Two-dimensional NMR Investigation of Solanascone

2-n-Heptyl-4-hydroxyquinoline-N-oxide (KF8940), isolated from Pseudomonas methanica, was a potent and selective inhibitor of the arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells. Kinetic analysis indicated that the inhibitory mode was non competitive. The Ki value was 3.5 × 10^?7 m. KF8940 also inhibited 12-lipoxygenase of bovine platelets in a non-competitive manner, but with a Ki value of 7 × 10^?5M. Ionophore A23187-stimulated SRS generation from rat peritoneal cells and antigen-stimulated SRS-A generation from sensitized rat lung were significantly inhibited by KF8940. KF8940 at a dose of 10 mg/kg (p.o.) suppressed the passive anaphylactic bronchoconstriction in guinea pigs.     

Digital image analysis of the flagellar beat of activated and hyperactivated suncus spermatozoa

The flagellar beat of hyperactivated Suncus spermatozoa was analyzed by digital imaging and was compared to that of the nonhyperactivated (activated) spermatozoa in order to examine the function of the accessory fibers during the flagellar beat and the sliding filament mechanism inducing the motility of the hyperactivated spermatozoa. Unusual large and long characteristics of the accessory fibers were involved in generating the gently curved bends and a low beat frequency. Examination of the motility parameters of the flagellar beat of the activated and hyperactivated spermatozoa attached to a slide glass by their heads revealed that there were two beating modes: a frequency-curvature dependent mode in the activated flagellar beat and a nearly constant frequency mode in the hyperactivated flagellar beat. The hyperactivated flagellar beat was characterized by sharp bends in the proximal midpiece and a low beat frequency. The sharp bends in the proximal midpiece were induced by the increase in the total length of the microtubule sliding at the flagellar base. The rate of microtubule sliding (sliding velocity) in the axoneme remained almost constant in the flagellar beat of both the activated and hyperactivated spermatozoa. Comparison of the sliding velocity in Suncus, golden hamster, monkey, and sea urchin sperm flagella with their stiffness suggests that the sliding velocity is determined by the stiffness at the flagellar base and that the same sliding microtubule system functions in both mammalian and echinoderm spermatozoa.     

Combined Inhibition of DYRK1A,SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells

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Differences between Spinocerebellar Ataxias and Multiple System Atrophy-Cerebellar Type on Proton Magnetic Resonance Spectroscopy

Purpose

A broad spectrum of diseases can manifest cerebellar ataxia. In this study, we investigated whether proton magnetic resonance spectroscopy (MRS) may help differentiate spinocerebellar ataxias (SCA) from multiple systemic atrophy- cerebellar type (MSA-C).

Material and Methods

This prospective study recruited 156 patients with ataxia, including spinocerebellar ataxia (SCA) types 1, 2, 3, 6 and 17 (N = 94) and MSA-C (N = 62), and 44 healthy controls. Single voxel proton MRS in the cerebellar hemispheres and vermis were measured. The differences were evaluated using nonparametric statistic tests.

Results

When compared with healthy controls, the cerebellar and vermis NAA/Cr and NAA/Cho were lower in all patients(p<0.002). The Cho/Cr was lower in SCA2 and MSA-C (p<0.0005). The NAA/Cr and Cho/Cr were lower in MSA-C or SCA2 comparing with SCA3 or SCA6. The MRS features of SCA1 were in between (p<0.018). The cerebellar NAA/Cho was lower in SCA2 than SCA1, SCA3 or SCA6 (p<0.04). The cerebellar NAA/Cho in MSA-C was lower than SCA3 (p<0.0005). In the early stages of diseases (SARA score<10), significant lower NAA/Cr and NAA/Cho in SCA2, SCA3, SCA6 or MSA-C were observed comparing with healthy controls (p<0.017). The Cho/Cr was lower in MSA-C or SCA2 (p<0.0005). Patients with MSA-C and SCA2 had lower NAA/Cr and Cho/Cr than SCA3 or SCA6 (p<0.016).

Conclusion

By using MRS, significantly lower NAA/Cr, Cho/Cr and NAA/Cho in the cerebellar hemispheres and vermis were found in patients with ataxia (SCAs and MSA-C). Rapid neuronal degeneration and impairment of membrane activities were observed more often in patients with MSA-C than those with SCA, even in early stages. MRS could also help distinguish between SCA2 and other subtypes of SCAs. MRS ratios may be of use as biomarkers in early stages of disease and should be further assessed in a longitudinal study.     

A human islet cell culture system for high-throughput screening

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.     

1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.     

Inbreeding depression of female fecundity by genetic factors retained in natural populations of a male-haploid social mite (Acari: Tetranychidae)

We previously determined that certain recessive genes decrease female fecundity in a haplo-diploid spider mite, Stigmaeopsis miscanthi (Saito). However, whether the depression was caused by the breakdown of heterosis or the expression of deleterious genes retained in a population could not be determined, because we had started our inbreeding experiment from a mixture of two isolated populations. In order to answer this basic question, inbreeding effects on survival and fecundity were measured for eight small populations occurring far from the two initial populations. There was little depression of immature survival of inbred lineages in all populations. On the other hand, in two inbred lineages, both originating from the smallest populations, female oviposition decreased significantly with the increase of Wrights f-value, showing that mildly deleterious genes are actually retained even in natural populations of haplo-diploid organisms.     

<Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strain 06TCa19 suppresses inflammatory chemokine induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in human gastric epithelial cells

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.     

Induction of phagocyte oxidase components during human myeloid differentiation: independent protein expression and discrepancy with the function.

We investigated the content of four components of the O2(-)-producing enzyme (p47, p67, p22, and gp91) and the O2(-)-producing capacity in human myeloid cell lines. The content of the four components of the phagocyte oxidase was minimal before differentiation induction. During differentiation, expression of p22 and gp91 was at consistently low levels, even when the O2(-)-producing capacity was equivalent to that of normal neutrophils. On the other hand, p47 was consistently and rapidly induced to the level comparable to normal neutrophils. The results indicate that low expression of p22 and gp91 is sufficient to obtain normal O2- production, and that p47 might play an important regulatory role in the functional differentiation.     

Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform

The plant secondary metabolites benzylisoquinoline alkaloids (BIAs) have diverse pharmaceutical activities, and some are used medicinally (e.g., morphine, codeine, berberine). Recently, we constructed a platform to produce BIAs using bioengineered Escherichia coli, which could be useful for bulk production. The E. coli strain used in this system produces the important intermediate (S)-reticuline from glucose or glycerol. Although the amount produced (40 mg/L) exceeded the amount that can be purified from plants, the conversion efficiency from glycerol was only 0.15%; thus, there was much room for improvement. Our production system was developed in a jar fermenter but it is difficult to work with multiple samples using this system. In contrast, many samples can be cultured in parallel using shake flask cultures, allowing optimization of production conditions. Here, we describe bench-top production of (S)-reticuline and optimization of culture conditions using shake flask cultures. The production of (S)-reticuline reached 33.9 mg/L.