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PubMed2005

MSAP enhances migration of C6 glioma cells through phosphorylation of the myosin regulatory light chain.

A key regulatory mechanism in cell motility is the control of myosin activity, which in non-muscle cells is determined by phosphorylation of the myosin regulatory light chain (MRLC). Here we show that MRLC-interacting protein (MIR)-interacting saposin-like protein (MSAP) enhances cell spreading in fibroblasts and migration of rat C6 glioma cells through increases in MRLC phosphorylation. Overexpression of MSAP enhanced the motility of glioma cells measured in matrigel invasion chambers and using a scratch assay. Downregulation of MSAP by RNA interference significantly decreased glioma cell migration and phosphorylation of MRLC. Inhibition of the corresponding MRLC kinase by ML-7 did not affect migration of MSAP-overexpressing cells. The present results show that MSAP controls glioma cell migration via enhancement of MRLC phosphorylation. This effect is independent of the activity of MRLC kinase. Thus, MSAP is a novel modulator of cell motility that influences migration of glioma cells and possibly other tumors.

3T3 Cells↗

PubMed1968

Hemoglobin synthesis in beta-thalassemia: the properties of the free alpha-chains.

The decrease in hemoglobin A (HbA, alpha(2)beta(2)) synthesis in the erythroid cells of patients with beta-thalassemia is due to a selective defect in beta-chain synthesis. Since alpha-chains continue to be formed at a normal rate in these cells, this results in a marked relative excess of alpha-chain synthesis over beta- and gamma-chain synthesis. The alpha-chains uncombined with beta- or beta-like-chains (delta, gamma) will be referred to as free alpha-chains. The experiments presented in this paper show that these free alpha-chains are capable of combining with beta-chains to form HbA and are, therefore, structurally normal. Alternatively, in the absence of added beta-chains, alpha-chains aggregates of various sizes are formed. Peripheral blood from patients with beta-thalassemia was incubated with radioactive amino acids and hemolysates were prepared. Column chromatography demonstrates that a majority of the free alpha-chains are not present in HbA. They are strongly bound to carboxymethylcellulose resin at pHs from 7.0 to 10.0, and do not elute with HbA. However, when chemically prepared hemoglobin H (Hbbeta(4)) is added to the fresh hemolysates, the free alpha-chains are readily recovered in the HbA peak. This indicates that the free alpha-chains are able to combine normally with beta-chains to form HbA. Freshly labeled hemolysates were also subjected to Sephadex G-100 chromatography. The free alpha-chains eluted as a broad peak migrating between myoglobin and hemoglobin, consistent with their forming alpha-chain aggregates of various mol wt between 16,000 and 64,000. It is suggested that the chromatographic behavior of the free alpha-chains reported herein simply reflects the chemical properties of normal alpha-chains in the absence of adequate numbers of beta- or gamma-chains. The tendency of these free alpha-chains to aggregate may lead to their intracellular precipitation and the subsequent destruction of the cells containing them.

Amino Acids↗

PubMed1965

Differences in cattle globins.

1. Comparisons have been made between electrophoretic mobilities of the cattle haemoglobins, HbA, HbB, HbC, HbD and HbF, at pH8.9. The fastest was HbB, then came in decreasing order HbF, HbC, HbA and HbD. 2. Globins were prepared from the main fractions of the five haemoglobins by CM-cellulose chromatography and investigated by starch-gel electrophoresis in four different buffer systems. In three of these (pH2.0 and 11.8) the globins appeared as two bands on stained starch gels. The slowest bands, the alpha-chains, showed the same rate of migration in all five globins. The faster bands, the non-alpha-chains, differed, that of HbF being the fastest and that from HbC the slowest. The other three were intermediate with, however, very small difference between the non-alpha-chains from HbA and HbD. 3. At pH1.8 in an acetate-phosphate-hydrochloric acid-urea buffer three bands appeared in all five globins of which the two slowest were indistinguishable in rates of migration, whereas the rates of migration of the third and fastest bands differed. Explanations for the occurrence of three bands are discussed.

Animals↗

PubMed1997

Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5.

Adenocarcinoma↗

PubMed2002

Prognostic significance of laminin-5 gamma2 chain expression in colorectal carcinoma: immunohistochemical analysis of 103 cases.

PURPOSE: The laminin-5 gamma2 chain plays an important role in cell migration during tumor invasion and tissue remodeling. Although this chain has been reported to be expressed in tumor-stroma interface of colorectal carcinoma, prognostic significance of its expression has not been elucidated in these tumors, so we investigated the clinicopathologic significance of Laminin-5 gamma2 chain expression in colorectal carcinoma. METHODS: Laminin-5 gamma2 chain expression was investigated immunohistochemically in 103 colorectal carcinoma patients with Stage II, III, and IV disease. The patients were categorized into three groups according to the number of immunopositive tumor cells in the sections containing the maximum diameter of the tumor as follows: +, less than 20 tumor cells were positive; ++, 20 to 500 tumor cells were positive; +++, more than 500 tumor cells were positive. RESULTS: Laminin-5 gamma2 chain expression was observed in cytoplasm of tumor cells, especially those in the invasive front of the tumor penetration. Eighteen (17 percent) of tumors showed +, 60 (58 percent) showed ++, and 25 (24 percent) showed +++. The increased number of immunopositive tumor cells was significantly associated with synchronous liver metastasis (P = 0.048). The univariate (P = 0.036) and multivariate (P = 0.001) analysis of the patients' survival revealed that the prognosis became significantly poorer in patients with the increased number of immunopositive tumor cells. CONCLUSIONS: Increased laminin-5 gamma2 chain immunoreactivity, suggesting a high invasive potential of tumor cells, was a significant poor prognostic indicator for the patients with colorectal carcinoma.

Adenocarcinoma↗

PubMed1976

Isolation and preliminary characterization of two varieties of low molecular weight immunoglobulin in the bullfrog, Rana catesbeiana.

Two varieties of low m.w. immunoglobulins have been isolated from the serum of Rana catesbeiana frogs. They are highly cross-reactive, although each also contains unique antigenic determinants. Since both low m.w. immunoglobulins were identified in the serum of 22 individual frogs, it was concluded that they are isotypic variants. The light chains of R. catesbeiana and mammalian high and low m.w. immunoglobulins are similar in electrophoretic mobility on polyacrylamide gels containing sodium dodecyl sulfate. The heavy chains of fropg high m.w. immunoglobulins have the mobility of mammalian mu-chains; the heavy chains of both variants of frog low m.w. immunoglobulins migrate between mammalian mu- and gamma-chains in approximately the position of mammalian alpha-chains. An unusual structural feature of the R. catesbeiana high ald low m.w. immunoglobulins is that the unreduced proteins are partially dissociated in sodium dodecyl sulfate.

Animals↗

PubMed1987

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain.

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.

Amino Acid Sequence↗

PubMed1998

The different mobility of complementary strands depends on the proportion AC/GT.

The electrophoretic mobility of DNA fragments on denaturing polyacrylamide gel depends on various factors. One of these is the base composition of a single-stranded DNA (ssDNA). We confirmed that one strand and its complementary strand of polymerase chain reaction (PCR) products migrated with different mobilities in all alleles detected at 12 out of the 13 short tandem repeat (STR) loci studied. The mobility differences between complementary strands (MD) were also observed regardless of end-polishing with Pfu DNA polymerase. MD was therefore not influenced by additional nucleotides to each strand of the PCR products. We then analyzed the relation between MD and the base composition using one representative allele at each of the 13 loci. The results indicated that MD was affected by the adenine plus cytosine (AC) content in the ssDNA and was proportional to the values of the AC content divided by the guanine plus thymine (GT) content in the AC-rich strand (the proportion AC/GT). When the proportion AC/GT was well-balanced, MD decreased. The same tendency was observed even in the end-polished strands. In this study, the electrophoretic mobility of an ssDNA on denaturing polyacrylamide gels was shown to depend on the proportion AC/GT. Unless the same side of the PCR products is labelled in the context of a PCR-based STR typing, distinct alleles may be mistaken for identical ones because of the different mobility of complementary strands. Accordingly, the labelled strand should be described if only one strand of the PCR products is detected. When using an allelic ladder marker as a size standard, the labelled side should be unified between STR alleles and the allelic ladder alleles.

Adenine↗

PubMed1987

Effects of castanospermine and 1-deoxynojirimycin on insulin receptor biogenesis. Evidence for a role of glucose removal from core oligosaccharides.

The insulin proreceptor is a 190-kDa glycoprotein that is processed to mature alpha (135-kDa) and beta (95-kDa) subunits. In order to determine the role of carbohydrate chain processing in insulin receptor biogenesis, we investigated the effect of inhibiting glucose removal from core oligosaccharides of the insulin proreceptor with glucosidase inhibitors, castanospermine and 1-deoxynojirimycin. Cultured IM-9 lymphocytes treated with inhibitors had 50% reduction in surface insulin receptors as demonstrated by ligand binding, affinity cross-linking with 125I-insulin, and lactoperoxidase/Na 125I labeling studies. Degradation rates of surface labeled receptors were similar in both control and inhibitor-treated cells (t1/2 = 5 h); thus, accelerated receptor degradation could not account for this reduction. Biosynthetic labeling experiments with [3H]leucine and [3H]mannose identified an apparently higher molecular size proreceptor (approximately 205 kDa) that failed to show the characteristic decline with time as seen in the normal 190-kDa proreceptor. Along with this finding, the biosynthetic label appearing in the mature subunits was reduced in these inhibitor-treated cells. Endoglycosidase H treatment of both precursors produced identical 170-kDa bands. Carbohydrate chains released from the 205-kDa precursor by endoglycosidase H migrated in the same position as the Glc2-3Man9GlcNAc standards when separated by high performance liquid chromatography, whereas the 190-kDa proreceptor oligosaccharides migrated similar to the Man7-9GlcNAc chains. Although the mature subunits of control and inhibitor-treated cells demonstrated equal electrophoretic mobility, the endoglycosidase H-sensitive oligosaccharides of the mature subunits in treated cells also contained residues that migrated similar to the Glc2-3Man9GlcNAc standards. Thus, glucose removal from core oligosaccharides is apparently not necessary for the cleavage of the insulin proreceptor, but does delay processing of this precursor, which probably accounts for the reduction in cell-surface receptors.

1-Deoxynojirimycin↗

PubMed1993

Serine for glycine substitutions in type I collagen in two cases of type IV osteogenesis imperfecta (OI). Additional evidence for a regional model of OI pathophysiology.

Serine for glycine substitutions in type I collagen have been described in seven cases of lethal type II osteogenesis imperfecta (OI), and six cases of nonlethal OI. We describe here two cases of moderately severe type IV OI with serine substitutions at alpha 1(I) Gly352 and alpha 2(I) Gly922, respectively. In both cases, G-->A point mutations were detected by RNase A cleavage of RNA/RNA and RNA/DNA hybrids. These cases extend the location for serine substitutions producing the moderately severe OI phenotype to the alpha 2(I) chain and the amino-terminal end of the alpha 1(I) chain. Their location supports a regional model of OI pathophysiology for serine substitutions. The proband with alpha 2(I) Gly922-->Ser has both normal and overmodified forms of both type I collagen chains. The overmodified form has delayed migration of all CNBr peptides. Helix thermal stability is decreased 4 degrees C. The fibroblast collagen protein and RNA of her unaffected parents are normal. However, the father was demonstrated to be a mosaic carrier using leukocyte DNA. The fibroblasts of the proband whose serine substitution is at alpha 1(I) Gly352 synthesize type I procollagen chains with delayed electrophoretic migration; normally migrating forms are difficult to detect. Only alpha 1(I) CB 8 displayed delayed migration. Helix thermal stability is reduced 2 degrees C. Parental genomic DNA was normal.

Amino Acid Sequence↗

PubMedDate not supplied

The regiospecific N-derivatization of histidine side chains: reinvestigation of a supposed N tau to N pi migration.

A report claiming that AcHisOMe reacts regiospecifically with 4-fluoronitrobenzene to give AcHis[tau Ph(NO2)]OMe, which on treatment with H2/Pd(C) undergoes a partial tau-pi shift to give some AcHis[pi Ph(NH2)]OMe, cannot be substantiated. 4(5)-Methylimidazole, a model for AcHisOMe, gives on reaction with 4-fluoronitrobenzene a 4:1 mixture of the regioisomers 1-(4-nitrophenyl),4-methylimidazole, corresponding to tau-substitution, and 1-(4-nitrophenyl),5-methylimidazole, corresponding to pi-substitution, each of which has been isolated and fully characterized, including proof of orientation. In both cases, treatment with H2/Pd(C) gives a single product, without any change of orientation in either case.

Histidine↗

PubMed2001

Proton mobility in protonated glycylglycine and N-formylglycylglycinamide: a combined quantum chemical and RKKM study.

Theoretical model calculations were performed to investigate the degree of validity of the mobile proton model of protonated peptides. The structures and energies of the most important minima corresponding to different structural isomers of protonated diglycine and their conformers, as well as the barriers separating them, were determined by DFT calculations. The rate coefficients of the proton transfer reactions between the isomers were calculated using the RRKM method in order to obtain a quantitative measure of the time scale of these processes. The proton transfer reactions were found to be very fast already at and above the threshold to the lowest energy decomposition pathway. Two possible mechanisms of b2+-ion formation via water loss from the dipeptide are also discussed. The rate-determining step of the proton migration along a peptide chain is also investigated using the model compound N-formylglycylglycinamide. The investigations revealed that this process very possibly occurs via the protonation of the carbonyl oxygens of the amide bonds, and its rate-determining step is an internal rotation-type transition of the protonated C=O-H group between two adjacent C=O-HellipsisO=C bridges.

Chemical Phenomena↗

PubMed2003

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry.

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.

Electrophoresis, Capillary↗

PubMed1980

The effect of "shearing" forces during ultracentrifugation on the membrane-bound polysomes associated with light rough microsomes of MPC-11 cells.

When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that [3H]-choline label and A260 nm absorption did not coincide in the LR region of the gradient. This was in contrast to the situation when microsomes were isolated from cells disrupted by nitrogen cavitation at 25 mM KCl. The A260 nm absorbing material that appeared in gradient fractions (1-5) below the position of LR membranes was found to consist of polysomal material. This material gave a "richer" polysome profile than that released from the LR membranes by addition of detergent. Labeling experiments with [3H]-leucine showed that nascent polypeptides associated with monosomes and polysomes in fractions 1-5 were of shorter length than the corresponding ones in the LR fraction. A mere contamination of LR microsomes by free polysomes appeared most unlikely. The results are consistent with an effect of "shearing" on the membrane-bound polysomes of the LR microsomes under specific experimental conditions. This effect results in the production of a 5' mRNA fragment (short polypeptide chains) and a 3' mRNA fragment (long polypeptide chains), the former fragment migrating further down the gradient tube free of LR membranes, whilst the latter remained attached to the LR membranes.

Animals↗

PubMed1989

Ferritin subunits in livers of siderotic mice.

The major ferritin species of mouse liver has been resolved by SDS-PAGE into two bands similar to the H and L subunits of rat liver ferritin with the L subunit predominating. Amino acid sequencing has confirmed the major, faster-migrating component as L chain. An additional, electrophoretically fast, minor ferritin was isolated from siderosome-containing subcellular fractions. In denaturing gels it gave a single 'F' subunit band of about 17 kDa, significantly smaller than the L and H subunits (about 20 and 21 kDa respectively). A small fragment isolated from the fast ferritin was sequenced. It corresponds to a 19-residue C-terminal peptide cleaved from L subunits in the assembled molecules. The F subunit must be derived from L subunits by loss of this peptide, and is not the expression product of a different gene. 'Fast' ferritins of siderotic mice and rats are thus analogous.

Amino Acid Sequence↗

PubMed2000

A spindle pole body-associated protein, SNAD, affects septation and conidiation in Aspergillus nidulans.

The nudA1 mutation in the cytoplasmic dynein heavy chain gene inhibits nuclear migration, colony growth and asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans. It also alters the location of the first cell division event (septation) and prevents nucleation of tip cells. We showed previously that a suppressor of nudA1, snaD290, partially reversed the nuclear migration defect and partially restored colony growth. We have now demonstrated that the snaD290 mutation also delays septation and restores the septum to its normal position, allowing tip cells to be nucleated. Although snaD290 does not affect nuclear migration or vegetative hyphal growth, it almost completely inhibits conidiation. We propose that the SNAD protein participates in septation, and is essential for asexual spore formation. SnaD encodes a novel 76-kDa coiled-coil protein (SNAD) that is located at the spindle pole body throughout the cell cycle. Therefore, our results suggest that proteins at the spindle pole body are likely to be involved in temporal regulation of septation in A. nidulans.

Actins↗

PubMed1975

Isolation and properties of the transcription complex of Escherichia coli RNA polymerase.

A procedure is described which permits complete separation of a transcription complex formed with template DNA, growing RNA chain and functioning RNA polymerase, from RNA polymerase molecules which have bound to DNA but not initiated RNA synthesis. The method is based on the marked stability of the transcription complex to dissociation by high concentrations of CsCl or CS2SO4 which enable banding the complex after equilibrium centrifugation. With use of the newly developed procedure, affinity of Escherichia coli RNA polymerase to T7 phage DNA was found to increase during initiation of RNA synthesis but then decrease concomitant with elongation of RNA chain presumably due to migration of the enzyme to DNA sites of weak affinity. Under the conditions of maximum affinity, the transcription complex contained one core polymerase for each T7 DNA if it was isolated by centrifugation in CsCl; in contrast, 2-6 enzyme molecules remained attached on the complex when the centrifugation was carried out in Cs2SO4. Thus, RNA polymerases bound to different sites of transcription initiation appear to be distinguished based on the affinity of interaction. Attempts are also described to isolate the transcription complex in vivo by Cs2SO4 centrifugation by Brij 58-deoxycholate lysate of lysozyme-treated Escherichia coli cells. The isolated complex contained approx. 50 polymerase molecules per Escherichia coli genome as well as other unidentified proteins.

Centrifugation, Density Gradient↗

PubMed1990

First purification and characterization of a sucrase-isomaltase from goose kidney microvillous membrane.

Goose (Anser anser) kidney microvillus sucrase-isomaltase (EC 3.2.1.48-EC3.2.1.10) was solubilized from isolated microvillus membranes using Emulphogen BC 720 or papain. Detergent-solubilized enzyme (D-SI) was purified 149 +/- 29 times with a yield of 15.7 +/- 2.6% by a two-step procedure which included chromatofocusing. The specific activity was 2.95 +/- 0.34 U/mg protein for sucrase, 1.02 +/- 0.13 for palatinase and 5.63 +/- 0.53 for maltase. D-SI was amphiphilic as indicated by its detergent-binding properties. These properties were not observed for sucrase-isomaltase released from the microvillus membrane by papain. The Mr of the enzyme purified after solubilization by Emulphogen and papain was 543,000 and 380,000, respectively, as determined by gel filtration. The difference in Mr indicates that an Emulphogen micelle is bound to the detergent-solubilized enzyme. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sucrase-isomaltase migrated as several polypeptide chains: a major band (Mr 280,000) and at least seven additional minor bands (Mr 220,000-100,000). It is suggested that the major band represents the precursor pro-sucrase-isomaltase and that the lower molecular weight bands are generated by PMSF or aprotinin-resistant proteinases during homogenisation and chromatography of the enzyme. Measured by chromatofocusing, the isoelectric point was found to be pH 4.6. Sucrase-isomaltase accounts for about 20% of total microvillus membrane proteins.

Animals↗