Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery.

Background: The identification of permeation enhancers has gained interest in the development of drug delivery systems. A six-mer peptide, H-FCIGRL-OH (AT1002), is a tight junction modulator with promising permeation-enhancing activity. AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity.

A Methanol Extract of Adansonia digitata L. Leaves Inhibits Pro-Inflammatory iNOS Possibly via the Inhibition of NF-κB Activation.

This study examined the total polyphenol content of eight wild edible plants from Ethiopia and their effect on NO production in Raw264.7 cells. Owing to its relatively high polyphenol concentration and inhibition of NO production, the methanol extract of Adansonia digitata L.

Tert-butylhydroquinone reduces lipid accumulation in C57BL/6 mice with lower body weight gain.

tert-Butylhydroquinone (tBHQ) is a commonly used antioxidant additive that is approved for human use by both the Food and Agriculture Organization and the World Health Organization (FAO/WHO). In this study, we examined the effect of tBHQ on body weight gain and found that food supplementation with 0.001 % (w/w) tBHQ inhibited 61.

Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha.

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha.

Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85.

1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp.

Evidences for correlation between the reduced VCAM-1 expression and hyaluronan synthesis during cellular senescence of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) undergo cellular senescence during in vitro expansion culture, which accompanies the loss of migration and homing abilities. In this study, we analyzed expression levels of several surface markers of human MSCs at different passages of expansion culture. It has been shown that expression of vascular cell adhesion molecule-1 (VCAM-1) was most markedly decreased among the tested markers in the senescent MSCs.

Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen.

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H.

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha.

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 degrees C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1).

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae.

The yapsin family of aspartic proteases, located at cell surface, has a common specificity for paired or single basic reside cleavage sites of proproteins. Our previous study reported that the aberrant proteolytic cleavage of secretory recombinant human parathyroid hormone (hPTH) protein was problematic at late stages of fed-batch cultivations, even in the Saccharomyces cerevisiae mutant strain deficient in yapsin 1 (yps1Delta). To overcome this problem, we constructed a set of S.

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens.

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of D: -glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for D: -glucose production and levan formation was 6.

An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression.

Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems.

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 muM Cd for 2 h.

Cyclization mechanism of amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis.

Cyclization of farnesyl diphosphate into amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) initiates biosynthesis of artemisinin, a clinically important antimalarial drug precursor. Three possible ring-closure mechanisms, two involving a bisabolyl carbocation intermediate followed by either a 1,3-hydride shift or two successive 1,2-shifts, and one involving a germacrenyl carbocation, were proposed and tested by analyzing the fate of farnesyl diphosphate H-1 hydrogen atoms through (1)H and (2)H NMR spectroscopy. Migration of one deuterium atom of [1,1-(2)H(2)]farnesyl diphosphate to H-10 of amorpha-4,11-diene singled out the bisabolyl carbocation mechanism with a 1,3-hydride shift.

Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation.

The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1.

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter.

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A.

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S.

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha.

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha.

Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene.

The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP).

Levan fructotransferase from Arthrobacter oxydans J17-21 catalyzes the formation of the di-D-fructose dianhydride IV from levan.

A new levan fructotransferase (LFTase) isolated from Arthrobacter oxydans J17-21 was characterized for the production of difructose dianhydride IV (DFA IV). LFTase was purified to apparent homogeneity by Q-Sepharose ion exchange chromatography, Mono-Q HR 5/5 column chromatography, and gel permeation chromatography. The enzyme had an apparent molecular mass of 54000 Da.

Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae.

In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome. Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S. cerevisiae.

Molecular characterization of the growth phase-dependent expression of the lsrA gene, encoding levansucrase of Rahnella aquatilis.

Expression of the lsrA gene from Rahnella aquatilis, encoding levansucrase, is tightly regulated by the growth phase of the host cell; low-level expression was observed in the early phase of cell growth, but expression was significantly stimulated in the late phase. Northern blot analysis revealed that regulation occurred at the level of transcription. The promoter region was identified by primer extension analysis.

Sequencing and functional analysis of the Hansenula polymorpha genomic fragment containing the YPT1 and PMI40 genes.

A 6.0 kb genomic DNA segment was isolated by its ability to rescue the temperature-sensitive growth defect and the hypersensitivity to sodium deoxycholate of a spontaneous vanadate-resistant mutant derived from Hansenula polymorpha DL-1. The genomic fragment contains four open reading frames homologous to the Saccharomyces cerevisiae genes YPT1 (which codes for a GTP-binding protein; 75% amino acid identity), PMI40 (encoding phosphomannose isomerase; 61% identity), YLR065c (30% identity) and CST13 (28% identity).

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium.