Periplasmic production of human pancreatic prokallikrein in Escherichia coli

Summary The human pancreatic prokallikrein gene has been fused to the DNA sequence coding for the signal peptide of the Escherichia coli major outer membrane protein F (OmpF) and expressed under the control of tac promoter in E. coli. By induction with isopropyl--d-thiogalactopyranoside, the cells produced prokallikrein very efficiently. The fused OmpF signal peptide was verified as being processed correctly at the cleavage site of the OmpF signal peptide, and the N-terminal amino acid sequence of the product was found to be identical to that of native human prokallikrein. However, the prokallikrein produced by E. coli formed insoluble aggregates and was always collected in the insoluble fraction. An electron micrograph of prokallikrein-producing cells indicated that the prokallikrein was secreted into the periplasmic space and formed insoluble inclusion bodies there. By treating the insoluble inclusion bodies with oxidized and reduced glutathione in 1 M guanidine-HCl solution, a portion of them could be solubilized in water and showed kallikrein activity of 8 units (approx. 264 g kallikrein) per litre of culture by trypsin activation.     

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication

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A Microfibril Formation from Depolymerized Chitosan by N-Acetylation

The geometrical isomers of O-4-tert-butylcyclohexyl methylthiocarbamate were prepared by way of the corresponding dithiocarbonates. The O-trans-isomer was obtained from a mixture of cis- and trans-4-tert-butylcyclohexanols via an alkoxide formation involving biassed equilibration, whereas the O-cis isomer was from the cis-cyclohexanol via an alkoxide formation free from equilibration.

The acid-catalyzed rearrangement of the O-cis thiocarbamate gave a 1:3 mixture of S-cis- and S-trans-4-tert-butylcyclohexyl methylthiocarbamates, whereas that of the O-trans thiocarbamate afforded a 9:1 mixture of S-cis and S-trans products.

These results, together with the data from a study on the reactions of O-cis-3,3,5-tri-methylcyclohexyl methylthiocarbamate and the O-neopentyl analog, indicate that the acid-catalyzed rearrangement proceeded mostly through an S_N2 type of transalkylating mechanism to give the S-isomer in the case where the approach of nucleophiles was not sterically blocked, otherwise the reaction gave S_N1-type products.     

Biodegradable nanogel formation of polylactide-grafted dextran copolymer in dilute aqueous solution and enhancement of its stability by stereocomplexation

Monodisperse stereocomplex nanogels were obtained through the self-assembly of an equimolar mixture of dextran-graft-poly(L-lactide) (Dex-g-PLLA) and dextran-graft-poly(D-lactide) (Dex-g-PDLA) amphiphilic copolymers with well-defined composition in a dilute aqueous solution. The stereocomplex nanogel possessed partially crystallized cores of hydrophobic polylactide (PLA) and the hydrophilic dextran skeleton by intra- and/or intermolecular self-assembly between PLLA and PDLA chains. The stereocomplex nanogels exhibited significantly lower critical aggregation concentration (CAC) value as well as stronger thermodynamic stability compared with those of the corresponding L- or D-isomer nanogels. The mean diameter of the stereocomplex nanogels was 70 nm with narrow size distribution, implying they were well-defined and presumably nanogels. Furthermore, stereocomplex nanogel exhibited strong kinetic stability. The tunable degradation properties of Dex-g-PLA nanogels were achieved by varying the number of grafted PLA chains as well as applying stereocomplexation. This study demonstrates the advantage of stereocomplexation in the design of biodegradable nanogels with enhanced stability.     

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.     

The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts

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Highlights? XEco2 loading and cohesin acetylation require pre-RC assembly but not DNA synthesis ? Two N-terminal motifs in XEco2 are essential for XEco2 loading and cohesion ? The PIP box in XEco2 contributes to cohesion ? Interaction of acetylated cohesin with DNA is stabilized after DNA replication     

Chemical characterization of acidic oligosaccharides in milk of the red kangaroo (Macropus rufus)

In the milk of marsupials, oligosaccharides usually predominate over lactose during early to mid lactation. Studies have shown that tammar wallaby milk contains a major series of neutral galactosyllactose oligosaccharides ranging in size from tri- to at least octasaccharides, as well as β(1-6) linked N-acetylglucosamine-containing oligosaccharides as a minor series. In this study, acidic oligosaccharides were purified from red kangaroo milk and characterized by (1)H-nuclear magnetic resonance spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be as follows: Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. These acidic oligosaccharides were shown to be sialylated or sulfated in the non-reducing ends to the major linear and the minor branched series of neutral oligosaccharides of tammar wallaby milk.     

Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2

Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 microM, which is comparable with that of PAPST1 (0.8 microM). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon.     

Association study on chromosome 20q11.21-13.13 locus and its contribution to type 2 diabetes susceptibility in Japanese

Several linkage studies have predicted that human chromosome 20q is closely related to type 2 diabetes, but there is no clear evidence that certain variant(s) or gene(s) have strong effects on the disease within this region. To examine disease susceptibility variant in Japanese, verified SNPs from the databases, with a minor allele frequency larger than 0.15, were selected at 10-kb intervals across a 19.31-Mb region (20q11.21-13.13), which contained 291 genes, including hepatocyte nuclear factor 4α (HNF4α). As a result, a total of 1,147 SNPs were genotyped with TaqMan assay using 1,818 Japanese samples. By searching for HNF4α as a representative disease-susceptible gene, no variants of HNF4α were strongly associated with disease. To identify other genetic variant related with disease, we designed an extensive two-stage association study (725 first and 1,093 second test samples). Although SNP1146 (rs220076) was selected as a landmark within the 19.31 Mb region, the magnitude of the nominal P value (P = 0.0023) was rather weak. Subsequently, a haplotype-based association study showed that two common haplotypes were weakly associated with disease. All of these tests resulted in non-significance after adjusting for Bonferroni’s correction and the false discovery rate to control for the impact of multiple testing. Contrary to the initial expectations, we could not conclude that certain SNPs had a major effect on this promising locus within the framework presented here. As a way to extend our observations, we emphasize the importance of a subsequent association study including replication and/or meta-analysis in multiple populations.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Association of single-nucleotide polymorphisms in the suppressor of cytokine signaling 2 (SOCS2) gene with type 2 diabetes in the Japanese

Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.