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Myosin heavy chain expression in zebrafish and slow muscle composition.

In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross-reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from "adaxial" myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti-fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co-expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome.

Animals↗

Characterization of neonatal cardiac myosin heavy chain mRNA and synthesis of complementary DNA.

A procedure for the extraction of intact RNA from whole heart tissue using guanidine hydrochloride has been accomplished. Subsequent chromatography on oligo(dT)-cellulose of the total RNA and translation of the poly(A)+ RNA in a cell-free reticulocyte translation system has shown that the myosin heavy chain mRNA from rat heart tissue was present. The myosin heavy chain mRNA was identified by several criteria: (i) sizing by sucrose density gradient centrifugation, (ii) migration of a polypeptide translation product coincided with a myosin heavy chain marker in one-dimensional sodium dodecyl sulfate-- polyacrylamide gel electrophoresis, and (iii) immunoprecipitation of the mRNA translation product with a myosin heavy chain specific antibody. A myosin heavy chain mRNA enriched fraction was used to direct cDNA synthesis in an in vitro system. An optimal condition has been elucidated in which large molecular weight cDNA strands are produced. Evidence is presented here for the production of cDNA strands in excess of 4000 nucleotides which includes the heavy chain myosin in a high potassium--low sodium transcription assay, as revealed by dot blot hybridization with a cDNA clone of the myosin heavy chain.

Animals↗

Saposin C stimulates growth and invasion, activates p42/44 and SAPK/JNK signaling pathways of MAPK and upregulates uPA/uPAR expression in prostate cancer and stromal cells.

AIM: To determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily. METHODS: We employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells. RESULTS: Saposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies. CONCLUSION: Saposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer.

Cell Division↗

Effectiveness of adjunctive low-dose doxycycline therapy on clinical parameters and gingival crevicular fluid laminin-5 gamma2 chain levels in chronic periodontitis.

BACKGROUND: Laminin-5 (Ln-5) is involved in the apical migration of epithelial cells during the development of periodontal pockets. Low-dose doxycycline (LDD) can therapeutically modulate the host response with its non-antimicrobial properties. In the present randomized, double-blind, placebo-controlled, parallel arm study, the effectiveness of LDD in combination with non-surgical periodontal therapy on gingival crevicular fluid (GCF) Ln-5 gamma2 chain fragment levels and clinical parameters in patients with chronic periodontitis was examined over a 12-month period. METHODS: GCF samples were collected and clinical parameters including probing depth (PD), clinical attachment level, gingival index (GI), and plaque index were recorded. Thirty chronic periodontitis patients were randomized either to low-dose doxcycline or placebo groups. LDD group received doxycycline (20 mg, b.i.d.) for 3 months plus scaling and root planing (SRP), while placebo group was given placebo capsules b.i.d. for 3 months plus SRP. The patients were evaluated every 3 months during the 12-month study period. All clinical parameters and GCF sampling were repeated at each visit. GCF Ln-5 gamma2 chain fragment levels were determined by Western immunoblotting using specific antibody and quantitated by computerized image analysis. Friedman test was used for intragroup comparisons followed by Wilcoxon signed rank test to analyze significance of changes over time. The Mann-Whitney test was used to determine differences between both LDD and placebo groups. RESULTS: Both groups revealed significant improvements in all clinical parameters over the 12-month period (P < 0.0125). LDD group showed a significantly greater reduction in the mean PD scores at 9 and 12 months and in the mean GI scores at all time points than the placebo group (P < 0.05). In the LDD group, GCF Ln-5 gamma2 chain fragment levels were significantly reduced at 3 months (P < 0.0125) and then slightly increased during the rest of the study period. In the placebo group, GCF 45 and 70 kDa Ln-5 gamma2 chain fragments tended to decrease at 3 months compared to baseline, but did not reach significance; these levels continued to increase throughout the remainder of the study period. GCF Ln-5 gamma2 chain fragment levels in LDD group were significantly lower than those of the placebo group during the study period (P < 0.05). CONCLUSIONS: The present data indicate that LDD therapy in combination with SRP therapy can reduce GCF Ln-5 gamma2 chain fragment levels and improve clinical periodontal parameters in patients with chronic periodontitis. Since matrix metalloproteinases (MMP)-mediated fragmentation of laminin-5 can contribute to pocket formation by stimulating epithelial cell migration, the reduction of Ln-5 gamma2 chain fragment levels could provide a new mechanism by which LDD, adjunctive to SRP, inhibits periodontal disease more effectively than SRP alone. Thus, these results provide extended and additional information about the effectiveness of the LDD therapy as an adjunct to non-surgical periodontal therapy in the long-term management of periodontal disease.

Adult↗

Cardiac surgery with extracorporeal circulation: neutrophil transendothelial migration is mediated by beta1 integrin (CD29) in the presence of TNF-alpha.

Cardiac surgery with extracorporeal circulation is associated with neutrophil activation, inflammation, and edema. Endothelial hyperpermeability elicited by the interaction of activated neutrophils and/or cytokines with endothelial cells may be critical in this regard. However, the immune and cellular mechanisms involved are not fully understood. Cocultures with human endothelial cells and neutrophils from cardiac surgery patients were used to evaluate the role of beta1 integrin activity and the proinflammatory cytokine tumor necrosis factor (TNF)-alpha in neutrophil transendothelial migration and in impairment of the integrity of endothelial cell-to-cell contacts. Blocking of CD29 (heavy chain of beta1 integrins) totally prevented neutrophil adhesion and transendothelial migration. Pretreatment of neutrophils with either a CD29-stimulating monoclonal antibody or the addition of TNF-alpha (0.1-10 U/ml) to the coculture failed to induce transendothelial migration. However, coculture of endothelial cells with CD29-stimulated neutrophils in the presence of 0.1-10 U/ml TNF-alpha strongly induced neutrophil transmigration. CD29/TNF-alpha-mediated transmigration was associated with intracellular redistribution of endothelial beta-catenin. We further showed that CD29/TNF-alpha-mediated effects involved PI3K and tyrosine kinase-dependent signaling via MAPK but were independent of nuclear transcription factor (NF)-kappaB activity. Inhibition of CD29/TNF-alpha might be a therapeutic option to limit endothelial dysfunction following cardiac surgery with extracorporeal circulation.

Cell Movement↗

Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: analysis of serum samples problematic by agarose gel electrophoresis.

The capabilities of capillary electrophoresis (CE) for serum protein electrophoresis and immunotyping have been demonstrated. CE-based systems specifically designed for serum protein electrophoresis and immunotyping via immunosubtraction (IS) are now available and are being evaluated for efficiency, specificity and sensitivity by several groups. The use of CE for serum protein electrophoresis and immunotyping (IS) in the clinical laboratory compares well with agarose gel electrophoresis (AGE) and immunofixation (IF) for the detection and characterization of monoclonal proteins. In addition to routine use, this technology is useful for a subset of serum samples that are difficult to interpret with conventional technology. In this study, sera abnormalities difficult to detect/interpret by AGE-IF are subdivided into four categories: (i) patients with polyclonal increases in immunoglobulin, (ii) point of application artifacts, (iii) abnormalities in the beta region, and (iv) patients with free light chains. CE is superior to AGE for evaluating samples characterized by the above abnormalities. Sera containing monoclonal proteins within a polyclonal increase are easier to detect by CE as well as being easier to type by IS than by IF. Point-of-application artifacts, periodically observed with AGE, do not exist on CE since the point of detection is remote from the point of application. Enhanced resolution in the beta region allows for increased detection of monoclonal proteins migrating in this region. Some free light chains are undetected by CE as a result of no apparent abnormalities on the CE serum protein profile and, thus, still require IF for detection. CE detects more serum electrophoretic abnormalities than AGE in this clinically important group of patients with Bence Jones proteinemia.

Antibodies, Monoclonal↗

Fibrinogen-mediated epidermal cell migration: structural correlates for fibrinogen function.

Previously we showed that epidermal cells are able to use fibrinogen (FGN) as a migration substratum during wound closure. The goal of the present study was to determine the structural features of FGN that allow this migration. Pieces of glass coated with native, fragmented, or other modified forms of FGN were implanted into full-thickness skin wounds of adult newts such that migrating epidermal cells would encounter the implant. In this system, a coating of FGN allowed considerably more migration than a coating of BSA. At high concentrations, heat-denatured FGN supported as much migration as the same amount of intact FGN. Fraction I-9, a circulating form of FGN missing a 20-30K (K = 10(3) Mr) carboxy-terminal segment of the A alpha chain, was no less effective than intact FGN. Comparison of the isolated D1 and E fragments of FGN showed migration only on D1, but never to the extent seen on intact FGN containing the same amount of D1. Plasmin digestion of D1 in the presence of EDTA, a process which produces D3, a fragment differing from D1 by the loss of the carboxy-terminal 109 amino acids of the gamma chain, caused a significant loss of activity in the D fragment. Migration was good on implants coated with relatively high concentrations of purified A alpha chains but gamma chains were inactive. Migration over intact FGN was almost totally blocked by 230 microM-Arg-Gly-Asp-Ser (RGDS), a peptide known to interact with integrin-type receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

[Evaluation of a new lid implant for rehabilitation of the paralyzed eye].

BACKGROUND: Since 1999 the platinum chain is being used as implant to correct lagophthalmos, which promises, due to its flexibility, lower postoperative complications. The lack of mandatory results reported in the literature, prompted us to survey all surgeons using the platinum chain in order to learn of the complications they had experienced. METHODS: A questionnaire was sent to 30 surgeons to survey results and complications associated with the implantation of the platinum chain to treat lagophthalmos. Complications like contouring, migration and extrusion of the implant occurred. RESULTS: Complications observed were bulging of the implant in 12 cases (5,7 %), 4 migrations of the implant (1,9 %), in 7 cases a frank extrusion (3,3 %), a ptosis of the upper eyelid in 8 cases (3,8 %), a corneal astigmatism in 3 cases (1,4 %) and in one case an infection of the upper eyelid (0,5 %). In 2 cases (0,9 %) the patients lamented a mechanical irritation of the upper eyelid. CONCLUSION: Implantation of eyelid weights is a straightforward and reversible procedure for the surgical therapy of lagophthalmos. Through the use of the platinum chain as implant the postoperative complications are to be regarded as small in total. With that the effectiveness of the procedure can be increased compared to rigid gold implants.

Blepharoptosis↗

Developmental patterning of carbohydrate antigens during early embryogenesis of the chick: expression of antigens of the poly-N-acetyllactosamine series.

This report describes a striking temporal and spatial patterning of specific carbohydrate sequences in the developing chick embryo. By using oligosaccharide sequence-specific monoclonal antibodies as immunohistochemical reagents in conjunction with neuraminidase, it was possible to visualize the occurrence, as well as the changes in distribution, of oligosaccharides of the poly-N-acetyllactosamine series. These were (a) long-chain unbranched sequences reactive with anti-i Den, (b) long-chain branched sequences reactive with anti-I Step and (c) short-chain branched sequences reactive with anti-I Ma and (d) their sialylated forms. The salient observations with serial sections of embryos from the unincubated to the 17th stage were as follows. (1) A pronounced anteroposterior patterning appeared during neuroectodermal development, such that the long-chain unbranched and long-chain branched sequences, which were abundant on the ectoderm of the earlier stages, were replaced by short-chain branched sialo-oligosaccharides in the developing brain and anterior neural tube. (2) A striking anteroposterior and mediolateral patterning developed in the subectodermal extracellular spaces. The long-chain linear and short-chain non-sialylated sequences demarcated regions favourable for migration of the lateral plate mesoderm. (3) A distinction was made between the dorsal and ventral routes of the trunk neural crest in that the extracellular matrix of the dorsal route only was associated with long-chain linear and short-chain sialylated branched sequences. (4) A circumscribed perinotochordal distribution of the short-chain sialylated branched sequences was observed in the region of the future centra of the vertebrae. (5) An abundance of long-chain linear and long-chain sialylated branched structures was detected in primordial germ cells which permitted their identification during migration. These observations suggest that oligosaccharides of the poly-N-acetyllactosamine series may have roles as short-range, region-specific information factors during morphogenetic events that take place in the developing embryo, and they open the way to the search for recognition proteins (e.g. endogenous lectins) specific for each of these oligosaccharide structures.

Animals↗

Radioautographic study of in vivo and in vitro incorporation of fucose-3H into thyroglobulin by rat thyroid follicular cells.

The incorporation of fucose-(3)H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-(3)H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-(3)H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-(3)H. At 3-5 min following fucose-(3)H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.

Animals↗

Overexpression of hyaluronan synthase 2 alters hyaluronan distribution and function in proximal tubular epithelial cells.

The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-alpha-inhibitor (IalphaI), a process that is known to be catalyzed by TNF-alpha-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IalphaI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IalphaI and pre-alpha-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IalphaI/pre-alpha-inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix.

Catalysis↗

Laminin 5 beta3 and gamma2 chains are frequently coexpressed in cancer cells.

Laminin 5 plays an important role in cell migration during tumor invasion and tissue remodeling. However, previous studies have not clarified whether laminin 5 beta3 chain is coexpressed with laminin 5 gamma 2 chain in cancer cells. The present immunohistochemical study investigated the distribution of the laminin 5 beta3 and gamma2 chains in 20 cases of squamous cell carcinoma of the tongue and 17 cases of colorectal carcinoma. Laminin 5 beta3 and gamma2 chains were expressed in the cytoplasm of tumor cells. Tumor cells at the cancer-stromal interface and at the invasive front frequently showed immunopositivity for laminin 5 beta3 and gamma2 chains in both squamous cell carcinoma of the tongue and colorectal carcinoma. Furthermore, strong expressions of these two proteins were observed in cancer cells invading in a scattered manner. Laminin 5 beta3 expression correlated significantly with laminin 5 gamma2 expression in 20 cases of squamous cell carcinoma of the tongue (P = 0.0002) and 17 cases of colorectal carcinoma (P < 0.0001). These results suggest that in squamous cell carcinoma of the tongue and colorectal carcinoma, laminin 5 gamma2 chain and beta3 chain are both important in the invasiveness of cancer cells.

Adenocarcinoma↗

Aberrant expression of laminin gamma 2 chain and its prognostic significance in intrahepatic cholangiocarcinoma according to growth morphology.

Laminin gamma 2 chain is an extracellular matrix protein that plays an important role in cell migration and tumor invasion. We report altered expression and characteristic localization of this chain in a series of 105 cases of intrahepatic cholangiocarcinomas examined immunohistochemically. All tumors were grossly classified into the following three types: intraductal growth type (n=9), periductal infiltrating type (n=8) and mass-forming type (n=88). The tumors exhibited three distinct staining types: basement membrane staining, cytoplasmic staining and stromal staining. The basement membranous staining of laminin gamma 2 chain was more frequent in biliary dysplasia, intraductal growth and periductal infiltrating type than in mass-forming type. The cytoplasmic staining of carcinoma cells was observed especially at the cancer-stromal interface or at the invasive front of tumors. Stromal staining of laminin gamma 2 chain was essentially localized in the stroma around cancer cells at the invasive area, and the expression was significantly correlated with tumor aggressive factors and a poor prognosis in patients with intrahepatic cholangiocarcinoma. We conclude that laminin gamma 2 chain exhibits aberrant expression in a stepwise manner through different aggressive stages of tumor progression.

Aged↗

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain.

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.

Animals↗

Modulation of intestinal immune system by dietary fat intake: relevance to Crohn's disease.

Gut-associated lymphoid tissue is the major inductive site of the mucosal immune system, which is functionally independent of the systemic immune system. Both the amount and type of dietary fat modulate intestinal immune function. Absorption of long-chain fatty acids stimulates lymphocyte flux and lymphocyte blastogenesis in intestinal lymphatics. Long-chain fatty acid absorption also significantly enhances migration of T lymphocytes to Peyer's patches, possibly due to up-regulation of adhesion molecules, such as alpha4-integrin and L-selectin. Lipoproteins are involved in stimulation of lymphocyte function by both receptor-dependent and independent mechanisms. However, unsaturated fatty acids at higher concentrations have a suppressive effect on cell-mediated immunity via eicosanoid release, receptor affinity changes or interactions with intracellular signal transduction. Fat absorption also influences various other cells in the intestinal mucosa: increased cytokine release from intestinal epithelial cells follows long-chain fatty acid absorption. In Crohn's disease, elemental diets and total parenteral nutrition often induce remission, possibly by reducing antigenic load on activated immune cells in the intestine and, thus, down-regulating hyperreactive CD4 cells. Dietary oleic acid supplements caused an immunological reversal effect in the intestinal immune system of animals fed an elemental diet. An excess of long-chain fatty acids in an elemental diet, therefore, may negate its beneficial effect on gut-associated lymphoid tissues in Crohn's disease. In contrast, supplemental dietary fish oil apparently tends to prevent relapse of Crohn's disease. Because dietary fat intake is closely associated with immunological function of the intestinal mucosa, careful manipulation of dietary fat can be important in management of this disease.

Adjuvants, Immunologic↗

Proteoglycans of human rotator cuff tendons.

Rotator cuff and biceps tendons that appeared grossly normal were procured from adult cadavers without a history of shoulder problems. These tendons were analyzed for the amount and type of glycosaminoglycan, type of proteoglycan, and histology. When compared with the distal/tensional region of biceps tendon, the glycosaminoglycan content of supraspinatus, infraspinatus, and subscapularis tendons was 2.5-fold higher and the glycosaminoglycan content of the proximal/compressed region of biceps tendon was 3-fold higher. The ratio of hyaluronic acid to chondroitin sulfate/dermatan sulfate in all three cuff tendons was approximately 1. Rotator cuff tendons contained large proteoglycan similar to aggrecan, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel migration elution from Sepharose CL-4B, and content of both chondroitin sulfate and keratan sulfate chains. Both decorin and biglycan were also present, as demonstrated by migration in sodium dodecyl sulfate-polyacrylamide gels and core protein immunoreactivity. In contrast decorin was the only proteoglycan prominent in distal/tensional regions of biceps tendon. Histological analysis showed layers of loosely organized alcian blue-stained material running between the longitudinal collagen fiber bundles. The proteoglycan content of rotator cuff tendons was similar to fibrocartilage in tendons that have been subjected to compressive loads in situ. This suggests that cells of normal adult rotator cuff tendons have adapted to loads distinct from pure tension. However, the histological organization did not resemble mature fibrocartilage. The increased amount of proteoglycan in rotator cuff tendons may serve to separate and lubricate collagen bundles as they move relative to each other during normal shoulder motion.

Adult↗

Neuronal migration depends on intact peroxisomal function in brain and in extraneuronal tissues.

Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairment of neuronal migration. Although the molecular mechanisms underlying the neuronal migration defect are at present unknown, the excess of very long chain fatty acids in brain, a consequence of peroxisomalbeta-oxidation deficiency, has often been hypothesized to play a major role. The purpose of the present study was to investigate the contribution of peroxisomal dysfunction in brain as opposed to peroxisomal dysfunction in extraneuronal tissues to the migration defect. Peroxisomes were selectively reconstituted either in brain or liver of Pex5 knock-out mice, a model for Zellweger syndrome, by tissue-selective overexpression of Pex5p. We found that both rescue strains exhibited a significant correction of the neuronal migration defect despite an incomplete reconstitution of peroxisomal function in the targeted tissue. Animals with a simultaneous rescue of peroxisomes in both tissues displayed a pattern of neuronal migration indistinguishable from that of wild-type animals on the basis of cresyl violet staining and 5',3'-bromo-2'-deoxyuridine birth-dating analysis. These data suggest that peroxisomal metabolism in brain but also in extraneuronal tissues affects the normal development of the mouse neocortex. In liver-rescued mice, the improvement of the neuronal migration was not accompanied by changes in very long chain fatty acid, docosahexaenoic acid, or plasmalogen levels in brain, indicating that other metabolic factors can influence the neuronal migration process.

Animals↗

Role of the small GTP-binding protein rho in epithelial cell migration in the rabbit cornea.

PURPOSE: To determine the role of the small guanosine triphosphate (GTP)-binding protein Rho in the migration of corneal epithelial cells. METHODS: The presence of the Rho target proteins Rho-associated coiled coil-containing protein kinase (ROCK)-1 and ROCK-2 in rabbit cornea was examined by immunohistochemical analysis, and that of the corresponding mRNAs in rabbit corneal epithelial cells was determined by reverse transcription-polymerase chain reaction analysis. The effects of various agents on epithelial cell migration were investigated by measuring the length of the migration path in rabbit corneal blocks in culture. RESULTS: Both ROCK-1 and ROCK-2 were detected in the rabbit corneal epithelium at both protein and mRNA levels. The Rho activator lysophosphatidic acid (LPA) stimulated corneal epithelial migration in a dose-dependent manner, whereas exoenzyme C3, a Rho inhibitor, inhibited epithelial migration also in a dose-dependent manner. The stimulatory effect of LPA on corneal epithelial migration was prevented by exoenzyme C3. Both cytochalasin B, an inhibitor of actin filament assembly, and ML-7, an inhibitor of myosin light chain kinase, also prevented LPA stimulation of epithelial migration. CONCLUSIONS: These results suggest that Rho mediates corneal epithelial migration in response to external stimuli by regulating the organization of the actin cytoskeleton.

Actins↗