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At least 217 records · Page 12Linked to original sources
PubMed1995

A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.

3T3 Cells↗
PubMed2002

Stage migration after biopsy of internal mammary chain lymph nodes in breast cancer patients.

BACKGROUND: Involvement of the internal mammary chain lymph nodes (IMNs) is associated with worsened prognosis in breast cancer. Use of lymphoscintigraphy to visualize sentinel nodes reveals that IMNs often receive lymph from the area containing the tumor. METHODS: We biopsied IMNs in 182 patients because there was radiouptake to the IMNs or because the tumor was located in the medial portion of the breast. After tumor removal, pectoralis major fibers were divided to expose intercostal muscle. A portion of intercostal muscle adjacent to the sternum was removed. Lymph nodes and surrounding fatty tissue in the intercostal space were freed, removed, and analyzed histologically. The pleural cavity was breached in four cases (2.2%), with spontaneous resolution. RESULTS: IMNs were found in 160 (88%) of 182 patients; 146 (94.4%) were negative and 14 (8.8%) were positive. The latter received internal mammary chain radiotherapy. The axilla was negative in 4 of 14 cases and positive in 10. CONCLUSIONS: IMNs can be quickly and easily removed via the breast incision with insignificant risk and no increase in postoperative hospitalization. The patients with a positive IMN migrated from N0 (4 cases) or N1 (10 cases) to N3, prompting modification of both local (radiotherapy to internal mammary chain) and systemic treatment; without IMN sampling, they would have been understaged.

Breast Neoplasms↗
PubMed1977

[Comparison of the cyanogen bromide peptides of vitreous body collagen and type II collagen (author's transl)].

Pepsin-soluble collagen was isolated from bovine vitreous humor. This collagen showed only one alpha-chain in disc electrophoresis, migrating in the alpha1-chain position and between the alpha- and beta-components some colored bands were visible. The disc electrophoretic patterns of the cyanogen bromide peptides of pepsin-soluble vitreous body collagen and pepsin-soluble type II collagen revealed no identity.

Animals↗
PubMed2005

A point mutation in the Ch3 domain of human IgG3 inhibits antibody secretion without affecting antigen specificity.

Immunoglobulins (Ig) require correct folding and assembly of both heavy (H) and light (L) chains to form a functional H2L2 dimer that is secreted from plasma cells. This process is dependent upon the endoplasmic reticulum (ER) chaperone BiP, which targets improperly, folded or assembled Ig molecules for degradation. While investigating the mechanism of low IgG3 secretion, we identified a missense mutation L368P in the Ch3 region of the human gamma3 H-chain that was associated with impaired secretion of intact and functional Ig. The non-secreted H-chains displayed slower electrophoretic migration than secreted H-chains, consistent with them being glycosylated in the ER but not fully processed in the golgi apparatus and secretory pathway. Reversion of the mutated codon to wild type restored secretion of the IgG3, which displayed the same fine specificity for antigen as non-secreted IgG3. However, the non-secreted IgG3 was not opsonic in an in vitro phagocytosis assay. The results indicate that correct IgG3 Ch3 domain folding is essential for secretion and effective function but does not affect specificity for antigen.

Amino Acid Substitution↗
PubMed1994

Humanization of KC4G3, an anti-human carcinoma antibody.

We have previously constructed a chimeric version of KC4G3, a murine antibody that reacts with several human epithelial cancers and binds to the human breast epithelial mucin. We have now successfully humanized KC4G3 using positional consensus data, previously compiled after examining several other antibody structures, listing residues in the VH and V kappa frameworks that could influence antigen binding. We have previously showed that a fraction of the kappa chains of murine and chimeric KC4G3 migrates abnormally on SDS-PAGE most likely due to N-linked glycosylation in V kappa. The glycosylation signal has now been removed from V kappa, as a consequence of humanization. As expected, the humanized kappa chain migrates normally on SDS-PAGE. We detected no significant differences either in the affinities (1.6 x 10(9) M-1 vs. 1.4 x 10(9) M-1, respectively) or in the ability to compete for antigen binding, between the murine and the humanized antibodies. The humanized version is an IgG1, kappa immunoglobulin produced by mouse myeloma SP2/0-Ag14 cells and is designated HuKC4v2. The HuKC4v2 frameworks conform to the V kappa II and VHIII human consensus in all but six positions in V kappa and three positions in VH.

Amino Acid Sequence↗
PubMed1988

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration.

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.

Animals↗
PubMed1981

Electrophoretic analyses of myofibrillar proteins from the body wall muscles of Ascaris suum.

A myofibrillar protein extract has been isolated from the muscle of Ascaris suum. Two-dimensional electrophoresis of this extract revealed that the myosin light chain 1 (ALC1) migrates as 3 components with approximate isoelectric points in the range of 5.3-5.6. The most acidic component of ALC1 appeared to be phosphorylated when the myofibrillar extract was incubated for 10 s with catalytic subunit of cAMP dependent protein kinase and [gamma-32P] ATP. The myosin light chain 2 (ALC2) migrated as a single component in isoelectric focusing with an approximate isoelectric point of 5.5 Actin was resolved into 2 components with identical molecular weight but isoelectric points differing by approximatley 0.2 pH units. A protein was tentatively identified in the myofibrillar extract as tropomyosin. It migrated as a single band with an approximate isoelectric point of 5.0 and a molecular weight of 39 000. None of the troponin components could be identified in the myofibrillar extract. It is postulated that muscle contraction in A. suum muscle could be controlled by phosphorylation of myosin.

Actins↗
PubMed1993

A de novo G+1-->A mutation at the alpha 2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI type IV proband.

We have investigated the procollagen, collagen, alpha 2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two alpha 2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast alpha 2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of alpha,alpha'-dipyridyl. The alpha 2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating alpha 2(I) chain was reduced 6 degrees C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to alpha 2(I) nt 236-1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+1-->A mutation at the exon 16 donor site in one allele. The mutation eliminates a StyI site.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed2000

Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14).

Recombinant calprotectin, consisting of 2 individual peptide chains also called migration inhibitory factor-related protein (MRP)-8 and MRP14, was tested for antimicrobial activity in a Candida albicans growth inhibition assay. Both chains contain HEXXH zinc-binding sites and might be expected to manifest zinc-reversible, antimicrobial activity similar to that of native calprotectin. When tested alone, neither MRP8 nor MRP14 showed activity in the Candida growth assay. A synthetic 20-amino acid peptide containing the HEXXH sequence of MRP14, along with a nearby HHH sequence, was also inactive in this assay. However, equimolar concentrations of MRP8 and MRP14 demonstrated a potent growth inhibitory effect that was reversible by 30 microM zinc. Truncated MRP14 (missing the C-terminal GHHHKPGLGEGTP tail) used in combination with MRP8 demonstrated zinc-reversible activity that was somewhat less than that with complete MRP14. These results suggest that intact calprotectin, consisting of a heterodimer of MRP8 and MRP14, is necessary to form a zinc-binding site capable of inhibiting microbial growth.

Amino Acid Sequence↗
PubMed2002

Comparative studies of osteoblast and fibroblast type I collagen in a patient with osteogenesis imperfecta type IV.

The expression of type I collagen has been compared in fibroblast and osteoblast cultures of a patient with moderately severe osteogenesis imperfecta (OI) type IV, with respect to control cells. Electrophoretic analysis of type I collagen showed that both OI osteoblasts and fibroblasts synthesized normal chains and chains with delayed migration. However, the osteoblasts contained a higher proportion of abnormal chains than fibroblasts from the proband. Pulse-chase experiments showed that the trimers containing abnormal chains were cleared more rapidly from osteoblasts than fibroblasts. Moreover, the collagen secreted by OI osteoblasts had thermal stability 1 degrees C higher than collagen secreted by OI fibroblasts. These results suggest that the abnormal collagen in osteoblasts may be more resistant to intra- and extracellular degradation and may thus have better survival than in fibroblasts. This finding could have implications for understanding the clinical phenotype of OI.

Case-Control Studies↗
PubMed1995

Onco-fetal/laminin-binding collagen from colon carcinoma: detection of new sequences.

We have recently identified an oncofetal-laminin binding collagen (OF/LB) composed of three alpha chains, with the apparent molecular mass of about 100 kDa each, but bearing different pI. One of the chains appears markedly acidic in a bidimensional electrophoretic system, where the NEPHGE is used as first dimension separating gel, while the two more basic chains have similar migration as alpha 1(III) and alpha 1(I) collagen chains, respectively. Sequence analyses have been performed on CNBr-peptides, derived from pepsinized triple helical molecules and on tryptic fragments obtained after in gel digestion of the acidic band. The research of sequence homology with computerized databases indicated that the acidic chain represents a gene product distinct from either type I, type III and other known collagen chains, while the identity of the other two chains remains to be fully determined.

Amino Acid Sequence↗
PubMed1993

Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation.

Granulosa cells from ovarian follicles were shown to express and secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH-dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen beta- and gamma-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical gamma-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno-precipitation analysis. Fibrinogen gamma-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fibrinogen gene expression by these cells. Fibrinogen secretion by granulosa cells was measured by a competitive enzyme-linked immunosorbent assay. Granulosa cells treated with FSH (100 ng/ml) secreted 2-3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high circulating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation.

Amino Acid Sequence↗
PubMed1994

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1999

Immunohistochemical localization of laminin-1 in the acellular nerve grafts is associated with migrating Schwann cells which display corresponding integrin receptors.

The presence of laminin-1, collagen-IV, alpha6 and beta1 integrin chains was detected by indirect immunohistochemistry using biotin/streptavidin/HRP or gold-conjugated secondary antibody at the light and electron microscope level, respectively. Cryo-treated segment of the peripheral stump without living Schwann cells (S-100-) did not display immunoreactivity for laminin-1 and integrin's chains, while the migrating Schwann cells in the marginal regions were immunostained for the antigens. Isolated acellular nerve segments protected from migration of Schwann cells (S-100-) exhibited laminin-1-, beta1-, and alpha6- integrin chains immunoreactivities. Position of the basal lamina was verified by collagen-IV+ immunoreactivity. Results indicate that presence of the laminin in the peripheral nerve is related with living Schwann cells.

Animals↗
PubMedDate not supplied

Myosin light chain compositions of the interatrial and interventricular septa of sheep heart.

The myosin light chain composition of sheep interatrial and interventricular septa were analysed by one- and two-dimensional polyacrylamide gel. The interventricular septum has myosin light chain composition indistinguishable from that of ventricular myosin. Myosin from the interatrial septum contains three light chains, two of which co-migrated with the two atrial light chains (ALC1 and ALC2), while the third co-migrated with ventricular light chain 2 (VLC2). ALC1 are more abundant than ALC2 or VLC2 suggesting a mixed myosin population. Myosin with ALC1 and VLC2 light chain composition may be present, and its possible relationship with cardiac "conducting" cells is discussed.

Animals↗
PubMed1991

Laminin synthesized by stationary and migrating rat liver epithelial cells lacks the A chain.

The expression of laminin chains was analyzed in normal and Ha-ras1-transformed rat liver epithelial cells. The normal, nontumorigenic cells were induced to migrate by epidermal growth factor, whereas the Ha-ras1-transformed, malignant derivatives migrate constitutively. None of these cells express a typical (EHS-like) laminin A chain. Immunoprecipitation of [35S]-methionine-labeled liver cell lysates with an antibody against EHS-laminin revealed B1 and B2 chains and, in addition, two high Mr polypeptides. These polypeptides were not recognized by the antibody in immunoblots, suggesting that they might constitute alternative laminin A chains. Analysis of the expression of all three laminin chains at the RNA and protein level revealed that the pattern of expression of the stationary cells does not differ from that of the migratory ones and is also not influenced by epidermal growth factor. These results indicate that expression of a typical laminin A chain by rat liver epithelial cells is not required for the secretion and deposition of the protein in the extracellular matrix. The data also indicate that an EHS-like laminin A chain is not required for the migration of these epithelial cells.

Animals↗
PubMed1987

Ascorbate effects on type I procollagen synthesis by human adult skin fibroblasts: different migration positions of type I procollagen chains on SDS polyacrylamide gel after incubation with ascorbate.

The effects of ascorbate and steroids on type I procollagen synthesis by human skin fibroblasts were studied. Ascorbate treatment (50 micrograms/ml) for 24 hours stimulated a 2-3 fold increase in type I procollagen synthesis and an unexpected shift in the mobility of type I procollagen on SDS polyacrylamide gels. The kinetics of the increase in procollagen synthesis (4 hours) and the shift in electrophoretic mobility (1 hour) were dissimilar, suggesting different controlling mechanisms. This was confirmed by the addition of alpha-alpha'-dipyridyl to ascorbate-treated cultures which eliminated the ascorbate-induced shift in electrophoretic mobility without altering the amount of procollagen synthesis. In contrast, hydrocortisone (1.5 microM) reduced the ascorbate-induced stimulation of type I procollagen synthesis by 80% but did not affect the ascorbate-induced shift in electrophoretic mobility. These studies indicate that the ascorbate-induced increase in type I procollagen synthesis is due to increased levels of type I procollagen mRNA and is independent of the level of hydroxylation of the procollagen.

2,2'-Dipyridyl↗