Isolation, cloning, and characterization of a novel phosphomannan-binding lectin from porcine serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.     

Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach

Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Stochastic thermodynamic limit on E. coli adaptation by information geometric approach

Biological systems process information under noisy environment. Sensory adaptation model of E. coli is suitable for investigation because of its simplicity. To understand the adaptation processing quantitatively, stochastic thermodynamic approach has been attempted. Information processing can be assumed as state transition of a system that consists of signal transduction molecules using thermodynamic approach, and efficiency can be measured as thermodynamic cost. Recently, using information geometry and stochastic thermodynamics, a relationship between speed of the transition and the thermodynamic cost has been investigated for a chemical reaction model. Here, we introduce this approach to sensory adaptation model of E. coli, and examined a relationship between adaptation speed and the thermodynamic cost, and efficiency of the adaptation speed. For increasing external noise level in stimulation, the efficiency decreased, but the efficiency was highly robust to external stimulation strength. Moreover, we demonstrated that there is the best noise to achieve the adaptation in the aspect of thermodynamic efficiency. Our quantification method provides a framework to understand the adaptation speed and the thermodynamic cost for various biological systems.     

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression

ObjectivesMammalian DNA methyltransferases are essential to re‐establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl‐CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear.Materials and MethodsThis study used the Cre‐loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real‐time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms.ResultsGermline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity.ConclusionsThese findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Heterozygous loss of Zbtb38 leads to aberrant epiblast cell proliferation and apoptosis shortly after implantation. Heterozygous loss of Zbtb38 reduced the expression of Nanog and Sox2 in ICM and epiblast.     

Effects of cytoskeleton-disrupting agents on tyrosine transport into cultured bovine adrenal chromaffin cells

The effects of various cytoskeleton-disrupting agents on tyrosine transport into chromaffin cells were examined to assess the possibility of cytoskeleton involvement in the regulation of precursor supply for catecholamine synthesis. Tyrosine transport was markedly increased by cytochalasin B. Vinblastine also stimulated tyrosine transport, although its effect was less pronounced than that of cytochalasin B. While colchicine failed to cause any significant increase in the transport under the same conditions. These results therefore suggest a possible role of microfilaments as a factor regulating tyrosine transport into chromaffin cells.     

Synthesis of branched cyclomaltooligosaccharide carboxylic acids (cyclodextrin carboxylic acids) by microbial oxidation

Novel branched cyclomaltooligosaccharide carboxylic acid (cyclodextrin carboxylic acid) derivatives were synthesized by microbial oxidation using Pseudogluconobacter saccharoketogenes to oxidize five types of branched cyclodextrins, including maltosyl beta-cyclodextrin (maltosyl-beta-CyD). For each novel cyclodextrin carboxylic acid derivative synthesized, the hydroxymethyl group of the terminal glucose residue in the branched part of the molecule was regiospecifically oxidized to a carboxyl group to give the corresponding uronic acid. In addition, the physicochemical properties of cyclomaltoheptaosyl-(6-->1)-alpha-D-glucopyranosyl-(4-->1)-alpha-D-glucopyranosiduronic acid (GUG-beta-CyD) (1) and its sodium salt were studied more extensively, as these compounds are most likely to have a practical application.     

Serine proteinase inhibitor 3 and murinoglobulin I are potent inhibitors of neuropsin in adult mouse brain

Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 +/- 0.22 x 10(6) M(-1) s(-1), showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively.     

Effects of antisense oligodeoxynucleotides against opioid receptors on the receptor mRNA contents

It was demonstrated in the previous study that the microinjection of antisense oligodeoxynucleotide (AS ODN) against mu-opioid receptor (MOR) into periaqueductal gray (PAG) of rat brain selectively decreased the MOR mRNA content in PAG, and the decrease in MOR mRNA content was enhanced by pretreatment of the PAG with MOR AS ODN. In the present investigation, effects of the pretreatment of PAG with AS ODN against kappa- or delta-opioid receptor (KOR or DOR) on the decrease in the MOR mRNA content induced by MOR AS ODN were examined. Both KOR and DOR AS ODNs significantly decreased the target mRNA contents, while they did not significantly change MOR mRNA content. The decrease in MOR mRNA content induced by MOR AS ODN, however, was significantly enhanced by the pretreatment of PAG with either KOR or DOR AS ODNs. Results show that the AS ODN has both the specific target mRNA decreasing action and the nonspecific enhancing action on the AS-induced decrease in the mRNA content.     

Disruption of clh-1, a chloride channel gene, results in a wider body of Caenorhabditis elegans

We cloned the clh-1 gene coding for a putative ClC chloride channel in Caenorhabditis elegans. The gene product exhibited a high degree of homology with human ClC-1 and ClC-2. The clh-1 gene was predominantly expressed in the hypodermis, including seam cells. Null mutations of clh-1 caused a significantly wider body and an abnormal alae structure. High osmolarity in the culture medium restored the normal body width of the clh-1 mutants. These results suggest that the clh-1 gene contributes to maintenance of the body width through regulation of osmolarity.