Altered expression of phospholipase D1 in spontaneously hypertensive rats.

To clarify the involvement of phospholipase D (PLD) in the mechanism underlying genetically-induced hypertension, we investigated the activity and expression levels of PLD in tissues taken from spontaneously hypertensive rats (SHR), and their normotensive controls, Wistar-Kyoto rats (WKY). The ADP-ribosylation factor 3 (ARF3)-dependent PLD activity and protein levels of PLD1 from SHR increased significantly in the brain and liver, but not in the heart and kidney, compared to those of WKY. The activity and expression of PLD were the same between the homogenated whole kidneys of the two strains; however, there were topographical differences in the expression and activity of PLD between the kidneys of the two strains. The activity and expression level of PLD gradually increased from the cortex to the inner medulla of WKY. The enzyme activity, and amount of PLD in the inner stripe of the outer medulla and in the inner medulla, was significantly lower in SHR than in WKY. Taken together, these results suggest that the distinctly distributed patterns of PLD in the kidney may be associated with differential signal transduction pathways that are involved in hypertension in conjunction with an increase of PLD activity in the brain and liver.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Daidzein administration in vivo reduces myocardial injury in a rat ischemia/reperfusion model by inhibiting NF-kB activation

AimsWe tested the hypothesis that daidzein may reduce myocardial damage by both inhibiting the release of cytokines and limiting the nuclear translocation of NF-kB.Main methodsMale Sprague–Dawley rats were anesthetized, and the left anterior descending coronary artery (LAD) was ligated for 25 min. Twenty-four hours after reperfusion was established, the hemodynamics and infarct size were examined.Key findingsTreatment with daidzein (10 mg/kg, i.p.) 1 h prior to the ischemia/reperfusion procedure (I/R) reduced the infarct size by 52.8% (P < 0.05). Daidzein also significantly improved I/R-induced myocardial contractile dysfunction by improving the left ventricular diastolic pressure and the positive and negative maximal values of the first derivative of the left ventricular pressure. In addition, daidzein reduced the plasma levels of TNF-α and IL-6 in I/R rats and decreased malondialdehyde levels, myeloperoxidase activity, catalase activity and neutrophil infiltration in I/R rat myocardium. Interestingly, daidzein inhibited I/R-induced myocardial apoptosis by decreasing DNA strand breaks and cleaved caspase-3 activity. Furthermore, daidzein inhibited both the nuclear translocation of NF-kB in I/R rat hearts and the H₂O₂-induced activation of NF-kB-luciferase activity in human umbilical vein endothelial cells.SignificanceThis study reveals that the administration of daidzein in vivo attenuates I/R-induced myocardial damage via inhibition of NF-kB activation, which in turn may suppress inflammatory cytokine expression.     

Role of interleukin-7 in degenerative and inflammatory joint diseases

IL-7 is known foremost for its immunostimulatory capacities, including potent T cell-dependent catabolic effects on bone. In joint diseases like rheumatoid arthritis and osteoarthritis, IL-7, via immune activation, can induce joint destruction. Now it has been demonstrated that increased IL-7 levels are produced by human articular chondrocytes of older individuals and osteoarthritis patients. IL-7 stimulates production of proteases by IL-7 receptor-expressing chondrocytes and enhances cartilage matrix degradation. This indicates that IL-7, indirectly via immune activation, but also by a direct action on cartilage, contributes to joint destruction in rheumatic diseases.     

Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor

The action of tyrphostin AG-1478, a potentprotein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels(Kv1.5) stably expressed in Chinese hamster ovary cells wasinvestigated using the whole cell patch-clamp technique. AG-1478rapidly and reversibly inhibited Kv1.5 currents at 50 mV in aconcentration-dependent manner with an IC₅₀ of 9.82 µM.AG-1478 accelerated the decay rate of inactivation of Kv1.5 currentswithout modifying the kinetics of current activation. Pretreatment withthe structurally dissimilar PTK inhibitors (genistein and lavendustinA) had no effect on the AG-1478-induced inhibition of Kv1.5 and did notmodify the AG-1478-induced current kinetics. The rate constants forbinding and unbinding of AG-1478 were 1.46 µM¹ · s¹ and 10.19 s¹, respectively. The AG-1478-induced inhibition of Kv1.5channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation timecourse, resulting in a tail crossover phenomenon. Inhibition of Kv1.5by AG-1478 was use dependent. The present results suggest that AG-1478acts directly on Kv1.5 currents as an open-channel blocker andindependently of the effects of AG-1478 on PTK activity.

     

Conjugative plasmid mediated inducible nickel resistance in<Emphasis Type="Italic"> Hafnia alvei</Emphasis> 5-5

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni²⁺ or 2 mM Co²⁺. The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni²⁺, and cobalt resistance to 2 mM Co²⁺. A 4.8-kb Sal–EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni²⁺) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni²⁺. By random TnphoA′-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A. Electronic Publication     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Optimization of dilute sulfuric acid pretreatment of corn stover for enhanced xylose recovery and xylitol production

Pretreatment steps are necessary for the bioconversion of corn stover (CS) to xylitol. In order to optimize the pretreatment parameters, the sulfuric acid concentration, sulfuric acid residence time, and solid slurry concentration were evaluated, based on the glucose and xylose recovered from CS at the relatively low temperature of 120°C. The optimum conditions were found to be pretreatment with 2.5% (w/v) sulfuric acid for 1.5 h, with a solid slurry concentration of 90 g/L. Under these conditions, the hydrolysis rates of glucan and xylan were approximately 26.0 and 82.8%, respectively. High xylitol production (10.9 g/L) and conversion yield (0.97 g/g) were attained from corn stover hydrolysate (CSH) without detoxification and any nutrient addition. Our results were similar for xylitol production in synthetic medium under the same conditions. The non-necessity of both the hydrolysate detoxification step and nutrient addition to the CSH is undoubtedly promising for scale-up application on an industrial scale, because this medium-based manufacturing process is expected to reduce the production cost of xylitol. The present study demonstrates that value-added xylitol could be effectively produced from CS under optimized pretreatment conditions, especially with CSH as the substrate material.     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.