Sensorimotor Modulation Differs with Load Type during Constant Finger Force or Position

During submaximal isometric contraction, there are two different load types: production of a constant force against a rigid restraint (force task), and maintenance of position against a constant load (position task). Previous studies reported that the time to task failure during a fatigue task was twice as long in the force task compared with the position task. Sensory feedback processing may contribute to these differences. The purpose of the current study was to determine the influence of load types during static muscle contraction tasks on the gating effect, i.e., attenuation of somatosensory-evoked potentials (SEPs) and the cortical silent period (cSP). Ten healthy subjects contracted their right first dorsal interosseus muscle by abducting their index finger for 90 s, to produce a constant force against a rigid restraint that was 20% of the maximum voluntary contraction (force task), or to maintain a constant position with 10° abduction of the metacarpophalangeal joint against the same load (position task). Somatosensory evoked potentials (SEPs) were recorded from C3′ by stimulating either the right ulnar or median nerve at the wrist while maintaining contraction. The cortical silent period (cSP) was also elicited by transcranial magnetic stimulation. Reduction of the amplitude of the P45 component of SEPs was significantly larger during the position task than during the force task and under control rest conditions when the ulnar nerve, but not the median nerve, was stimulated. The position task had a significantly shorter cSP duration than the force task. These results suggest the need for more proprioceptive information during the position task than the force task. The shorter duration of the cSP during the position task may be attributable to larger amplitude of heteronymous short latency reflexes. Sensorimotor modulations may differ with load type during constant finger force or position tasks.     

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca(2+) concentration ([Ca(2+)]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K(+) selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na(+) or Ca(2+) channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca(2+)]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca(2+)]i influx through specific Ca(2+) channels and not on the overall [Ca(2+)]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca(2+) regulates cilia and flagella beating is a fundamental question.     

Successful Treatment of Pulmonary Mucormycosis Caused by <Emphasis Type="Italic">Cunninghamella bertholletiae</Emphasis> with High-Dose Liposomal Amphotericin B (10 mg/kg/day) Followed by a Lobectomy in Cord Blood Transplant Recipients

Infection caused by Cunninghamella bertholletiae carries one of the highest mortality rates among mucormycosis, and there are no reported cases that survived from the infection in allogeneic hematopoietic stem cell transplantation recipients occurring before neutrophil engraftment. Here, we present two cases of pulmonary mucormycosis caused by C. bertholletiae occurring before neutrophil engraftment after cord blood transplantation. Both were successfully treated with high-dose liposomal amphotericin B (10 mg/kg/day) combined with micafungin, which was then followed by neutrophil recovery, reduction in immunosuppressive agents, and a subsequent lobectomy. The intensive antifungal therapy immediately administered upon suspicion of mucormycosis greatly suppressed the infection in its early stage and was well tolerated despite its prolonged administration and simultaneous use of nephrotoxic agents after transplantation. Although the synergic effect of micafungin remains unclear, these cases highlight the importance of prompt administration of high-dose lipid polyene when suspecting mucormycosis in highly immunocompromised patients, which enables subsequent diagnostic and therapeutic interventions, resulting in a favorable outcome.     

Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH₂CONH₂) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH₂CH₂CONH₂) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.     

Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse,a Model of Krabbe's Disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.     

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.     

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.