Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.     

Establishment and characterization of monoclonal antibody against androgen receptor

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.     

Detection of maltose fermentation genses in the baking yeast strains of Saccharomyces cerevisiae

Y.ODA AND K. TONOMURA. 1996. The presence of any one of the five unlinked MAL loci ( MALI, MAL2, MAL3, MAL4 AND MAL6 ) confers the ability fo ferment maltose on the yeast Saccharomyces cerecvisiae . Each locus is composed of three genes encoding maltose permease, α-glucosididase and MAL activator. Chromosomal DNA of seven representative baking strains has been separated by pulse-field gel electrophoresis and probed with three gense in MAL6 locus. The DNA bands to which all of the three MAL derived probes simultaneously hybridized were chromosome VII carrying MAL1 in all of the strains tested, chromosome XI carrying MAL4 in six strains, chromosome III carrying mal2 in three strains and chromosomes II and VIII carrying MAL3 and MAL6 , respectively, in the one strain. The number of MAL loci in bakig strains was comparable to those of brewing strins.     

Group I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, contains thirty-two introns. Twenty-five of these introns possess the characteristic secondary structures and consensus sequences of group II introns. The remaining seven are group I introns, six of which happen to interrupt the gene coding for subunit 1 of cytochrome oxidase (cox1). Interestingly, the insertion sites of one group II and four group I introns in the cox1 gene coincide with those of the respective fungal mitochondrial interns. Moreover, comparison of the four group I introns with their fungal counterparts shows that group I introns inserted at identical genomic sites in different organisms are indeed related to one another, in terms of the peptide sequences generated from the complete or fragmental ORFs encoded by these introns. At the same time, the liverwort introns turned out to be more divergent from their fungal cognates than the latter are from one another. We therefore conclude that vertical transmission from a common ancestor organism is the simplest explanation for the presence of cognate introns in liverwort and fungal mitochondrial genomes.     

Mechanomyographic and electromyographic responses of the triceps surae during maximal voluntary contractions.

The objective of this study was to examine the effect of joint angle on the electromyogram (EMG) and mechanomyogram (MMG) during maximal voluntary contraction (MVC). Eight subjects performed maximal isometric plantar flexor torque productions at varying knee and/or ankle angles. Maximal voluntary torque, EMG, and MMG from the soleus (Sol), medial (MG) and lateral gastrocnemius (LG) muscles were measured at different joint angles. At varying knee angles, the root mean squared (rms) MMG amplitude of the MG and LG increased with knee joint extension from 60 degrees to 180 degrees (full extension) in steps of 30 degrees, whereas that of the Sol was constant. At varying ankle angles, the rms-MMG of all muscles (Sol, MG, and LG) decreased with torque as ankle joint extending from 80 degrees (10 degrees dorsiflexion position) to 120 degrees (30 degrees plantar flexion position) in steps of 10 degrees. In each case, changes in the rms-MMG of the three muscles were almost parallel to those in torque. In contrast, there were no significant differences in the rms-EMG of all muscles among all joint angles. Our data suggest that the MMG amplitudes recorded from individual muscles during MVCs can represent relative torque-angle relationships that cannot be represented by the EMG signals.     

Mycoplasma orale infection affects K+ and Cl− currents in the HSG salivary gland cell line

Summary The relations between K⁺ channel and Cl⁻ channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K⁺ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K⁺ channels and K⁺ channel expression as estimated by ionomycin- or hypotonically induced K⁺ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl⁻ channels, but only the latter decrease was statistically significant. Also, Cl⁻ currents could be elicited more frequently than K⁺ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K⁺ channels relatively more than Cl⁻ channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurredin vivo.     

Force-dependent changes in movement-related cortical potentials

The purpose of this study was to compare movement-related cortical potentials (MRCPs) associated with different levels of isometric contractions by elbow flexors. Eight healthy, right-handed male subjects participated in this study and performed different levels (10 and 50% of maximal voluntary contraction) of isometric contractions by the right elbow flexors. Electroencephalogram (EEG) signals were recorded from Fz, C3, Cz and C4 of the international 10/20 system. Motor potential (MP) amplitudes (from −200 to approximately −50 ms before force onset) for C3 associated with both force generations was significantly greater (P < 0.01) than those for C4, indicating that contralateral predominance of MRCP was observed in the right arm flexion. In Fz, the potentials of negative slope (NS′) (from −600 to approximately −200 ms) and MPs for 50% MVC were significantly greater than those of 10% MVC. In Cz, the MP associated with 50% MVC revealed a significantly greater (P < 0.05) value than that with 10% MVC. In C3 and C4, the MP associated with 50% MVC tended to be greater than that with 10% MVC, but no statistically significant differences were found. These force-dependent changes in MRCPs imply increased activation of neural circuits involved in motor preparation and initiation. It is therefore suggested that the larger potentials from Fz and Cz for 50% MVC compared with 10% MVC reflect a greater activation of supplementary motor area for the preparation of the larger force generation.     

Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.