Cardiac abnormalities cause early lethality of jumonji mutant mice

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.     

Phylogenetic analysis of honey bee behavioral evolution

DNA sequences from three mitochondrial (rrnL, cox2, nad2) and one nuclear gene (itpr) from all 9 known honey bee species (Apis), a 10th possible species, Apis dorsata binghami, and three outgroup species (Bombus terrestris, Melipona bicolor and Trigona fimbriata) were used to infer Apis phylogenetic relationships using Bayesian analysis. The dwarf honey bees were confirmed as basal, and the giant and cavity-nesting species to be monophyletic. All nodes were strongly supported except that grouping Apis cerana with A. nigrocincta. Two thousand post-burnin trees from the phylogenetic analysis were used in a Bayesian comparative analysis to explore the evolution of dance type, nest structure, comb structure and dance sound within Apis. The ancestral honey bee species was inferred with high support to have nested in the open, and to have more likely than not had a silent vertical waggle dance and a single comb. The common ancestor of the giant and cavity-dwelling bees is strongly inferred to have had a buzzing vertical directional dance. All pairwise combinations of characters showed strong association, but the multiple comparisons problem reduces the ability to infer associations between states between characters. Nevertheless, a buzzing dance is significantly associated with cavity-nesting, several vertical combs, and dancing vertically, a horizontal dance is significantly associated with a nest with a single comb wrapped around the support, and open nesting with a single pendant comb and a silent waggle dance.     

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.     

Glycosaminoglycan affinity of the complete fibroblast growth factor family

BACKGROUND: Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype. METHODS: To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins. RESULTS: We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood. GENERAL SIGNIFICANCE: This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.     

Monocarboxylate transporter-1 is required for cell death in mouse chondrocytic ATDC5 cells exposed to interleukin-1beta via late phase activation of nuclear factor kappaB and expression of phagocyte-type NADPH oxidase

Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.     

Bisphenol A Does Not Affect Memory Performance in Adult Male Rats

Bisphenol A (BPA) is an estrogenic endocrine disruptor used for producing polycarbonate plastics and epoxy resins. This study investigated the effects of oral BPA administration on memory performance, general activity, and emotionality in adult male Sprague Dawley rats using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial memory performance. In addition, in order to confirm the effects of BPA on spatial memory performance, we examined whether intrahippocampal injection of BPA affects spatial memory consolidation. In the MAZE test, although oral BPA administration at 10 mg/kg significantly altered the number of entries into the incorrect area compared to those of vehicle-treated rats, male rats given BPA through either oral administration or intrahippocampal injection failed to show significant differences in latencies to reach the reward. Also, oral BPA administration did not affect fear-motivated memory performance in the step-through passive avoidance test. Oral BPA administration at 0.05 mg/kg, the lowest dose used in this study, was correlated with a decrease in locomotor activity in the open-field test, whereas oral administration at 10 mg/kg, the highest dose used in this study, was correlated with a light anxiolytic effect in the elevated plus-maze test. The present study suggests that BPA in adulthood has little effect on spatial memory performance in male rats.     

Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.     

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily,Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266^∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.     

Purification, cloning and characterization of egg lectins from the teleost Tribolodon brandti

Three L-rhamnose-binding egg lectins, TBL1, TBL2 and TBL3, were isolated from the eggs of the Far East dace Tribolodon brandti by a combination of affinity chromatography on L-rhamnose-Sepharose 6B gel and reversed-phase HPLC. L-rhamnose is a common inhibitor of the purified lectins and strongly inhibited the hemagglutinating activity of TBL2 and TBL3, but less weakly that of TBL1. L-arabinose, which has the same hydroxyl group orientation at C2 and C4 as L-rhamnose, and D-galactose showed no inhibitory activity against TBL1 but showed weak inhibitory activity against TBL2 and TBL3. The open reading frames of the cDNAs of TBL1, TBL2 and TBL3 encoded for mature proteins of 207, 189, and 293 amino acid residues, respectively. A BLAST homology search showed that the TBLs have about 40% homology to the carbohydrate recognition domains of rhamnose-binding lectins in salmonid eggs. The tandem repeated domains present in TBL1, TBL2 and TBL3 were two, two and three, respectively. TBL2 was exclusively expressed in ovary, while TBL1 and TBL3 were expressed mainly in ovary and weakly in various tissues including gill, heart, kidney, liver, spleen and testis.     

Production of anti-amyloid β antibodies in mice fed rice expressing amyloid β

The main signs of Alzheimer's disease (AD) are cognitive impairment and senile plaques composed of amyloid beta (Aβ) observed in patients' brains. Therefore, therapy for AD focuses on the removal of Aβ. We developed an "edible vaccine" that employs intestinal immunity with little to no side effects. Rice was utilized as an edible vaccine. It expressed GFP-Aβ42. Aβ rice was administered orally to wild-type (WT) mice causing production of anti-Aβ antibodies. Since Aβ rice was mixed with the cholera toxin B subunit (CTB), antibody against the rice seed protein was also produced. Then, mice were caused to develop immune tolerance against the rice seed protein by oral administration of Aβ rice mixed with CTB. The results indicated that only anti-Aβ antibodies were produced.