THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.     

Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes

Floral organ identity is largely controlled by the spatially restricted expression of several MADS-box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE . Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF , has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.     

Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog,Erinaceus europaeus,and the phylogenetic position of the Lipotyphla

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches. Correspondence to: U. Arnason     

Microfiltration of yeast suspensions with self-cleaning spiral vortices: possibilities for a new membrane module design

A novel method of producing controlled vortices was used to reduce both concentration polarization and membrane fouling during microfiltration of Saccharomyces cerevisiae broth suspensions. The method involves flow around a curved channel at a sufficient rate so as to produce centrifugal instabilities (called Dean vortices). These vortices depolarize the build-up of suspended particles such as yeast cells at the membrane-solution interface and allow for increased membrane permeation rates. Various operating conditions under which such vortices effectively reduced cake build-up of suspended particles such as yeast cells at the membrane-solution interface and allow for increased membrane permeation rates. Various operating conditions under which such vortices effectively reduced cake build-up during microfiltration of 0 to 0.55 dry wt% yeast broth were investigated. Flux improvements of over 60% for 0.25 dry wt% yeast broth for flow with over that without Dean vortices were observed. This beneficial effect increased with increasing retentate flow rate and increasing transmembrane pressure and decreased with increasing concentration of suspended matter. Similar behavior was observed whether the cells were viable of killed. the improvement in flux in the presence over that in the absence of vortices correlated well with centrifugal force or azimuthal velocity squared. The relative cake resistances increased with reservoir yeast concentration. These values with vortices increased from 62% to 75% of that without vortices with increasing yeast concentration. The ratio of the cake thicknesses in the limiting case (at high feed concentration) was 3.25. These results suggest that self-cleaning spiral vortices could be effective in maintaining good and steady microfiltration performance with cell suspensions other than those tested. (c) 1995 John Wiley & Sons, Inc.     

Latent Transforming Growth Factor-β Binding Protein Domains Involved in Activation and Transglutaminase-dependent Cross-Linking of Latent Transforming Growth Factor-β

Transforming growth factor-β (TGF-β) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-β, the TGF-β propeptide, and the latent TGF-β binding protein (LTBP). To interact with its cell surface receptors, TGF-β must be released from the latent complex by disrupting noncovalent interactions between mature TGF-β and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-β. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S–transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in crosslinking and formation of TGF-β by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-β generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (ΔN293) or 441 (ΔN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that ΔN293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas ΔN441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.     

Chromosomal Proteins and Cytokinesis: Patterns of Cleavage Furrow Formation and Inner Centromere Protein Positioning in Mitotic Heterokaryons and Mid-anaphase Cells

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.