is present in tobacco smoke and automotive exhaust gases, is produced by the oxidation of ethanol, and causes respiratory organ cancers in animals. We show both the types and spectra of acetaldehyde-induced mutations in supF genes in double- and single-stranded shuttle vector plasmids replicated in human cells. Of the 101 mutants obtained from the double-stranded plasmids, 63% had tandem base substitutions, of which the predominant type is GG to TT transversions. Of the 44 mutants obtained from the single-stranded plasmids, 39% had tandem mutations that are of a different type than the double-stranded ones. The GG to TT tandem substitutions could arise from intra-strand crosslinks. Our data indicate that acetaldehyde forms intra- as well as inter-strand crosslinks between adjacent two-guanine bases. Based upon the following observations: XP-A protein binds to acetaldehyde-treated DNA, DNA excision repair-deficient xeroderma pigmentosum (XP) cells were more sensitive to acetaldehyde than the repair-proficient normal cells, and a higher frequency of acetaldehyde-induced mutations of the shuttle vectors was found in XP cells than in normal cells, we propose that the DNA damage caused by acetaldehyde is removed by the nucleotide excision repair pathway. Since treatment with acetaldehyde yields very specific GG to TT tandem base substitutions in DNA, such changes can be used as a probe to identify acetaldehyde as the causal agent in human tumors.     

The 21-kDa Polypeptide (VAP21) in the Rabies Virion Is a CD99-Related Host Cell Protein

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.