Number of hits necessary for complement-mediated hemolysis

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.     

Development of novel carrier using natural zeolite and continuous ethanol fermentation with immobilized Saccharomyces cerevisiae in a bioreactor

A natural zeolite, easily vitrified and blown at 1300 °C with a high porosity and diam. of 5–100 m, was used to immobilize Saccharomyces cerevisiae at 3.6 × 10⁸ cells ml^–1 carrier. When the abilities of natural zeolite carrier were compared with glass beads, the capacity for immobilization and alcohol fermentation activity were, respectively, 2-fold higher and 1.2-fold higher than that of glass beads. Continuous alcohol fermentation was stable for over 21 d without breakage of the carrier.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.     

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

Amelioration of pneumonia with Streptococcus pneumoniae infection by inoculation with a vaccine against highly pathogenic avian influenza virus in a non-human primate mixed infection model

Background  Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria.
Methods  H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles .
Results  Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae . Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant.
Conclusions  Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.     

The temperature sensitive mutant 72c

Summary The spontaneous temperature sensitive mutant 72c is shown to be more tolerant to fusidic acid, but less tolerant to trimethoprim on plates at permissive temperature, than is the parental strain. The poor growth of the mutant on amino acids supplemented plates, as well as its inability to grow on broth plates at 40°, can be compensated by sublethal amounts of chloroamphenicol. Also some mutations to Rif-R or Str-R improve growth of the mutant under certain conditions.Reversion and other genetic analysis strongly suggest, that the pleiotropic behaviour of the mutant is due to a single mutation in a gene, which is designated fusB and is closely cotransducible with lip at min 14 of the E. coli chromosome. The gene order is lip-fusB-supE.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Site‐specific rapid deamidation and isomerization in human lens αA‐crystallin in vitro

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens‐specific αA‐crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA‐crystallin with Asn by using site‐directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA‐crystallin was subjected to enzymatic digestion followed by liquid chromatography–MS/MS to evaluate the ratio of modifications in Asn151‐containing peptides. The Asp151Asn αA‐crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit–subunit interactions between αA‐crystallin and αB‐crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA‐crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L‐Asp to D‐Asp residues in vivo.