Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.     

Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen.

Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.     

Human megakaryocytic progenitors (CFU-M) assayed in methylcellulose: Physical characteristics and requirements for growth

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony-forming cells (CFU-M) from normal human marrow growing in methylcellulose. Ficoll-hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single-cell origin of the colonies was provided by analysis of the glucose-6-phosphate dehydrogenase (G-6-PD) enzyme type of individually harvested colonies grown from a G-6-PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet-release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU-M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU-M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony-forming cells (CFU-GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA-LCM dose-response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G-100. The resulting conditioned medium increased the cloning efficiency for CFU-M compared with that with crude PHA-LCM (15.3 ± 7.0 and 8.2 ± 5.3/10⁵ marrow cells, respectively).     

The 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides: application to the synthesis of Alzheimer's disease-related amyloid beta peptide (Abeta) 1-42.

An efficient 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides was applied successfully to the synthesis of amyloid beta peptide (Abeta) 1-42 via a water-soluble O-acyl isopeptide of Abeta1-42, i.e. '26-O-acyl isoAbeta1-42' (6). This paper describes the detailed synthesis of Abeta1-42 focusing on the importance of resin selection and the analysis of side reactions in the O-acyl isopeptide method. Protected '26-O-acyl isoAbeta1-42' peptide resin was synthesized using 2-chlorotrityl chloride resin with minimum side reactions in comparison with other resins and deprotected crude 26-O-acyl isoAbeta1-42 was easily purified by HPLC due to its relatively good purity and narrow elution with reasonable water solubility. This suggests that only one insertion of the isopeptide structure into the sequence of the 42-residue peptide can suppress the unfavourable nature of its difficult sequence. The migration of O-acyl isopeptide to intact Abeta1-42 under physiological conditions (pH 7.4) via O--N intramolecular acyl migration reaction was very rapid and no other by-product formation was observed while 6 was stable under storage conditions. These results concluded that our strategy not only overcomes the solubility problem in the synthesis of Abeta1-42 and can provide intact Abeta1-42 efficiently, but is also applicable in the synthesis of large difficult sequence-containing peptides at least up to 50 amino acids. This synthesis method would provide a biological evaluation system in Alzheimer's disease research, in which 26-O-acyl isoAbeta1-42 can be stored in a solubilized form before use and then rapidly produces intact Abeta1-42 in situ during biological experiments.     

Electrophysiology and kinesiology for health and disease.

This paper summarizes my Basmajian keynote presentation at the 2004 International Society of Electrophysiology and Kinesiology Conference. I dedicate this paper to Dr. Herbert A. deVries, the mentor of my research career. The following topics will be covered from the standpoint of Electrophysiology and Kinesiology for health and disease: (1) electromechanical manifestations of neuromuscular fatigue and muscle soreness, (2) cardiac depolarization-repolarization characteristics of normal and patients, (3) etiology of obesity and diabetes and autonomic nervous system, and (4) functional electrical stimulation for health and disease, respectively.     

Analysis of HLA-DQ α sequences for prenatal diagnosis in single fetal cells from maternal blood

We have extended a previously developed method that allows prenatal DNA diagnosis of female fetuses through the isolation of single nucleated erythrocytes from maternal blood by developing a method that can distinguish between maternal and fetal nucleated erythrocytes. Nucleated erythrocytes were separated by a density-gradient method and then collected by micromanipulation. Sex was determined after primer extension preamplification (PEP) of the entire genome of a single cell, and human leukocyte antigen (HLA)-DQ α type was determined after further amplification of this gene. The HLA-DQ α genotype of fetal erythrocytes in maternal blood samples and their corresponding paternal and maternal lymphocytes were successfully determined in all cases. The accuracy of the method was determined by using single nucleated erythrocytes from umbilical cord blood from five normal deliveries. This is the first demonstration that the fetal HLA-DQ α gene sequences can be identified in a small aliquot of a single nucleated erythrocyte in maternal blood. We believe that this method ushers in a new era in which the reliability and accuracy of noninvasive prenatal DNA diagnosis from maternal blood is markedly improved. Received: 18 April 1997 / Accepted: 1 October 1997     

Heterogeneity of phenotypic and genotypic traits including organic-acid resistance in Escherichia coli O157 isolates

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing Escherichia coli (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.