A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.     

Modulation of control of muscle sympathetic nerve activity during orthostatic stress in humans

We tested the hypothesis that orthostatic stress would modulate the arterial baroreflex (ABR)-mediated beat-by-beat control of muscle sympathetic nerve activity (MSNA) in humans. In 12 healthy subjects, ABR control of MSNA (burst incidence, burst strength, and total activity) was evaluated by analysis of the relation between beat-by-beat spontaneous variations in diastolic blood pressure (DAP) and MSNA during supine rest (CON) and at two levels of lower body negative pressure (LBNP: -15 and -35 mmHg). At -15 mmHg LBNP, the relation between burst incidence (bursts per 100 heartbeats) and DAP showed an upward shift from that observed during CON, but the further shift seen at -35 mmHg LBNP was only marginal. The relation between burst strength and DAP was shifted upward at -15 mmHg LBNP (vs. CON) and further shifted upward at -35 mmHg LBNP. At -15 mmHg LBNP, the relation between total activity and DAP was shifted upward from that obtained during CON and further shifted upward at -35 mmHg LBNP. These results suggest that ABR control of MSNA is modulated during orthostatic stress and that the modulation is different between a mild (nonhypotensive) and a moderate (hypotensive) level of orthostatic stress.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Jasmonate-dependent plant defense restricts thrips performance and preference

Background  

The western flower thrips (Frankliniella occidentalis [Pergande]) is one of the most important insect herbivores of cultivated plants. However, no pesticide provides complete control of this species, and insecticide resistance has emerged around the world. We previously reported the important role of jasmonate (JA) in the plant's immediate response to thrips feeding by using an Arabidopsis leaf disc system. In this study, as the first step toward practical use of JA in thrips control, we analyzed the effect of JA-regulated Arabidopsis defense at the whole plant level on thrips behavior and life cycle at the population level over an extended period. We also studied the effectiveness of JA-regulated plant defense on thrips damage in Chinese cabbage (Brassica rapa subsp. pekinensis).     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.