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PubMed1997

The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products.

This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.

Buffers↗
PubMed1988

Large-scale fractionation of S-form lipopolysaccharide from Salmonella abortus equi. Chemical and serological characterization of the fractions.

The S-form lipopolysaccharide of Salmonella abortus equi was separated by a newly elaborated extraction method with organic solvents into three fractions of different chain length of the O-polysaccharide they contained. The three fractions were designated long-chain (20-50 repeating units), short-chain (0-6) and R-fraction (no repeating units) according to their migration pattern in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. The nature of the fractions as long- and short-chain and as R-fraction was confirmed by chemical analysis. The concentration of O-specific sugars was highest in the long-chain fraction, where their molar ratio to glucosamine was ca. 25:1. In the short-chain fraction the ratio of O-sugars to glucosamine was 2.5:1, and in the R-fraction O-specific sugars were absent. The serological properties of the three fractions were in good agreement with their chemical composition.

Carbohydrates↗
PubMed2004

Growth factors improve the in vivo migration of human skeletal myoblasts by modulating their endogenous proteolytic activity.

BACKGROUND: A main technological problem related to the clinical application of myoblast transplantation is the poor migration of transplanted cells. In this study, we investigated a new physiologic approach that consists of coinjecting motogenic factors insulin growth factor (IGF)-1 or basic fibroblast growth factor (bFGF) to enhance the migration of human skeletal myoblasts. Among the different ways by which those factors can induce the cell migration processes, we investigated their capacity to enhance cell endogenous proteolytic activity that will help transplanted cells to migrate through the extracellular matrix. METHODS: In vitro, myoblasts were coincubated with bFGF or IGF-1. Growth factors effects on cell migration were evaluated using invasion chambers, and their effects on proteolytic systems were evaluated by zymography, Western blot, and reverse transcription polymerase chain reaction. In vivo, myoblasts were coinjected with growth factors and the intramuscular migration capacity was assessed using the microtube technique. RESULTS: In vitro, the presence of IGF-1 or bFGF significantly enhanced the expression of the gelatinase matrix metalloproteinase-9 and focalized the fibrinolytic system activity at the cell membrane. In vitro and in vivo, both bFGF and IGF-1 showed strong chemokinetic potentials and improved the migration of human myoblasts. Moreover, the implication some proteinases in the in vivo enhanced migration was confirmed using specific inhibitors (BB94 or amiloride). CONCLUSIONS: These results suggest that IGF-1 or bFGF coinjection with human myoblasts increased their proteolytic activities and consequently their migratory capacity. This study may help to develop approaches that will reduce the number of injection sites for the treatment of Duchenne muscular dystrophy patients.

Amiloride↗
PubMed1995

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Base Sequence↗
PubMed1989

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis.

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.

Amino Acid Sequence↗
PubMed2004

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly.

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.

Actins↗
PubMed1992

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.

Animals↗
PubMed1996

Characterization of Aeromonas hydrophila strains of clinical, animal, and environmental origin expressing the O:34 antigen.

A collection of Aeromonas strains of different origins were characterized for isolates expressing the O:34 somatic antigen. Of over 200 strains tested, approximately 14% belonged to serogroup O:34 with >85% of these strains identified as A. hydrophila regardless of source. A subset of 14 A. hydrophila O:34 strains were further analyzed for a number of structural and pathogenic features. Most O:34 strains expressed similar whole-cell protein profiles with regards to minor bands, but major band differences were noted in outer membrane proteins (OMPs) migrating between the 31K and 58K region. OMP profiles could be subdivided into three distinct patterns. All O:34 strains expressed a heterogeneous O polysaccharide side chain profile in their lipopolysaccharide (LPS), although some variation in the electrophoretic migration of lower and higher molecular weight LPS bands was noted. Polyclonal antisera raised against a 45-K OMP-associated protein of one O:34 strain (AH-195) reacted in immunoblot assays with a major 43 to 46-K OMP in 11 of 14 (79%) O:34 strains tested. Most O:34 strains (69%) were found to be pathogenic in mice with LD-50 values (i.p.) of <1.0 x 10(7) CFU; pathogenicity appeared to correlate best with elevated protease activity. The collective results suggest significant differences in both structural and pathogenic properties between some members of the O:34 group originating from human and nonhuman (fish, water) sources.

Aeromonas hydrophila↗
PubMed1979

Migratory patterns of cloned neural crest melanocytes injected into host chicken embryos.

Cloned quail melanocytes grown in tissue culture for 8 days or more were injected into 2 1/2-day-old chicken embryos. The pigment cells were placed directly into the somitic lumen by means of an injection micropipette. This technique for introducing marked neural crest cells into host embryos causes far less damage than previous methods which require extirpation and replacement of the neural tube. In addition, small numbers of homogeneous cells can be implanted by this procedure. When injected into one of the posterior somites, cultured pigment cells migrated along the ventral neural crest pathway. Three days after injection the melanocytes had migrated ventral to the dorsal root ganglia and prevertebral and primary sympathetic chain ganglia and were seen associated with the adrenal gland and aortic plexi. Melanocytes were frequently found in or adjacent to the gonads and often had migrated as far as the gut.

Animals↗
PubMed2006

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression.

BACKGROUND: Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E2 (PGE2). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression. METHODS: Breast cancer cells (Hs578T, MDA-MB-231 and MCF-7) were treated with selective COX-2 inhibitors (NS-398 and Niflumic acid, NA). Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer. Cell migration and invasion were measured using migration and invasion chamber systems. MMP expression was determined by enzyme immunoassay (secreted protein) and real-time quantitative polymerase chain reaction (mRNA). RESULTS: Our results show that there is a decline in proliferation, migration and invasion by the Hs578T and MDA-MB-231 breast cancer cell lines in the presence of either low concentrations (1 microM or lower) NA or NS-398. We also report that MMP mRNA and protein expression by Hs578T cells is inhibited by NS-398; there was a 50% decrease by 100 muM NS-398. PGE2 completely reversed the inhibitory effect of NS-398 on MMP mRNA expression. CONCLUSION: Our data suggests that COX-2-dependent activity is a necessary component for cellular and molecular mechanisms of breast cancer cell motility and invasion. COX-2 activity also modulates the expression of MMPs, which may be a part of the molecular mechanism by which COX-2 promotes cell invasion and migration. The studies suggest that COX-2 assists in determining and defining the metastatic signaling pathways that promote the breast cancer progression to metastasis.

Breast Neoplasms↗
PubMed1989

Glial cells of mammalian brain produce a variant form of laminin.

Glial cells of mammalian brain transiently produce laminin, an extracellular matrix protein and a potent inducer of neurite outgrowth of cultured neurons. We demonstrate that the glial laminin is a variant with the 200-kDa B chains present and the 400-kDa A chain missing. Immunoprecipitation of cell extracts of purified cultured astrocytes, using antibodies to native laminin, revealed the B chains only, although the same antibody immunoprecipitated both A and B chains from the cell extracts of a basement membrane producing embryonal carcinoma cell line (PYS). This result was further obtained if cell extracts of embryonic mouse brain or the PYS cells were immunoblotted using antibodies to native laminin. Northern analysis of the poly(A)+ RNA from the mouse brain revealed no A chain transcript, although the same probe detected the A chain message in a basement membrane producing F9 cell line. No differences in the sizes of the B chain transcripts in different tissues were seen. Immunostaining of embryonic mouse optic nerve and other areas of the brain, using affinity-purified chain-specific antibodies to the major subunits of laminin, showed that the glial cells expressed the B chains only. It was apparent that the punctate, extracellular deposits of laminin, found in association with neuronal migration and nerve pathway formation in vivo were due to the glial expression of the B chains. The A chain immunoreactivity in the brain was confined to basement membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

[Biomechanical principles in diarthroses and synarthroses. IV: the mechanics of lumbar vertebrae. A pilot study].

UNLABELLED: Theoretical considerations help define the requirements for an apparatus that is to localize the instantaneous helical axes (IHA) of axial rotations of lumbar segments. RESULT: Since the range of axial rotation of an L3/4 segment is in only approximately +/- 1.5 degrees the rotational angle intervals have to be smaller than 0.3 degree with an resolution less than 0.03 degree in order to be able to determine the loci of the IHAs. For the first time in vitro measurements are presented that satisfy this requirement. The data prove that the guidance by the artt. zygapophysiales critically influence the possible positions of the IHA. Comparatively, ligaments and intervertebral disk play a marginally role. During axial torques Tz the IHAs lie dorsal to the intervertebral disk and migrate from one joint to the other depending on axial rotation (length of migration: approximately 3-4 cm). The IHAs lie almost parallel to the axial torque vector. When the joints are removed the IHA is stationary and almost perpendicular to the intervertebral disk and intersects the disk's central region. The screw inclination (pitch) of the instantaneous screw movement is proportional to the rotational angle. Therefore, depending on the direction of rotation, one obtains left or right handed screw movements. This means: axial torsional load leads to an increase in thickness of the intervertebral disk. During preloads that produces extensions the fixed centrodes (paths of axis migration) of intact segments are dorsally beaten out, whereas during flexional loads they are ventrally beaten out. Then, the IHAs migrate through the canalis vertebralis. By the concept "dimeric link chain" the different shapes of the fixed centrode are traced back to the morphology of curvature of the articulating surfaces. The measurements suggest the hypothesis that the distinct nonlinearity of the load displacement curves (s-shape of alpha = alpha (Tz) funktion is an affection of IHA migration. Comparatively, the influence of ligaments can be neglected. The measurements suggest the hypothesis that the hysteresis of the load displacement curves (neutral zone) is an artefact that does not appear in vivo. Altogether, the experiments prove that the loci of the IHAs are determined by the interplay of preload, structure of the applied force system and morphology of curvature of the articulating surfaces. By that the possibility is clinically given to calculate the multitude of possible movements as function of muscle activity when in the individual the shape and the position of the articulating surfaces are measured in vivo (by NMR-methods e.g.). A physically based classification of pathological cases seems to be possible.

Biomechanical Phenomena↗
PubMed2003

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta.

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.

Aspartic Acid↗
PubMed1996

Anionic biopolymers as blood flow sensors.

The finding of flow-dependent vasodilation rests on the basic observation that with an increase in blood flow the vessels become wider, with a decrease the vascular smooth muscle cells contract. Proteoheparan sulphate could be the sensor macromolecule at the endothelial cell membrane-blood interface, that reacts on the shear stress generated by the flowing blood, and that informs and regulates the vascular smooth muscle cells via a signal transduction chain. This anionic biopolyelectrolyte possesses viscoelastic and specific ion binding properties which allow a change of its configuration in dependence on shear stress and electrostatic charge density. The blood flow sensor undergoes a conformational transition from a random coil to an extended filamentous state with increasing flow, whereby Na+ ions from the blood are bound. Owing to the intramolecular elastic recoil forces of proteoheparan sulphate the slowing of a flow rate causes an entropic coiling, the expulsion of Na+ ions and thus an interruption of the signal chain. Under physiological conditions, the conformation and Na+ binding proved to be extremely Ca(2+)-sensitive while K+ and Mg2+ ions play a minor role for the susceptibility of the sensor. Via counterion migration of the bound Na+ ions along the sensor glycosaminoglycan side chains and following Na+ passage through an unspecific ion channel in the endothelial cell membrane, the signal transduction chain leads to a membrane depolarization with Ca2+ influx into the cells. This stimulates the EDRF/NO production and release from the endothelial cells. The consequence is vasodilation.

Biosensing Techniques↗
PubMed2003

Polymerase chain reaction amplification of samples from Japanese monkeys using primers for human short tandem repeat loci.

We examined the species specificity of six commercially available human short tandem repeat systems (for CSF1PO, TPOX, TH01, F13A01, FESFPS and vWA) using six samples obtained from the Japanese monkey Macaca fuscata. Macaque genes were amplified with human TPOX and F13A01 primers. During electrophoresis, macaque polymerase chain reaction (PCR) products amplified with the TPOX primer migrated more slowly than human ones, and migrated close to human CSF1PO bands. Macaque PCR products amplified with the F13A01 primer also migrated more slowly than the human equivalent. The six macaque samples were classified into three types by the TPOX primer and into six types by the F13A01 primer. These results suggest that the primers for human TPOX and F13A01 loci can be used to distinguish between human and Japanese macaque samples.

Animals↗
PubMed2006

Anisotropic optical properties in electroluminescent conjugated polymers based on grazing angle photoluminescence measurements.

Grazing angle photoluminescence (GPL) originates from a waveguided light emitted at grazing angle to the substrate due to the total internal reflections, and the light emission is polarized with enhanced intensity at selective mode wavelength. GPL measurements reveal the optical anisotropy of luminescent conjugated polymers, in particular, the alignment of emitting dipoles from which emission occurs, in contrast to spectroscopic ellipsometry measurements that give the anisotropy in the absorption. Based on the GPL emission intensities and spectra, we investigate the anisotropic optical properties in electroluminescent poly(9,9'-di-n-octylfluorene-alt-benzothiadiazole) (F8BT) conjugated polymer thin films of different molecular weights (M(n) = 9-255 kg/mol), both in the pristine and annealed states. The optical anisotropy in F8BT films generally increases with molecular weight, suggesting that higher molecular weight polymers with longer chains are more likely to lie in-plane to the substrate. Upon annealing, high molecular weight F8BT films show even a higher degree of anisotropy, in contrast to low molecular weight F8BT films that become more isotropic. Annealing causes the polymer chains to rearrange and adopt a configuration in which the interchain exciton migration to better ordered low energy (LE) emissive states is strongly suppressed. We observe that the emissive states in F8BT are strongly affected by the local polymer chain arrangement, producing the less ordered high energy (HE) emissive states near the substrate interface where there is a higher degree of chain disorder and the LE states in the bulk of the film. When spin coated onto a quartz substrate precoated with a poly(styrenesulfonate)-doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) layer, films of F8BT show severe luminescence quenching near the PEDOT:PSS interface for both the LE and HE emissive states, but a selective quenching of the LE states in the bulk of the film. These observations have important implications for fabricating efficient electronic devices using conjugated polymers as an active material, since the performance of these devices will strongly depend on anisotropic optical properties of electroluminescent conjugated polymers.

Journal Article↗
PubMed1978

Bovine tendons. Aging and collagen cross-linking.

The level of cross-linking between the polypeptide chains of the collagen molecules in bovine tendons of different ages has been assessed by measuring quantitatively through densitometry the changes in the ratios of individual cyanogen bromide peptides separated on polyacrylamide gels. An increase in the number of cross-links in mature, as compared to young, tendons correlates with a depletion in the proportion of the free, COOH-terminal peptides alpha1-CB6 and alpha2-CB3,5 and with an increase in a broad distribution of peptides moving slowly in the gels: these peptides are not seen in digests of acid-soluble collagen. Some of these peptides which are presumably cross-linked migrate more slowly than beta components and collagen alpha chains and are apparently of a higher molecular weight. No increase in cross-linked peptides is detectable beyond the age of maturation; this analysis refutes, at least in this tissue, the common presumption that progressive cross-linking occurs in collagen through an animal's lifetime.

Aging↗