Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice.Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages.Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver.SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.     

A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E₂ induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.     

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3'), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.     

Maternal inheritance of mitochondrial DNA (mtDNA) in the Pacific oyster (Crassostrea gigas): a preliminary study using mtDNA sequence analysis with evidence of random distribution of MitoTracker-stained sperm mitochondria in fertilized eggs

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.     

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis or tumor invasion. To clarify the relationship of the expression patterns of mucins in human neoplasms with their biological behavior, we examined the expression profiles of MUC1, MUC2, and MUC4 mucins in various human neoplasms using immunohistochemistry and in situ hybridization, and compared them with clinicopathologic factors including outcome of the patients. MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients. In contrast, MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent pancreatobiliary neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas. The expression of MUC2 mucin in indolent pancreatobiliary neoplasms coincided with expression of MUC2 mRNA. Our recent studies to clarify the MUC2 gene regulation mechanism disclosed that DNA methylation and histone modification in the 5' flanking region of the MUC2 promoter may play an important role. Further studies of the epigenetics also in MUC1 and MUC4 gene expression may be needed to understand the relationship between the expression of mucins in human neoplasms with their biological behavior.     

Seasonal dynamics of Callibaetis willineri (Ephemeroptera, Baetidae) associated with Eichhornia azurea (Pontedericeae) in Guaraná Lake of the Upper Paraná River, Brazil

The micro-anatomy of the cephalon is described in the troglobic asellotan isopod Craseriella anops from the Nohoch Nah Chich anchialine cave system in southeast Mexico. The cephalon is entirely covered by cuticular scales bordered by marginal spines. The anterior end of the cephalon is bordered by a carina that is wider medially. The isopod is eyeless. The distal seventh portion of the cephalon is characterized by the presence of two sutures and six setae. A suture is found on each side of the distal margin of the cephalon.Each suture is bordered by microtrichs. Two simple setae with a sensory hair, articulated on the base by a socket, are found one on each side of each of the sutures. Two additional setae, similar in shape and size, occur medially on the cephalon. A terminal pore is absent on the sensory hairs of all setae. These setae are suggested to be mechanoreceptors that provide directional sensitivity and enhance the sensibility of turbulent motion, viscosity and changes of hydrostatic pressure.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

Ultrafast Red Light Activation of Synechocystis Phytochrome Cph1 Triggers Major Structural Change to Form the Pfr Signalling-Competent State

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.     

Phospholipid Acyl-hydrolases from a Mold,Corticium centrifugum

The activities of phospholipids acyl-hydrolases in an enzyme preparation from a mold, Corticium centrifugum, were examined. Lecithin acyl-hydrolase had an optimal pH at 3.5. The reaction proceeded beyond the range of 50%. Sigmoidal curves observed suggested the presence of lysophospholipase in the preparation. The latter enzyme activity was found to be seven times as strong as the former at the same pH. Fractionation by DEAE-Sephadex chromatography and analysis of the reaction products demonstrated that the main component of lecithin acyl-hydrolase was phospholipase B, which hydrolyzed both of fatty acyl ester groups of lecithin. This activity was found to be present as a separate enzyme from most of lysophospholipase.