Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences

We cloned two genes for alkaline pectate lyase, pel-4A and pel-4B, from alkaline pectinase-producing alkaliphilic Bacillus sp. strain P-4-N. The pel-4B gene product Pel-4B was purified to homogeneity and characterized. The purified enzyme had an isoelectric point of pH 9.6 and a molecular mass of 35 kDa, values close to those of the pel-4A gene product Pel-4A. The pH and temperature optima for activity were as high as 11.5 and 70 degrees C, respectively, which are the highest among the pectate lyases reported to date. The mature Pel-4B (304 amino acids; 33,868 Da) was structurally related to the enzymes in the polysaccharide lyase family 1 and showed 35.6% identity with Pel-4A on the amino acid level. It showed significant homology to other pectate lyases in the same family, such as the enzymes from alkaliphilic Bacillus sp. strains KSM-P7 and KSM-P103 and the fungi Aspergillus nidulans and Colletotrichum gloeosporioides f. sp. malvae.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori

To investigate the pathogenicity of Pseudomonas aeruginosa in insects, a gacA mutant of P. aeruginosa PA01 was constructed by site-directed mutagenesis. The mutant was designated as C1. C1 was less virulent to Bombyx mori than the parent strain. To complement the gacA gene, P. aeruginosa C1 was transformed with the broad host range plasmid pJB3Km1 carrying a 3.9-kbp gacA fragment. The expression of the gacA mRNA in C1 (pgacA) was detected. In addition, the complemented mutant restored the level and timing of pyocyanin production, indicating that functional GacA is produced in the complemented strain. However, no significant difference was observed between C1 and C1 (pgacA) with respect to the killing of B. mori larvae.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.     

Volatile Components of Roasted Almonds: Basic Fraction

Roasted almond volatiles were isolated by vacuum carbon dioxide distillation of acetone extracts and separated into basic and non-basic fractions. The basic fraction was analyzed by combination gas chromatography-mass spectrometry utilizing open tubular column. Seventeen pyrazines and 2-formyl pyrrole were identified by comparison of their mass spectra with reference spectra. Besides them, five compounds were tentatively identified by mass spectrometry.     

Heligmosomoides polygyrus infection reduces severity of type 1 diabetes induced by multiple low-dose streptozotocin in mice via STAT6- and IL-10-independent mechanisms

Some parasitic helminths are known to protect their hosts from allergic and autoimmune disorders. Here, we tested the effects of a gastrointestinal nematode, Heligmosomoides polygyrus (Hp), on streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. Hp infection significantly suppressed hyperglycemia induced by multiple low-dose administration of STZ, but did not affect hyperglycemia induced by single high-dose administration of STZ. In the multiple low dose model, Hp infection prevented a decrease in pancreatic islet size. The augmentation of TNF-α and IL-1β expression in the pancreas was abrogated by Hp infection. The genetic absence of IL-10 or STAT6 did not abrogate the anti-hyperglycemic effect of Hp. Hp has a suppressive effect on immune mechanism-mediated experimental T1D via Th2 polarization-independent mechanisms.     

Active site of deblocking aminopeptidase from Pyrococcus horikoshii.

New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion.