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PubMed2001

Dysferlin protein analysis in limb-girdle muscular dystrophies.

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Adult↗
PubMed2025

Twisted Sister1: an agravitropic mutant of bread wheat (Triticum aestivum) with altered root and shoot architectures.

We identified a mutant of hexaploid wheat (Triticum aestivum) with impaired responses to gravity. The mutant, named Twisted Sister1 (TS1), had agravitropic roots that were often twisted along with altered shoot phenotypes. Roots of TS1 were insensitive to externally applied auxin, with the genetics and physiology suggestive of a mutated AUX/IAA transcription factor gene. Hexaploid wheat possesses over 80 AUX/IAA genes, and sequence information did not identify an obvious candidate. Bulked segregant analysis of an F2 population mapped the mutation to chromosome 5A, and subsequent mapping located the mutation to a 41 Mbp region. RNA-seq identified the TraesCS5A03G0149800 gene encoding a TaAUX/IAA protein to be mutated in the highly conserved domain II motif. We confirmed TraesCS5A03G0149800 as underlying the mutant phenotype by generating transgenic Arabidopsis thaliana. Analysis of RNA-seq data suggested broad similarities between Arabidopsis and wheat for the role of AUX/IAA genes in gravity responses, although there were marked differences. Here we show that the sequenced wheat genome, along with previous knowledge of the physiology of gravity responses from other plant species, gene mapping, RNA-seq, and expression in Arabidopsis have enabled the cloning of a key wheat gene that defines plant architecture.

Triticum↗
PubMed2005

A high-throughput inducible RNAi vector for plants.

We describe here a vector system that allows dexamethasone-inducible RNAi against plant genes. The system utilizes a modified pHELLSGATE vector, under the control of the pOp6 promoter, and the synthetic transcription factor, LhGR. We demonstrate that the production of RNAi-inducing hairpin RNA from this system can be regulated by the application and removal of dexamethasone. Silencing of a target gene encoding phytoene desaturase was highly effective 24 h after application of dexamethasone. In the presence of the hormone silencing was maintained for at least 5 days while removal of the inducer resulted in significant recovery within 24 h. A transgene encoding luciferase was silenced with similar speed and efficiency following application of dexamethasone but unlike phytoene desaturase, mRNA levels did not recover within 10 days after dexamethasone was removed. Insertion of target gene sequences into this vector is mediated by Gateway recombination, facilitating its use for high-throughput applications, such as gene discovery or validation. The inducibility of RNAi from this system may be useful in helping to identify the functions of genes which when constitutively silenced give embryo lethality or pleiotropic phenotypes. A modified version of this system may also be used for tissue-specific hairpin RNA expression.

Journal Article↗
PubMed1994

Modifications of the notch function by Abruptex mutations in Drosophila melanogaster.

The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.

Alleles↗
PubMed2006

Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase.

The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes.

Arabidopsis↗
PubMed2000

Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells.

Cellular phenotype is determined not only by genetic transcription but also by subsequent translation of mRNA into protein. Extracellular signals trigger intracellular pathways that distinctly activate translation. The 70/85-kDa S6 kinase (pp70(S6k)) is a central enzyme in the signal-dependent control of translation, but its regulation in endothelial cells is largely unknown. Here we show that fluid flow (in the absence of an exogenous mitogen) as well as humoral agonists activate endothelial pp70(S6k). Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), and wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked flow-induced pp70(S6k) activation; FK-506, a rapamycin analog with minimal mTOR inhibitory activity, and PD-98059, an inhibitor of the flow-sensitive mitogen-activated protein kinase pathway, had no effect. Synthesis of Bcl-3, a protein whose translation is controlled by an mTOR-dependent pathway, was induced by flow and inhibited by rapamycin and wortmannin. Transcriptional blockade did not abolish the flow-induced upregulation of Bcl-3. Fluid forces may therefore modify endothelial phenotype by specifically regulating translation of certain mRNA transcripts into protein.

3T3 Cells↗
PubMed1974

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae.

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.

Antifungal Agents↗
PubMed1999

Influence of the fluoroquinolone ofloxacin on the intrinsic expression of multidrug resistance phenotype in HCT-8 human colon carcinoma cells.

The influence of antibiotics, particularly ofloxacin (OF), a commonly used antimicrobial fluoroquinolone, on the multidrug resistance (MDR) phenotype of the HCT-8 cell line was studied. This cell line was grown in OF containing medium for several months and the expression of the MDR phenotype was followed through the analysis of the expression and functionality of the P-glycoprotein (Pgp), the chemosensitivity to daunorubicin (DNR), and the mRNA expression of mdr-1, multidrug resistance-associated protein (MRP), and topoisomerase IIalpha and IIbeta genes. Replacement of OF by penicillin streptomycin (PS) resulted in a significant decrease in mdr-1 mRNA expression, which was found to correlate with a decrease in the expression and functionality of the Pgp. After antibiotic starvation for 4 weeks, cells grown in antibiotic-free medium were then exposed to PS or OF; these cells showed an increase in mdr-1 mRNA/Pgp and MRP mRNA expression without a decrease in DNR cytotoxicity. OF cultured cells exhibited a significant increase in Pgp expression without evidence of the functionality of the Pgp. An increase in topoisomerase IIalpha mRNA expression was observed with time and with the number of passages of the cell line without any relationship to the presence of antibiotics in the culture medium. These results showed that extensive use of antibiotics, particularly the quinolones, can modify the phenotype of the HCT-8 colon adenocarcinoma cell line.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway.

Animals↗
PubMed2004

Calcium-regulated changes in mitochondrial phenotype in skeletal muscle cells.

Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca(2+) in muscle cells. To characterize the potential role of Ca(2+) as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca(2+) ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 microM A-23187 for 48-96 h increased nuclear-encoded beta-subunit F(1)ATPase and malate dehydrogenase (MDH) mRNA levels by 50-100% (P < 0.05) but decreased mRNA levels of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially encoded subunits COX II and COX III were decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca(2+) differentially regulates the mRNA level of nuclear and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam levels by 48 h. A-23187 induced time-dependent increases in Egr-1 mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also increased Egr-1, serum response factor, and Sp1 protein expression, transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or beta-subunit F(1)ATPase mRNA. These results indicate that changes in intracellular Ca(2+) can modify mitochondrial phenotype, in part via the involvement of Egr-1.

AMP-Activated Protein Kinases↗
PubMed2005

EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis.

Summary EgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway, whereas core phenylpropanoid genes were not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation.

Amino Acid Sequence↗
PubMed2000

Biology of hypertensive cardiopathy.

Available data suggest that hypertensive cardiopathy is principally determined by the phenoconversion that allows the myocyte to adapt to the new working conditions by re-expressing a fetal program. Nevertheless, in clinical conditions, the scheme is different. The above phenotype is modified by trophic factors, which originate from ischemia, senescence, diabetes, genetics, or neurohormonal reactions. This review only focuses on some of the most recent advances concerning the permanent changes in the myocyte. Changes in extracellular matrix have been excluded. Recently, emphasis has been on the kinetic basis of the myocardial dysfunction at the myosin level, the potential therapeutic utilization of transferring the adrenergic receptor gene, the participation of NO synthases in the adaptational process, the existence of an abnormal excitation-contraction coupling due to a redistribution of Ca2+ sparks, the role of the microtubule as a determinant of sarcomere motion, and the multifactorial origin of cell death by apoptosis.

Adenosine Triphosphate↗
PubMed2007

The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations.

Germline mutations in the DNA mismatch repair (MMR) gene MLH1 are associated with a large percentage of hereditary non-polyposis colorectal cancers. There are approximately 250 known human mutations in MLH1. Of these, one-third are missense variants that are often difficult to characterize with regards to pathogenicity. We analysed 28 alleles of baker's yeast MLH1 that correspond to non-truncating human mutant alleles listed in online HNPCC databases, 13 of which had not been previously studied in functional assays. Using the highly sensitive lys2::InsE-A(14) reversion rate assay, we determined the MMR proficiency conferred by each allele in the S288c strain of Saccharomyces cerevisiae. Seven alleles conferred a null phenotype for MMR and eight others showed significant MMR defects, suggesting that all 15 are likely to be pathogenic in humans. In addition, we observed a strong correlation between these results, limited results from previous functional assays and clinical data. To test whether the potential pathogenicity of certain alleles depends on the genetic background of the host, we examined the mutation rates conferred by the mlh1 alleles in a second yeast strain, SK1, which is approximately 0.7% divergent from S288c. Many alleles displayed a difference in MMR efficiency between strain backgrounds with decreasing differences as the severity of the MMR defect increased. These findings suggest that genetic background can play an important role in determining the pathogenicity of MMR alleles and may explain cases of atypical colorectal cancer inheritance.

Adaptor Proteins, Signal Transducing↗
PubMed2007

The disaccharide trehalose inhibits proinflammatory phenotype activation in macrophages and prevents mortality in experimental septic shock.

Proinflammatory phenotype activation in macrophages (MPhis) after sepsis orchestrates an inflammatory response leading to multiple organ dysfunction. Trehalose preserves cell viability during exposure to a range of environmental stresses. We investigated whether trehalose may inhibit endotoxin-induced activation of the inflammatory phenotype in MPhis. Rat peritoneal MPhis were stimulated with 50 microg/mL of Salmonella enteritidis lipopolysaccharide (LPS). Stimulated MPhis were coincubated with trehalose (25, 50, and 100 mmol), sucrose (100 mmol), or RPMI alone. Macrophages cultures were used for Western blot analysis of extracellular-regulated kinase, c-jun-N terminal kinase, and inducible nitric oxide synthase; interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) gene expression by real-time reverse transcriptase-polymerase chain reaction, and supernatants for measuring the release of inflammatory cytokines and nitrite content. In vitro trehalose significantly blunted LPS-induced extracellular-regulated kinase (LPS = 21 +/- 6 integrated intensity; LPS + trehalose 100 mmol = 2 +/- 0.3 integrated intensity), c-jun-N terminal kinase (LPS = 15 +/- 5 integrated intensity; LPS + trehalose 100 mmol = 3.5 +/- 0.9 integrated intensity), and inducible nitric oxide synthase activation (LPS = 12 +/- 3 integrated intensity; LPS + trehalose 100 mmol = 1 +/- 0.09 integrated intensity), blunted IL-1beta (LPS = 5 +/- 1.9 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.5 +/- 0.8 n-folds/beta-actin), IL-6 (LPS = 4 +/- 1.5 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.4 +/- 0.5 n-folds/beta-actin), and TNF-alpha (LPS = 4.2 +/- 1.6 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.1 +/- 0.7 n-folds/beta-actin) gene expression, and markedly reduced the release of inflammatory cytokines and nitrite content. Furthermore, in vivo trehalose prevented mortality in rats challenged with a lethal dose (20 mg/kg; LD90) of LPS (80% survival rate and 70% survival rate 24 and 72 h after LPS injection, respectively) and reduced serum TNF-alpha. Sucrose did not modified inflammatory phenotype in vitro nor in vivo protected against endotoxin-induced mortality. Our study suggests that trehalose inhibits proinflammatory phenotype activation in MPhis and prevents endotoxin-induced mortality.

Animals↗
PubMed2001

Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes.

OBJECTIVE: To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in osteoarthritic (OA) cartilage compared with normal, age-matched articular cartilage. METHODS: Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL-1beta, TNFalpha, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography. RESULTS: Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP-3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL-1beta- and TNFalpha-positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, full-thickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL-1beta, TNFalpha, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL-1 with MMP-8 by individual chondrocytes in situ. CONCLUSION: These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL-1beta, TNFalpha, and 6 different MMPs. These observations support the concept that cytokine-MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.

Aged↗
PubMed1988

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity.

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.

Age Factors↗
PubMed1991

Hemoglobin Redondo [beta 92(F8) His----Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms.

Hb Redondo [beta 92(F8) His----Asn] illustrates how post-translational structural modifications may modify the phenotypic expression of an unstable hemoglobin. This variant was found in a Portuguese patient suffering from a chronic hemolytic anemia. The electrophoretic pattern demonstrated that it occurred in two forms, both being semi-hemoglobins: the fastest one migrating like HbS and the other like HbA2; after a few days of storage at 4 degrees C the intensity of the slowest Hb fraction decreased while that of the other increased proportionally. In both cases, the RP-HPLC analysis of the tryptic digest of the aminoethylated beta chains demonstrated the presence of an abnormal beta T10 peptide carrying a His----Asx substitution. Microsequence study of these two peptides demonstrated that the slowest abnormal Hb fraction had a beta 92 His----Asn substitution and the fastest a His----Asp at the same site. All these results suggest that the beta 92 His----Asn variant loses readily its heme group and that a deamidation occurs rapidly in vitro, yielding a beta 92 Asp semi-hemoglobin. The oxygen affinity of the patient's red blood cells was increased, leading to a stimulation of erythropoiesis and to a macrocytic hemolytic disease.

Amino Acid Sequence↗