Muscle‐specific calpain‐3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction

CAPN3 (also called p94/calpain‐3) is a skeletal muscle‐specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb‐girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril‐based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

cDNA microarray analysis of individual Duchenne muscular dystrophy patients

We have developed a novel cDNA microarray encompassing 3500 genes expressed in skeletal muscle. With this system, we have performed the first study of gene expression in samples from individual patients. We analyzed muscle specimen from individuals with Duchenne muscular dystrophy to identify differences among patients. Among the variably expressed genes, we focused on the expression of the genes encoding HLA-related proteins, myosin light chains and troponin Ts as markers of muscle necrosis and regeneration. The expression patterns of these genes correlated with the severity of dystrophic changes on histological examination. Our cDNA microarray provides a new tool to investigate molecular muscle pathology.     

Acute effects of zolpidem on daytime alertness, psychomotor and physical performance

In a double-blind cross-over study, seven athletes received zolpidem (10mg) or placebo in two sessions over two nights. Residual effects on subsequent daytime functions were evaluated objectively by measuring psychomotor and physical performance using a combined test of finger dexterity, a simple discriminatory reaction test, critical flicker fusion test (CFF), vertical jump, and 50-m sprint, as well as subjectively, by visual analog scales. Zolpidem shortened self-estimated sleep latency and increased total sleep at nighttime. There was no change in alertness and fatigue scales on the following day in the zolpidem session, but realm of daytime well-being was slightly worsened. The CFF test showed significantly better results in the zolpidem group than in the placebo group. Zolpidem did not have effects in athletic evaluation. Zolpidem has a hypnotic activity without disturbing psychomotor and physical performance on the following day when given to healthy adults, suggesting zolpidem may be used in healthy athletes to adjust their extrinsic sleep disturbances and their consecutive psychomotor and physical impairments.     

CCAAT/enhancer binding proteins are expressed in the gerbil hippocampus after transient forebrain ischemia

We analyzed CCAAT/enhancer binding protein (C/EBP) family protein levels during reperfusion after a single episode of sublethal forebrain ischemia in the gerbil hippocampus to investigate their expression after ischemia and correlation with neuronal cell death. The common carotid arteries were surgically exposed bilaterally and occluded for 10 min to induce forebrain ischemia in adult Mongolian gerbils. C/EBPalpha, beta, delta, epsilon, zeta protein immunoreactivity was expressed in the hippocampal layer of the CA1 region at 72 h after ischemia and peaked at 96 h. These results appear to correlate with neuronal degeneration as shown by hematoxylin and eosin staining and DNA fragmentation in the terminal transferase biotinylated-UTP nick end labeled-method. The present results demonstrate that C/EBP family proteins appear in the selectively vulnerable CA1 pyramidal cell layer in gerbils during neuronal degeneration, and may serve as a signal that neurons are progressing to neuronal cell death and DNA fragmentation.     

Clinical application of aquaporin research: aquaporin-1 in the peritoneal membrane

Peritoneal dialysis (PD) is an established mode of renal replacement therapy based on the exchange of fluid and solutes between blood and a dialysate that has been instilled in the peritoneal cavity. The dialysis process involves osmosis, as well as diffusive and convective transports through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores responsible for the selective transport of water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water, fit with the characteristics of the ultrasmall pore. Peritoneal transport studies using AQP1 knockout mice demonstrated that the osmotic water flux across the peritoneal membrane is mediated by AQP1. This water transport accounts for 50% of the ultrafiltration during PD. Treatment with high-dose corticosteroids upregulates the expression of AQP1 in peritoneal capillaries, resulting in increased water transport and ultrafiltration in rats. AQP1 may also play a role during inflammation, as vascular proliferation and leukocyte recruitment are both decreased in mice lacking AQP1. These data illustrate the potential of the peritoneal membrane as an experimental model in the investigation of the role of AQP1 in the endothelium at baseline and during inflammation. They emphasize the critical role of AQP1 during PD and suggest that manipulating AQP1 expression could be clinically useful in PD patients.     

An activation of parvocellular oxytocinergic neurons in the paraventricular nucleus in oxytocin-induced yawning and penile erection

Intracerebroventricular (ICV) or PVN local injections of oxytocin induce yawning and penile erection, for which a positive feedback mechanism for the PVN oxytocinergic activation is suggested, but this had not been directly substantiated in vivo. We have assessed the behavioral effects and activity of oxytocinergic neurons with double-staining for c-Fos and oxytocin in the PVN after ICV administration of oxytocin in adult male rats. ICV oxytocin injections (50 and 200 ng) dose-dependently induced yawning and penile erection and significantly increased the percentage of c-Fos positive oxytocin neurons in the medial, dorsal and lateral parvocellular subdivision of the PVN. However, increases in the magnocellular portion were not significant. We also found that lithium chloride (LiCl, 0.5 and l.0 mEq), a compound known to activate oxytocinergic neurons, also significantly increased the percentage of c-Fos positive oxytocin neurons in all PVN portions. However, LiCl did not induce yawning and penile erection, but counteracted the oxytocin-induced yawning and penile erection. These results suggest that if the activation of oxytocinergic neurons in the PVN is important for mediating oxytocin-induced yawning and penile erection, a selective activation of parvocellular oxytocinergic neurons in the PVN is likely to be involved.     

Studies on respiratory infections in primary care clinic (IV). Antibiotic sensitivity of bacteria isolated from patients with respiratory infections visiting 21 private clinics in the Tohoku District of Japan

We determined the MICs of ampicillin, methicillin, cefaclor, cefixime, cefteram, ofloxacin and ciprofloxacin against a total of 1,448 strains from 11 species: 464 strains of Staphylococcus aureus, 306 strains of Streptococcus pneumoniae, 114 strains of Streptococcus pyogenes, 37 strains of Branhamella catarrhalis, 329 strains of Haemophilus influenzae, 32 strains of Escherichia coli, 66 strains of Klebsiella pneumoniae, 26 strains of Enterobacter cloacae, 20 strains of Serratia marcescens, 12 strains of Pseudomonas aeruginosa and 42 strains of Acinetobacter calcoaceticus, isolated from the throat swab and the sputum of 2,539 patients with respiratory infections who visited 21 private clinics in Tohoku district of Japan during the period from January to April in 1989. Ciprofloxacin and ofloxacin were more active against S. aureus, B. catarrhalis, P. aeruginosa and A. calcoaceticus than other antibiotics. Ampicillin and cefteram were more active against S. pneumoniae and S. pyogenes than other antibiotics. New-quinolones and cephems of new-generation were active against H. influenzae, E. coli, K. pneumoniae, E. cloacae and S. marcescens. Of 30 strains of S. aureus which were resistant (MIC greater than or equal to 12.5 micrograms/ml) to ampicillin, only one strain was resistant (MIC greater than or equal to 12.5 micrograms/ml) to methicillin. Twenty strains (6.5%) of S. pneumoniae and 49 strains (14.9%) of H. influenzae were resistant (MIC greater than or equal to 1.56 micrograms/ml) to ampicillin. Of 101 strains of H. influenzae of which their beta-lactamase activity was determined by Nitrocephin-method, 27 (26.7%) were beta-lactamase-positive strains. The above results indicated that MRSA is only rarely found in primary care clinics but the incidence of ampicillin-resistant H. influenzae in primary care clinics is almost the same as that of the intensive care clinic, i.e. medical school-affiliated hospitals. Therefore caution should be exercised as regards antibiotic resistance of the causative organism even in primary care clinics.     

Imaging of lung cancer in the era of molecular medicine

Recent discoveries characterizing the molecular basis of lung cancer brought fundamental changes in lung cancer treatment. The authors review the molecular pathogenesis of lung cancer, including genomic abnormalities, targeted therapies, and resistance mechanisms, and discuss lung cancer imaging with novel techniques. Knowledge of the molecular basis of lung cancer is essential for radiologists to properly interpret imaging and assess response to therapy. Quantitative and functional imaging helps assessing the biologic behavior of lung cancer.     

Divergent effects of lithium and sodium valproate on brain-derived neurotrophic factor (BDNF) production in human astrocytoma cells at therapeutic concentrations

Mood stabilizers such as lithium (Li) or valproic acid (VPA) are used in the therapy of bipolar disorders, but the mechanisms by which these medicines work is unclear. Recently, neuroprotection has attracted attention as a potential action for VPA and Li. The close spatial relationship of the pre- and post-synapse with an astrocyte process within a 'tripartite synapse' suggests that mood stabilizer actions on astrocytes may be important. Therefore, we examined the effect of Li and VPA, at therapeutic concentrations, on brain-derived neurotrophic factor (BDNF) production in cultured human astrocytoma cells over an extended period of exposure. Released (extracellular) and intracellular BDNF was measured with sandwich-ELISA. Intracellular BDNF mRNA was also quantified using RT-PCR. VPA treatment potentiated the level of extracellular BDNF, whereas Li reduced it. Furthermore, VPA caused increased intracellular levels of BDNF protein and mRNA, while exposure to Li led to no significant differences compared to control cells. We suggest the possibility that VPA and Li have divergent effects on astrocyte BDNF production. Mood stabilizers play an essential role in regulating BDNF not only in neurons, but also in astrocytes. These findings could form the basis of a new astrocyte-targeted approach towards developing effective medications to treat bipolar disorders.