Glycosylation of the OMP85 homolog of Porphyromonas gingivalis and its involvement in biofilm formation

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.     

ATP-induced shrinkage of DNA with MukB protein and the MukBEF complex of Escherichia coli

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-gammaS, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.     

Unique anti-apoptotic activity of EAAC1 in injured motor neurons

Injured motor neurons of the adult rat can survive, whereas similar axotomy causes gradual motor neuron death in the adult mouse. We report that the decreased expression of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) following nerve injury is associated with motor neuron death in the mouse. Glutamate transporters play a crucial role in prevention of neuronal death by suppressing glutamate toxicity. However, the possible functional role of EAAC1 in preventing neuron death has not been resolved as compared with glial glutamate transporters such as GLT-1. Here, we have revealed a unique 'rescue' function of EAAC1, which is independent of removal of extracellular glutamate. During apoptotic stimuli, a mitochondrial protein, holocytochrome c synthetase (HCCS), translocates to outside the mitochondria, binds to and suppresses the X-linked inhibitor of apoptosis protein (XIAP), leading to activation of caspase-3. The N-terminus of EAAC1 can bind to HCCS, which interferes with the HCCS-XIAP association, and thereby maintain XIAP activity. This unique anti-apoptotic mechanism of EAAC1 functions in rescuing PC12 cells and motor neurons from NGF deprivation and nerve injury, respectively.     

Quality evaluation of rice crackers based on physicochemical measurements

The processing suitability as a material for rice crackers was characterized in the present study, based on physicochemical measurements and sensory testing of high-quality premium rice, low-amylose rice, Japonica-Indica hybrid rice, and red rice as the rice cultivar samples. Puffed rice crackers were prepared and the relationship between the physicochemical properties of the rice grains and the quality of the resulting products was investigated. It was possible to estimate the physical properties of a rice cracker by using multiple-regression analysis based on the chemical components, pasting properties and physical properties of the constituent rice. A formula for estimating the amylose content of the constituent rice was developed from the results of physicochemical measurements of the rice crackers. We assayed the quality of commercial rice crackers and examined the deterioration during the storage by measuring the physicochemical properties. The hardness and fat acidity of crackers increased markedly during storage for 20 d at 35 °C. The novel method of a one-bite test with a Tensipresser was useful to assay the quality of a rice cracker and made it possible to evaluate the quality deterioration of the rice cracker during storage.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.     

Characterization of p-chloroamphetamine-induced penile erection and ejaculation in anesthetized rats

Methodological shortcomings present in elicitation of male sexual reflexes in anesthetized animals. The present study has demonstrated, however, that intraperitoneal (i.p.) injection of p-chloroamphetamine (PCA), an indirect serotonin (5-HT) agonist, elicited simultaneously both penile erection and ejaculation in anesthetized rats. PCA (2.5-10.0 mg/kg, i.p.) caused an intermittent cluster of genital responses consisting of penile erection, glans erections, and penile cups, which closely resembles the response observed during the ex copula tests in unanesthetized rats. Measurements of intracavernous penile pressure showed that rhythmic changes in penile pressure were produced by PCA, together with glans erections and penile cups. PCA also caused a frequent ejaculations and the weighing of ejaculate accumulated over 0.5 hr was increased in a bell-shaped pattern, and the maximum effect was observed at 5.0 mg/kg. Pretreatment with p-chlorophenylalanine, a serotonin (5-HT)-synthesis inhibitor, significantly inhibited the expression of PCA-induced penile erection and ejaculation, while acute spinal transection at thoracic level did not affect the sexual responses. These results indicate that PCA-induced penile erection and ejaculation in anesthetized rats are mainly produced by the release of 5-HT, which is limited to the lower spinal cord and/or the peripheral sites. Furthermore, the sexual responses can be easily and reliably elicited by administration of PCA, which may be useful for the study of the mechanisms underlying male sexual functions.     

The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7's interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.     

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)