Interaction of iodinated vinculin, metavinculin and α-actinin with cytoskeletal proteins

Iodinated vinculin, metavinculin and α-actinin were used to probe the interaction of these proteins with electrophoretically separated cytoskeletal proteins. Using the gel overlay technique, we detected strong binding of ¹²⁵I-vinculin and ¹²⁵I-metavinculin to α-actinin, 175 kDa polypeptide, talin, vinculin and metavinculin themselves, and moderate binding to actin.¹²⁵I-α-actinin was capable of interacting with vinculin and metavinculin. The specific binding of ¹²⁵-I-α-actinin to vinculin and metavinculin immobilized on a polysterene surface was also demonstrated. We suggest that the ability of vinculin and α-actinin to form a complex may be realized in microfilament-membrane linkages.     

The physical map ofMus musculus chromosome 11 reveals evolutionary relationship with different syntenic groups of genes inHomo sapiens

Summary The physical localization of sequences homologous to three cloned genes was determined by in situ hybridization to metaphase chromosomes. Previous work had assigned the skeletal myosin heavy chain gene cluster (Myh), the functional locus for the cellular tumor antigen p53 (Trp53-1), and the cellular homologue of the viral erb-B oncogene (Erbb) toMus musculus chromosome 11 (MMU11). Our results provide regional assignments ofMyh andTrp53-1 to chromosome bands B2C, and ofErbb to bands A1A4. Taken together with in situ mapping of three other loci on MMU 11 (Hox-2 homeobox-containing gene cluster, theSparc protein, and theColla-1 collagen gene), which have been reported elsewhere, these data allowed us to construct a physical map of MMU11 and to compare it with the linkage map of this chromosome. The map positions of the homologous genes on human chromosomes suggest evolutionary relationships of distinct regions of MMU11 with six different human chromosome arms: 1p, 5q, 7p, 16p, 17p, and 17q. The delineation of conserved chromosome regions has important implications for the understanding of karyotype evolution in mammalian species and for the development of animal models of human genetic diseases.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on -mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl--d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.     

Fucosyltransferase expression in human platelets and leucocytes

The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.