Low-shear modeled microgravity affects metabolic networks of Escherichia coli O157:H7 EDL933: Further insights into space-microbiology consequences.

Escherichia coli O157:H7 EDL933 exposed to low-shear modeled microgravity (LSMMG) and normal gravity (NG) was used for a transcriptomic analysis. The modified Gompertz model (R2 = 0.81-0.99) showed an increased growth rate of E. coli O157:H7 under LSMMG. The mechanism of this active growth was associated with highly upregulated genes in nutrient and energy metabolism, including the TCA cycle, glycolysis, and pyruvate metabolism. Green fluorescent protein-labeled E. coli O157:H7 also formed significantly thick biofilms (fluorescent unit: NG, 1,263; LSMMG, 1,533; P = 0.0473) under LSMMG, whereas bacterial mobility decreased slightly (P = 0.0310). The transcriptomic analysis revealed that genes encoding glycogen biosynthesis (glgCAP operon) were upregulated (1.40 to 1.82 of log fold change [FC]) due to the downregulation of csrA (2.17 of log FC), which is the global regulator of biofilm formation of E. coli. We also identified 52 genes in E. coli O157:H7 EDL933 that were involved in the secretion pathway, 32 of which showed ≥2-fold significant changes in transcription levels after cultivation under LSMMG. Notably, all downregulated genes belonged to the type III and VI secretion systems, indicating that host cell contact secretion was dysregulated in the LSMMG cultures compared to the NG cultures. LSMMG also stimulates the pathogenicity of E. coli O157:H7 via transcriptional upregulation of Shiga toxin 1 (1.36 to 2.81 log FC) and toxin HokB (6.1 log FC). Our results suggest LSMMG affects bacterial growth, biofilm formation, and E. coli O157:H7 pathogenicity at some transcriptional levels, which indicates the importance of understanding biological consequences.

Whitening and antioxidant activities of bornyl acetate and nezukol fractionated from Cryptomeria japonica essential oil.

OBJECTIVES: The aim of this study was to investigate the whitening and antioxidant activities of essential oils from Cryptomeria japonica by determining their tyrosinase inhibition, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and superoxide dismutase (SOD)-like activities. METHODS: Essential oils of C. japonica leaves were extracted with distilled water, and after condensation of volatile constituents, the condensates were extracted with ethyl acetate. Crude essential oils of C. japonica were divided into six fractions by thin layer chromatography and open column chromatography, and their chemical analysis was performed by GC/MS. Major compounds of fractions were composed of kaurene, bornyl acetate, nezukol, (-)-4-terpineol, δ-cadinene, α-terpineol, γ-eudesmol, α-eudesmol and elemol. RESULTS: For tyrosinase inhibitory activity using two substrates, l-tyrosine and 3,4-dihydroxyphenylalanine (l-DOPA), kaurene, bornyl acetate and nezukol were highly effective. In antioxidant activity, (-)-4-terpinenol and δ-cadinene showed high DPPH radical scavenging activity, and bornyl acetate and nezukol indicated extremely high SOD-like activity. CONCLUSION: Therefore, bornyl acetate and nezukol fractionated from C. japonica essential oil, which showed highly active whitening and antioxidant activities, have potential applications in cosmeceutical materials.

Neuregulin 3 does not confer risk for schizophrenia and smooth pursuit eye movement abnormality in a Korean population.

Located on chromosome 10q22-q23, the human neuregulin3 (NRG3) is considered to be a strong positional and functional candidate gene for schizophrenia pathogenesis. Several case-control studies examining the association of polymorphisms in NRG3 with schizophrenia and/or related traits such as delusion have been reported recently in cohorts of Han Chinese, Ashkenazi Jews, Australians and white Americans of Western European ancestry. Thus, this study aimed to comprehensively investigate the association of NRG3 genetic variations with the risk of schizophrenia and smooth pursuit eye movement (SPEM) abnormality in a Korean population. Using TaqMan assay, six single-nucleotide polymorphisms (SNPs) in the intronic region of NRG3 were genotyped and two major haplotypes were identified in 435 patients with schizophrenia as cases and 393 unrelated healthy individuals as controls. A total of 113 schizophrenia patients underwent an eye tracking task, and degree of SPEM abnormality was measured using the logarithmic values of the signal/noise (Ln S/N) ratio. Differences in frequency distributions were analyzed using logistic and regression models following various modes of genetic inheritance and controlling for age and sex as covariates. Subsequent analysis revealed that the frequency distributions of NRG3 polymorphisms and haplotypes were similar between schizophrenia patients and healthy controls of Korean ethnicity. Furthermore, no significant differences were observed between the genetic variants tested for SPEM abnormality. By elucidating a lack of association in a Korean population, findings from this study may contribute to the understanding of the genetic etiology focusing on the role of NRG3 in schizophrenia pathogenesis.

The roles of fractalkine/CX3CR1 system in neuronal death following pilocarpine-induced status epilepticus.

Although fractalkine is one of chemokines involved in mediation of neuronal/microglial interaction, it is not known whether fractalkine/CX3CR1-mediated pathogenesis occurs in the rat brain following epileptogenic insults. In order to elucidate the roles of the fractalkine/CX3CR1 system in microglial activation and neurodegeneration induced by status epilepticus (SE), we investigated changes in fractalkine/CX3CR1 system within the rat hippocampus following SE. In non-SE induced animals, fractalkine and CX3CR1 immunoreactivity was detected in neurons and microglia, respectively. Following SE, fractalkine immunoreactivity was transiently increased in neurons and astrocytes. CX3CR1 immunoreactivity was also transiently detected in neurons (particularly in CA1 pyramidal cells). Intracerebroventricular infusions of recombinant rat fractalkine aggravated SE-induced neuronal damage, while fractalkine IgG or CX3CR1 IgG infusion alleviated it, compared to saline-infused animals. These findings suggest that fractalkine/CX3CR1 system may play an important role in SE-induced neuronal damages via neuron-microglial interactions.

Association of CHRM2 polymorphisms with severity of alcohol dependence.

The cholinergic muscarinic 2 receptor (CHRM2) gene has been considered a candidate gene for the alcohol dependence in that it might underpin certain risk factors for this condition. This study examined variations in the CHRM2 between the patients with alcohol dependence and population controls in Korean and explored the associations between CHRM2 polymorphisms and severity of symptoms in the patients with alcohol dependence. One hundred and fifty-five patients with alcohol dependence, defined by the Alcohol Use Disorders Identification Test (AUDIT) and the Alcohol Dependence Scale (ADS) to measure the severity of symptoms, and one hundred and ninety-five population controls were drawn in the study. Three single nucleotide polymorphisms (SNPs) of CHRM2 were genotyped using the TaqMan assay and analyzed with the severity of symptoms of alcohol dependence. We found that although SNP rs324650 showed marginal association with the risk of alcohol dependence (P = 0.03), the significance of the result was not sustained after multiple corrections. SNP rs1824024 was significantly associated with the AUDIT and ADS scores in patients (P = 0.005 and 0.003, respectively). These findings suggested that the muscarinic acetylcholine function might be related not with alcohol dependence itself but with the severity of alcohol dependence in Korean population.

Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats.

In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus.

Impact of the tryptophan hydroxylase 1 gene A218C polymorphism on amygdala activity in response to affective facial stimuli in patients with major depressive disorder.

Tryptophan hydroxylase-1 (TPH1) is the rate-limiting enzyme in serotonin biosynthesis, and allelic variations at the TPH1 locus have been implicated in the pathophysiology of depression. Using 1.5-Tesla functional magnetic resonance imaging, we investigated the possible relationship between TPH1 A218C polymorphism and amygdala response to negative facial stimuli in 26 right-handed female subjects with major depressive disorder (MDD). Genotyping was performed with the polymerase chain reaction. We found a significant association between A allele of the TPH1 A218C polymorphism and neural activations in response to negative facial stimuli. Subjects with the A allele of the TPH1 A218C polymorphism showed greater brain activity in the bilateral amygdala under the sad vs. the neutral condition compared with subjects homozygous for the C allele. Our results suggest that the A218C polymorphism of the TPH1 gene serves as a modulator of amygdala activity in patients with MDD.

Effect of citral, eugenol, nerolidol and alpha-terpineol on the ultrastructural changes of Trichophyton mentagrophytes.

The antifungal effects of citral, eugenol, nerolidol and alpha-terpineol on Trichophyton mentagrophytes were investigated. Citral over 0.1 mg/ml strongly inhibited the hyphal growth of T. mentagrophytes, and the antifungal activity of alpha-terpineol was less effective. The morphological changes of the fungus exposed to the terpenes were observed by electron microscopy. The hyphae were distorted and collapsed at 0.2, 0.4 and 1 mg/ml of eugenol, nerolidol and alpha-terpineol respectively, and cell membrane and organelles were irreversibly damaged at 0.2 mg/ml citral. These suggested that four terpenes possess antifungal activity against T. mentagrophytes, and the activity might lead to irreversible cellular disruption.

Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution.

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus.

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The Km and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 +/- 0.4mM and 0.79 +/- 0.06s(-1) or 0.50 +/- 0.07mM and 0.25 +/- 0.01s(-1), respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85 degrees C for 90 min.

Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization.

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 degreesC, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

Central GABAA and GABAB receptor modulation of basal and stress-induced plasma interleukin-6 levels in mice.

To investigate the modulatory roles of central gamma-aminobutyric acid (GABA)A and GABAB receptors in the regulation of basal and stress-induced plasma interleukin-6 (IL-6) levels, we examined the effects of i.c.v. injection of GABA receptor agonists and antagonists on basal and restraint stress-induced plasma IL-6 levels in mice. Muscimol (20-200 ng), a GABAA receptor agonist, and baclofen (5-20 ng), a GABAB receptor agonist, injected i.c.v. did not affect the basal levels of plasma IL-6. In the restraint-stressed animals, muscimol and baclofen inhibited the stress-induced plasma IL-6 levels from the dose of 50 and 15 ng, respectively. 2-(3-Carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide (SR-95,531; 0.3-10 ng), a GABAA receptor antagonist, and 2-hydroxysaclofen (1-10 microgram), a GABAB receptor antagonist, injected i.c.v. increased both the basal and the restraint stress-induced plasma IL-6 levels. The i.p. pretreatment of animals with 6-hydroxydopamine (100 mg/kg) for 3 days significantly inhibited SR-95,531 (3 ng i.c.v.)- but not 2-hydroxysaclofen (10 microg i.c.v.)-induced increase in the basal plasma IL-6 levels. These data suggest that central GABAA and GABAB receptors are involved in the suppressive modulation of basal and restraint stress-induced plasma IL-6 levels in mice.

Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus.

Aquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95 degrees C. To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5-2 kb genomic DNA fragments of A. pyrophilus. Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identified proteins in the PIR or Genebank databases. These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate. Although the GC ratio of the genome is about 40%, the codon usage of A. pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine. A higher ratio of positively charged amino acids in A. pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability.

Cloning and expression of superoxide dismutase from Aquifex pyrophilus, a hyperthermophilic bacterium.

A superoxide dismutase (SOD) gene of Aquifex pyrophilus, a marine hyperthermophilic bacterium, was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized. This is the first SOD from a hyperthermophilic bacterium that has been cloned. It is an iron-containing homo-oligomeric protein with a monomeric molecular mass of 24.2 kDa. The DNA-derived amino acid sequence is more similar to those of known Mn- and Fe-SODs from thermophilic archaea than of Cu, Zn-SODs. The metal binding residues found in all SOD sequences from different species are also conserved in A. pyrophilus SOD. The protein is biochemically active only as an oligomer and is resistant to thermal denaturation.

Prostacyclin-to-thromboxane A2 ratio in arteriogenic impotence.

It has been suggested that penile hypercoagulability predisposes to aging penile vascular changes and impotence, and that elevated thromboxane A2 during erection may contribute to hypercoagulability and atherosclerosis. Since the ratio of the prostacyclin concentration to the thromboxane A2 concentration is constantly maintained in normal hemostatic responses, an imbalance between thromboxane A2 and prostacyclin may be a factor to initiate vascular diseases and decrease blood flow. We assess the usefulness of the prostacyclin-to-thromboxane A2 ratio in penile blood during erection for diagnosis of arteriogenic impotence. The ratio in the arteriogenic impotence group was significantly lower (p less than 0.01) than in the psychogenic and venogenic impotence groups. Therefore, the prostacyclin-to-thromboxane A2 ratio seems to be useful to diagnose arteriogenic impotence.