bluuebunny/crossref_metadata_2025 huggingface dataset:
A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6.
Authors: Sullivan M L , Vierstra R D
Published: May 2006

The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.
MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily.
Authors: Lehmann J M , Riethmüller G , Johnson J P
Published: May 2006

The MUC18 antigen is an integral membrane glycoprotein of 113 kDa whose expression on primary human melanomas correlates with poor prognosis and the development of metastatic disease. MUC18 is expressed only sporadically in benign melanocytic nevi and thin primary melanomas that have a low probability of metastasizing. However, with increasing tumor thickness, MUC18 expression becomes more frequent and it is found on 80% of advanced primary tumors and metastases. MUC18-encoding cDNA clones were obtained by screening a human melanoma phage lambda expression library with monoclonal antibodies produced against the denatured antigen. The deduced sequence of 603 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The highest sequence similarity is with a group of nervous system cell adhesion molecules, which includes neural cell adhesion molecule (N-CAM). The close structural relationship with these molecules suggests that MUC18 may also be a developmentally regulated cell adhesion molecule.
The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others.
Authors: Elices M J , Hemler M E
Published: May 2006

The integrin heterodimer VLA-2, previously known as a collagen receptor, is now shown also to be a laminin receptor. Adhesion of the human melanoma cell line LOX to laminin was inhibited by anti-VLA alpha 2 antibodies. Because VLA-2-mediated LOX cell attachment to laminin was not inhibited by digestion with collagenase, collagen contamination of laminin was not a factor. In addition, VLA-2 from LOX cells bound to immobilized laminin, and binding was disrupted by EDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 also bound to laminin-Sepharose, although less avidly than VLA-2. Thus, at least four separate members of the integrin beta 1 subfamily serve as laminin receptors--i.e., VLA-2 and VLA-3 (this study) together with VLA-1 and VLA-6 (other reports). Whereas LOX and other cell lines used VLA-2 as both a laminin and collagen receptor, fibroblast VLA-2 mediated collagen but not laminin binding. Likewise, VLA-2 from platelets did not interact with laminin. Despite this functional discordancy, VLA-2 from laminin-binding and nonbinding sources was indistinguishable by all immunochemical and biochemical criteria examined. Thus, functional differences in VLA-2 may be due to cell type-specific modulation.
I/Lyn mouse phosphorylase kinase deficiency: mutation disrupts expression of the alpha/alpha'-subunit mRNAs.
Authors: Bender P K , Lalley P A
Published: May 2006

A cDNA encoding the alpha subunit of mouse skeletal muscle phosphorylase kinase was used to compare the expression of alpha mRNAs in normal and phosphorylase kinase-deficient tissues of the I/Lyn mouse. The results demonstrate that two different molecular weight species of poly(A)+ RNA in normal mouse heart and skeletal muscle hybridize to the alpha cDNA. These two mRNAs direct the synthesis of alpha protein and its isoform alpha' in a cell-free translation system. Thus, alpha and alpha' are encoded by two distinct mRNAs. The abundance of both of these mRNAs is reduced dramatically in the phosphorylase kinase-deficient skeletal muscle and heart tissues from the I/Lyn mouse strain. This result indicates that a mechanism common to both alpha and alpha' expression is disrupted by the I/Lyn mutation. The I/Lyn deficiency is inherited as an X chromosome trait. By Southern mapping of Chinese hamster-mouse cell hybrids the alpha gene was localized to the mouse X chromosome, supporting the possibility that the I/Lyn mutation is in the alpha gene. These results are discussed in terms of a cis or trans mutation influencing the expression of either a single alpha/alpha' gene or two genes encoding alpha and alpha'.
Protooncogene expression identifies a transient columnar organization of the forebrain within the late embryonic ventricular zone.
Authors: Johnston J G , van der Kooy D
Published: May 2006

Immunocytochemical studies using monoclonal antibodies directed against oncogenic peptides revealed a heterogeneous distribution of the peptides within the ventricular zone of the embryonic day 18 rat forebrain. The sis-, src-, ras-, and myc-encoded peptides were concentrated in the same isolated clusters of 5-25 radial glial cells (also identified by vimentin staining), providing a transient columnar compartmentalization to the ventricular zone. An increased number of [3H]thymidine-labeled ventricular zone cells were observed within the protooncogene stained radial glial cell columns as compared to noncolumn areas. The columnar heterogeneity of radial glial cells reveals the mosaicism of the embryonic ventricular zone and the differential proliferation of its cells.
Individual microglia move rapidly and directly to nerve lesions in the leech central nervous system.
Authors: McGlade-McCulloh E , Morrissey A M , Norona F , Muller K J
Published: May 2006

Small cells called microglia, which collect at nerve lesions, were tracked as they moved within the leech nerve cord to crushes made minutes or hours before. The aim of this study was to determine whether microglia respond as a group and move en masse or instead move individually, at different rates, and whether they move along axons directly to the lesion or take another route, such as along the edges of the nerve cord. Cell nuclei in living nerve cords were stained with Hoechst 33258 dye and observed under dim ultraviolet illumination using fluorescence optics, a low-light video camera, and computer-assisted signal enhancement. Muscular movements of the cord were selectively reduced by bathing in 23 mM MgCl2. Regions of nerve cord within 300 microns of the crush were observed for 2-6 hr. Only a fraction of microglia, typically less than 50%, moved at any time, traveling toward the lesion at speeds up to 7 microns/min. Cells were moving as soon as observation began, within 15 min of crushing, and traveled directly toward the lesion along axons or axon tracts. Movements and roles of leech microglia are compared with their vertebrate counterparts, which are also active and respond to nerve injury.
Retinal melatonin is metabolized within the eye of xenopus laevis.
Authors: Cahill G M , Besharse J C
Published: May 2006

Retinal synthesis of melatonin, a potent modulator of rhythmic retinal processes, is elevated at night as a result of regulation by a circadian clock. Despite high nocturnal synthetic capacity, both melatonin content and release are low in the retina of the frog Xenopus laevis. We report here that cultured eyecups from Xenopus have the capacity for rapid metabolic breakdown of melatonin. Pharmacological analysis indicates that the initial step in this degradation pathway is deacetylation of melatonin by the enzyme aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13). This produces 5-methoxytryptamine, which is then deaminated by monoamine oxidase [amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4], producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Inhibition of aryl acylamidase with eserine dramatically increases the release of endogenous melatonin by eyecups cultured at night, indicating that this pathway is the normal fate of retinal melatonin. Metabolism within the eye suggests a local neuromodulatory role for retinal melatonin, in contrast to the hormonal role of pineal melatonin.
Inhibitor of hematopoietic pluripotent stem cell proliferation: purification and determination of its structure.
Authors: Lenfant M , Wdzieczak-Bakala J , Guittet E , Prome J C , Sotty D , Frindel E
Published: May 2006

We report here a five-step purification procedure that led to the isolation from fetal calf bone marrow extract of a tetrapeptide, Ac-Ser-Asp-Lys-Pro (Mr 487), exerting a high inhibitory activity on the proliferation of hematopoietic pluripotent stem cells [defined here as spleen colony-forming units (CFU-S)]. The structure of this molecule was established from amino acid analysis, fast atom bombardment mass spectrometry, and 1H nuclear magnetic resonance spectral data. This structure was confirmed by comparison with the corresponding synthetic molecule, which presents identical physiochemical characteristics and biological properties. Natural and synthetic peptides administered to mice (at a dose of 100 ng per mouse) after one injection of cytosine arabinonucleoside prevent CFU-S recruitment into DNA synthesis.
Purification and characterization of a newly identified growth factor specific for epithelial cells.
Authors: Rubin J S , Osada H , Finch P W , Taylor W G , Rudikoff S , Aaronson S A
Published: May 2006

A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phase HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of approximately 28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by greater than 500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing revealed an amino-terminal sequence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation.
A single DNA response element can confer inducibility by both alpha- and gamma-interferons.
Authors: Reid L E , Brasnett A H , Gilbert C S , Porter A C , Gewert D R , Stark G R , Kerr I M
Published: May 2006

Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by alpha- and gamma-interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5' flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-alpha, beta. Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-gamma as well as IFN-alpha. Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5' side of a marker gene. Since the 6-16 gene does not normally respond to IFN-gamma, the context of the ISRE must determine the specificity of the response.
Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.
Authors: Zaitlin D , Hu J , Bogorad L
Published: May 2006

Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk of the RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology.
Induction of class I major histocompatibility complex antigen expression by West Nile virus on gamma interferon-refractory early murine trophoblast cells.
Authors: King N J , Maxwell L E , Kesson A M
Published: May 2006

Primary murine trophoblast giant cells (TGC) do not express detectable major histocompatibility complex (MHC) antigens and are refractory to the MHC-increasing effects of alpha and beta (virus-induced) interferons and gamma (immune type) interferon during early implantation (postcoital days 3.5-6). West Nile virus infection of primary TGC monolayers from postcoital-day-3.5 preimplantation blastocysts induced paternal MHC antigen expression within 16 hr, as detected by immunogold labeling for electron microscopy. Induction is unlikely to have been mediated by secreted virus-induced interferons or other factors, as it occurred in the presence of high concentrations of anti-alpha/beta interferon antibodies and was not induced by virus-inactivated supernatants from MHC-induced primary TGC cultures. Attempts to induce MHC antigen expression with poly(I.C) or recombinant tumor necrosis factor alpha in primary TGC cultures also failed. Thus, the apparent inhibition of MHC antigen expression in primary TGC during early implantation and their refractoriness to induction of de novo MHC antigen expression is not absolute. This may represent a maternal-and/or species-protective evolutionary device. As such, manipulation of this phenomenon may allow a conclusive assessment of the significance of inhibition of MHC antigen expression on trophoblast cells in the implanting semiallogeneic embryo.
Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.
Authors: Honegger A M , Kris R M , Ullrich A , Schlessinger J
Published: May 2006

Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.
A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing.
Authors: Giblin P , Ledbetter J A , Kavathas P
Published: May 2006

The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. We demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8+ T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern we observe differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.
Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.
Authors: Ohkawa J , Okada N , Shinmyo A , Takano M
Published: May 2006

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.
Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression.
Authors: Rubin D C , Ong D E , Gordon J I
Published: May 2006

We have examined the pattern of differentiation of the small intestinal epithelium in fetal rats during the 17th through 21st days of gestation. Five genes expressed in late fetal, neonatal, and adult enterocytes were used as markers of differentiation. They encode three homologous small cytoplasmic hydrophobic ligand binding proteins--liver fatty acid binding protein (L-FABP), intestinal fatty acid binding protein (I-FABP), and cellular retinol binding protein II (CRBP II)--and two apolipoproteins--apoAI and apoAIV. RNA blot hybridization studies indicated that gradients in mRNA concentration from the proximal small intestine to colon appear coincident with the initiation of rapid epithelial cell proliferation and villus formation (days 17-19 of the 22-day gestation period). Immunocytochemical studies disclosed a remarkably heterogeneous pattern of cell-specific expression of the three hydrophobic ligand binding proteins that was not apparent with either apoAIV or apoAI. This "mosaic" staining pattern was observed in morphologically similar cells occupying identical topographic positions along nascent villi in 17- to 18-day fetuses. The onset and resolution of this mosaicism varies between I-FABP, L-FABP, and CRBP II in the proximal small bowel, although it completely resolves by the first postnatal day. The distal small intestine exhibits a developmental delay of 1-2 days in the appearance of this heterogeneous pattern of initial gene expression. Double-label immunofluorescent analyses using L-FABP and I-FABP antibodies indicated that on the 18th day of gestation the proximal small intestinal columnar epithelium contains several populations of enterocytes expressing neither, one, or both proteins. The potential significance of this mosaic pattern of intestinal epithelial differentiation is discussed in light of recent studies with transgenic and chimeric mice.
Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice.
Authors: Chapman V M , Miller D R , Armstrong D , Caskey C T
Published: May 2006

We have used elevated levels of plasma creatine phosphokinase activity to identify muscular dystrophy phenotypes in mice and to screen the progeny of chemical mutagen-treated male mice for X chromosome-linked muscular dystrophy mutations. We were not successful in identifying heterozygous carriers of these induced muscular dystrophy mutations in greater than 8000 progeny. However, we were highly successful in identifying three additional alleles of the characterized mdx locus. These alleles of mdx were recovered from various mutagen-treated males and they occur on an X chromosome that carries flanking markers that allow us to follow the mutations in genetic crosses and in the development of corresponding mutant stocks. These alleles have been designated as mdx2Cv, mdx3Cv, and mdx4Cv. Preliminary data show that mice with mdx2Cv and mdx3Cv mutations have muscular dystrophic phenotypes that do not grossly differ from the characterized mdx mutation. These additional mdx mutations expand the value of mouse models of X chromosome-linked muscular dystrophy and potentially define additional sites of mutation that impair dystrophin expression.
Anomalous and selective DNA mutations of the Old World monkey alpha-globin genes.
Authors: Shaw J P , Marks J , Shen C C , Shen C K
Published: May 2006

It has been a widely accepted hypothesis that the molecular clock slows down during evolution of higher primates. By molecular cloning and nucleotide sequence comparison of a rhesus macaque alpha-globin gene to its homologs in man, orangutan, olive baboon, and other mammals, we demonstrate a burst of evolution of the baboon alpha-globin gene since its separation from the rhesus macaque. This mutation burst has occurred only at the nonsynonymous sites but not the synonymous sites. Its magnitude is at least 10-fold higher than the synonymous substitution rates in higher primates and as high as the synonymous substitution rates of the rodent lineage. On the contrary, the rate of synonymous site substitutions in the alpha-globin genes of either the rhesus macaque or the olive baboon is several times slower than that of human. Our data demonstrate an anomalous exception to the slow rates of molecular evolution in higher primates and provide strong evidence for a recently accelerated evolution of a primate globin gene under an as yet unknown selective force(s).
Low density lipoprotein undergoes oxidative modification in vivo.
Authors: Palinski W , Rosenfeld M E , Ylä-Herttuala S , Gurtner G C , Socher S S , Butler S W , Parthasarathy S , Carew T E , Steinberg D , Witztum J L
Published: May 2006

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component of the fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conjugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to greater than 4096) can be demonstrated in rabbit and human sera.
Complementation of recombinant baculoviruses by coinfection with wild-type virus facilitates production in insect larvae of antigenic proteins of hepatitis B virus and influenza virus.
Authors: Price P M , Reichelderfer C F , Johansson B E , Kilbourne E D , Acs G
Published: May 2006

We describe the coinfection of insects with wild-type and recombinant baculoviruses in which the polyhedrin gene promoter is used to express hepatitis B virus envelope protein (hepatitis B virus surface antigen; HBsAg) or influenza A virus neuraminidase (NA). Viruses were administered per os to larvae of the cabbage looper, Trichoplusia ni, causing an infection that within 5 days resulted in the production of approximately 0.15 mg of HBsAg per insect, representing 1.5% of the total extracted protein, or approximately 2.8 mg of NA per insect, representing 28% of the total extractable protein. The HBsAg and NA produced by infected larvae were purified from insect lysates. These proteins were antigenic as determined by conformation-dependent immunoassays. The NA was enzymatically active with conventional substrates. The method of infection described allows genetic complementation by wild-type virus of recombinant viruses lacking the polyhedrin gene essential for infection per os and has implications for the high-yield production in insect larvae of other recombinant proteins of baculoviruses.
Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.
Authors: Chaen S , Oiwa K , Shimmen T , Iwamoto H , Sugi H
Published: May 2006

To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass microneedle (tip diameter, approximately 7 microns; elastic coefficient, approximately 40 pN/microns), which was coated with skeletal muscle myosin at the tip and extended from a micromanipulator at right angles with the actin cables. When the myosin-coated tip of the microneedle was brought into contact with the exposed surface of the actin cables, it exhibited ATP-dependent movement along the actin cables over a distance of 20-150 microns in 20-200 s (20-23 degrees C) and eventually stopped due to a balance between forces generated by the actin-myosin interaction (800-6000 pN) and by the bent microneedle. Since the load on the force-generating myosin molecules increased with the bending displacement of the microneedle (auxotonic condition), the relation between the load and the sliding velocity of the myosin heads past the actin cables was determined from the time course of the microneedle movement recorded with a video system. The shape of the force-velocity curve thus obtained was convex upwards, similar to that of the force-velocity curve of intact frog muscle fibers obtained under the auxotonic condition.
Regulation of intestinal epithelial cell growth by transforming growth factor type beta.
Authors: Barnard J A , Beauchamp R D , Coffey R J , Moses H L
Published: May 2006

A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.
Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics.
Authors: Schwerzmann K , Hoppeler H , Kayar S R , Weibel E R
Published: May 2006

The oxidative capacity of cat skeletal muscles (soleus, gracilis, and gracilis chronically stimulated for 28 days) was derived from the total mitochondrial content in the muscle, the surface area of mitochondrial inner membranes, and respiratory activities of isolated mitochondria. Mitochondrial content was estimated by standard morphometry. The surface area of mitochondrial inner membranes per unit volume of mitochondria was estimated by a stereological method. The respiratory activities of isolated mitochondria were measured biochemically, using pyruvate/malate, glutamate/malate, succinate, or cytochrome c as substrate. Structurally and functionally, mitochondria from the three muscle types showed nearly identical characteristics. Oxidative activity was dependent on substrate; with succinate, 5.8 ml of O2 per min per ml of mitochondria was the rate most likely to represent physiological conditions. Oxidative activities of 3.1 ml.min-1.ml-1 with pyruvate/malate and 14.5 ml.min-1.ml-1 with cytochrome c as substrates were theoretical lower and upper bounds. The oxidative capacity of each of the three muscles was thus in direct proportion to the total volume of mitochondria in the muscle. The respiratory capacity of isolated mitochondria was very near to the maximal oxygen uptake rate of mitochondria that is commonly estimated in intact muscles of a wide variety of animals.
Yeast artificial chromosomes with 200- to 800-kilobase inserts of human DNA containing HLA, V kappa, 5S, and Xq24-Xq28 sequences.
Authors: Little R D , Porta G , Carle G F , Schlessinger D , D'Urso M
Published: May 2006

Sequences hybridizing to several human gene probes have been recovered as cloned inserts in yeast artificial chromosomes (YACs). Among 2300 YACs made from human leukocyte DNA (totaling about 0.1 genomic equivalent of human DNA) we have found two, 200 and 780 kilobases (kb), containing sequences of V kappa I immunoglobulin (V = variable); one, 240 kb, with class I HLA; and 11, 200-800 kb, with 5S rRNA-encoding DNA (rDNA). Fifty human YACs from a hamster-human cell hybrid with only the Xq24-Xq28 portion of the X chromosome include one that contains two anonymous probe sequences, DX13 and St14, previously inferred by indirect means to lie within about 70 kb of one another in Xq28. The YACs specific for human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human-specific. Furthermore, the human YACs, formed in a 280-fold excess of hamster DNA, do not hybridize to a hamster DNA probe, indicating that individual YACs do not contain a combination of human and hamster DNA. To confirm that sequences are not scrambled, the YACs containing V kappa I or DX13 and St14 sequences were shown to produce restriction fragments identical in mobility to fragments detected by the same probes in total human DNA digested with the same enzymes. YACs may therefore provide large clones to bridge gene mapping at the chromosome level to molecular analyses of small fragments of genomic DNA.
Tumorigenic methylcholanthrene transformants of C3H/10T1/2 cells have a common nucleotide alteration in the c-Ki-ras gene.
Authors: Chen A C , Herschman H R
Published: May 2006

The polymerase chain reaction was used to amplify DNA surrounding the codon 12 region of the c-Ki-ras gene from C3H/10T1/2 cells and from a number of 3-methylcholanthrene (MCA)-transformed derivatives of these cells. Sequence analysis demonstrated that tumorigenic MCAC116/39 cells, known by DNA-mediated transfection to contain an activated c-Ki-ras oncogene, had a G----T transversion in the first position of codon 12 of this gene, resulting in a Gly12----Cys mutation. A combination of polymerase chain-reaction amplification and oligonucleotide hybridization demonstrated that three additional tumorigenic MCA transformants of C3H/10T1/2 cells had an identical mutation in the c-Ki-ras gene. In contrast, this mutation was not present in an MCA-induced C3H/10T 1/2 transformant that was not tumorigenic. The molecular specificity of this MCA-induced mutation resulting in C3H/10T1/2 tumorigenic transformants should provide an excellent system in which to study the roles of transcription, replication, repair, and exogenous factors in the establishment and expression of transformation and tumorigenicity.
Human T cells expressing the gamma/delta T-cell receptor (TcR-1): C gamma 1- and C gamma 2-encoded forms of the receptor correlate with distinctive morphology, cytoskeletal organization, and growth characteristics.
Authors: Grossi C E , Ciccone E , Migone N , Bottino C , Zarcone D , Mingari M C , Ferrini S , Tambussi G , Viale O , Casorati G
Published: May 2006

BB3 and delta-TCS1 monoclonal antibodies identify two distinct nonoverlapping populations of T-cell receptor (TcR) gamma/delta (TcR-1)-positive cells, which express a disulfide-linked and a nondisulfide-linked form of TcR, respectively. BB3+ cells represented the majority of circulating TcR-1+ cells, but they were virtually undetectable in the thymus. On the other hand, delta-TCS1+ cells were largely predominant among TcR-1+ thymocytes but represented a minority in peripheral blood (PB). Similar distributions were observed by clonal analysis of thymocytes or PB TcR-1+ populations. The use of joining region (J)-specific probes indicated that BB3+ and delta-TCS1+ clones displayed different patterns of J rearrangement. Thus, the disulfide-linked form of TcR-1 (BB3+ clones) was associated with the expression of J segments upstream to the C gamma 1 gene segment, whereas the nondisulfide-linked form (delta-TCS1+ clones) was associated with the expression of J segments upstream to C gamma 2. delta-TCS1+ clones, in most instances, exhibited a growth pattern different from that of BB3+ or conventional TcR alpha/beta+ clones as they adhered promptly to surfaces, spread, and emitted long filopodia ending with adhesion plaques. Ultrastructural analyses showed, exclusively in delta-TCS1+ cells, nuclear deformations, uropod formation, and abundant cytoskeletal structures. In addition, immunofluorescence studies of this subset of TcR-1+ cells revealed the presence of abundant microtubules, intermediate filaments, and submembranous microfilaments. Thus, our findings suggest that delta-TCS1+ cells are capable of active motility.
Ischemic and reperfusion injury of rat peripheral nerve.
Authors: Schmelzer J D , Zochodne D W , Low P A
Published: May 2006

A rat model of severe nerve ischemia was used to study the effects of ischemia and reperfusion on nerve conduction, blood flow, and the integrity of the blood-nerve barrier. Conduction failure was consistently found in the sciatic-tibial nerve during 1- and 3-hr ischemic periods. Recovery of the compound muscle action potential was prompt and complete upon reperfusion following 1 hr of ischemia. However, after 3 hr of ischemia, recovery in the proximal portion of the sciatic nerve was less than 10%, and conduction block occurred in the distal portion of the nerve. Nerve blood flow was restored to only 55% and 45% of resting values following 1 and 3 hr, respectively, of ischemia and did not recover even after 2 hr of reperfusion. The blood-nerve barrier was not statistically impaired to the passage of [14C]sucrose following 1 hr of ischemia but was significantly impaired after 3 hr of ischemia. The permeability-surface area product was consistently greater following 1 hr of reperfusion than during the immediate reperfusion period. These data indicate that severe ischemia of peripheral nerve results in reperfusion injury, conduction block, and blood-nerve barrier disruption. Microvascular events, which may occur during reperfusion, may be important in amplifying the nerve fiber damage that began during ischemia.
Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.
Authors: Llinás R , Sugimori M , Lin J W , Cherksey B
Published: May 2006

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.
The basal ganglia, the deep prepyriform cortex, and seizure spread: bicuculline is anticonvulsant in the rat striatum.
Authors: Turski L , Cavalheiro E A , Calderazzo-Filho L S , Bortolotto Z A , Klockgether T , Ikonomidou C , Turski W A
Published: May 2006

The gamma-aminobutyric acid antagonist, bicuculline methiodide (BMI), induces myoclonic seizures in rats when injected into the deep prepyriform cortex at concentrations lower than those that induce convulsions from the amygdala, hippocampus, or neocortex. This observation prompted the suggestion that the deep prepyriform cortex was responsible for seizure generation regardless of the neurotransmitter and neuronal circuits involved. Bilateral intrastriatal application of BMI protects rats against seizures induced by (i) local application of BMI into the deep prepyriform cortex and (ii) systemic application of bicuculline, pilocarpine (a cholinergic agonist), or kainic acid (a glutamate receptor agonist). The region of the striatum sensitive to the previously unknown anticonvulsant action of BMI is located in the immediate vicinity of the deep prepyriform cortex and is 100-150 times more sensitive to the anticonvulsant action relative to the sensitivity of the deep prepyriform cortex to the convulsant action of BMI. These data suggest a powerful gamma-aminobutyric acid-dependent gating role of the basal ganglia in determining the seizure threshold in the forebrain. This argues against the suggestion that the deep prepyriform cortex plays a crucial role in the generation of seizures following systemic administration of convulsants. The discovery of an anticonvulsant action of BMI in the rat striatum contradicts the gamma-aminobutyric acid theory of epilepsy, which implies that deficits in the gamma-aminobutyric acid-mediated inhibition in the central nervous system lead to the emergence of seizures.
Crystal lattice packing is important in determining the bend of a DNA dodecamer containing an adenine tract.
Authors: DiGabriele A D , Sanderson M R , Steitz T A
Published: May 2006

The crystal structure of a DNA duplex dodecamer d(CGCAAAAATGCG) and its complementary strand has been determined at 2.6-A resolution. Although our goal was to deduce the structural features of the static bending of the helical axis exhibited by adenine-tract structures in solution, we conclude that the overall bend of 20 degrees in the direction of the major groove observed here arises from the forces associated with crystal packing. An isomorphous dodecamer brominated on one strand provides experimental evidence that this asymmetric sequence is positioned in two orientations in the crystal lattice that are related by a 180 degrees rotation around the pseudodyad axis of the sequence. The bend in these two differently positioned DNA molecules depends on their orientation in the crystal, not on their sequence. As with previously determined structures containing adenine tracts, the adenine and thymine base pairs are highly propeller twisted. The N-6 of the adenine comes within hydrogen bonding distance of the O-4 of thymine one step down the helix, facilitating the formation of a series of bifurcated hydrogen bonds within the adenine tract. The adenine tract is relatively straight and the bending is localized outside this region.
Phosphorylation and disassembly of intermediate filaments in mitotic cells.
Authors: Chou Y H , Rosevear E , Goldman R D
Published: May 2006

As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of Pi per mol of protein in interphase to 1.9 mol of Pi per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of Pi per mol of protein to 1.5 mol of Pi per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins.
Site-specific antibodies against the PrlA (secY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins.
Authors: Watanabe M , Blobel G
Published: May 2006

Genetic evidence indicates that the PrlA (SecY) protein of Escherichia coli functions as a membrane integrated signal sequence receptor in protein "export"--i.e., in protein translocation across (or integration into) the plasma membrane. We have raised antibodies in rabbits against two synthetic peptides representing the hydrophilic N- or C-terminal region of PrlA. Using these antibodies as probes in cell fractionation experiments, we confirm that PrlA is an integral membrane protein of the plasma membrane of E. coli. Fab fragments prepared from each of the two antisera specifically inhibit protein export by interfering with the binding of preproteins to the plasma membrane. Inhibition of preprotein binding and export by Fab fragments was shown in a cell-free translocation system: precursors for LamB (an integral membrane protein) and for a truncated form of MalE (a periplasmic protein) were first synthesized in a membrane-depleted E. coli-derived cell-free translocation system followed by posttranslational incubation with inverted vesicles derived from the plasma membrane of E. coli. Our data thus indicate that the N and C termini of PrlA are exposed to the cytoplasm. We discuss the possibility that the transmembrane segments of PrlA could be arranged in the lipid bilayer in a cylindrical fashion, thereby delimiting a protein conducting channel, with a signal sequence binding domain represented, at least in part, by the N and C termini of PrlA. Such a channel would also contain a "stop-transfer" sequence binding domain that in response to a stop-transfer sequence would open the cylindrical channel to the lipid bilayer and permit displacement of the polypeptide from the channel to the lipid bilayer, resulting in membrane integration.