Circulating soluble intercellular adhesion molecule-1 (sICAM-1) after exercise-induced muscular damage: Does the use of whole-body cryostimulation influence its concentration in blood?

As soluble intercellular adhesion molecule-1 (sICAM-1) was recently hypothesized to be a key player in the mechanisms involved in exercise-induced muscular damage (EIMD), we investigated its circulating concentration changes in athletes before and after EIMD with and without the use of whole-body cryostimulation (WBC; 3 min at −110 °C) at the exercise end and repeated once a day during 4 days.We previously characterized plasma specimens from 11 endurance athletes who performed twice (randomized crossover design) strenuous running leading to EIMD, followed by passive recovery or WBC. Muscle soreness and inflammatory response were observed in both cases but the use of WBC induced a significant reduction in these responses (PlosOne 2011; 6:e22748). We now found that sICAM-1 concentration slightly increased in both circumstances and remained elevated for 24 h (p < 0.01). However, no significant WBC effect was observed concerning sICAM-1 changes indicating that this compound is not a major player both in EIMD and WBC physiological impacts.     

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.     

Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928-1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor.     

The nuclear envelope in muscular dystrophy and cardiovascular diseases

Considerable interest has been focused on the nuclear envelope in recent years following the realization that several human diseases are linked to defects in genes encoding nuclear envelope specific proteins, most notably A-type lamins and emerin. These disorders, described as laminopathies or nuclear envelopathies, include both X-linked and autosomal dominant forms of Emery–Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system defects, limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances, and Dunnigan-type familial partial lipodystrophy. Certain of these diseases are associated with nuclear structural abnormalities that can be seen in a variety of cells and tissues. These observations clearly demonstrate that A-type lamins in particular play a central role, not only in the maintenance of nuclear envelope integrity but also in the large-scale organization of nuclear architecture. What is not obvious, however, is why defects in nuclear envelope proteins that are found in most adult cell types should give rise to pathologies associated predominantly with skeletal and cardiac muscle and adipocytes. The recognition of these various disorders now raises the novel possibility that the nuclear envelope may have functions that go beyond housekeeping and which impact upon cell-type specific nuclear processes.     

Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

The protease core of the muscle-specific calpain,p94, undergoes Ca2+-dependent intramolecular autolysis

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.     

Localization of the muscular dystrophy AM locus using a chicken linkage map constructed with the Kobe University resource family

A chicken linkage map, constructed with the Kobe University (KU) resource family, was used to locate the genetic locus for muscular dystrophy of abnormal muscle type (AM). The KU resource family is a backcross pedigree with 55 offspring produced from the mating of a White Leghorn F-line (WL-F) male and a hybrid female produced from a cross between the WL-F male and a female of the Fayoumi OPN line who was homozygous for the AM gene. In total, 872 loci were genotyped on the pedigree; 749 (86%) were informative and mapped to 38 linkage groups. These informative loci included 649 AFLPs, 93 MS, three functional genes, the AM locus, sex phenotype, and two red blood cell loci. The remaining 123 markers were unlinked. Nineteen of the 38 KU linkage groups were assigned to macrochromosomes 1-8 and 11 microchromosomes including chromosome W, while 19 linkage groups were unassigned. The total map was 3569 cM in length, with an average marker interval of 4.8 cM. The AM locus was mapped 130 cM from the distal end of chromosome 2q.     

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses

Background

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Methods

Replication‐defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7‐cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre‐terminal protein, and with a cre‐recombinase plasmid.

Results

We show that C7‐cre cells allow efficient production of gutted Ad using ΔE1 + ΔE2b + ΔE3 helper viruses whose growth can be limited by cre‐loxP‐mediated excision of the packaging signal. Gutted Ad vectors carrying ～28 kb cassettes expressing full‐length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%.

Conclusions

These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b‐deleted vectors display a profound reduction in viral gene expression. Copyright © 2002 John Wiley & Sons, Ltd.

     

Expanded CTG repeats inhibit neuronal differentiation of the PC12 cell line

Myotonic dystrophy (DM) is a dominant neuromuscular disorder caused by the expansion of trinucleotide CTG repeats in the 3-untranslated region (3'-UTR) of the MtPK gene. Although DM-associated mental retardation suggests that neuronal functions are disturbed by the expansion mutation, the effect of this alteration in neuronal cells has not been approached. In this study we established stable transfectans of PC12 neuronal cell line expressing the reporter gene CAT alone (empty-vector clone) or fused to the MtPK 3'-UTR with 5, 60, or 90 CTG repeats (CTG5, CTG60, and CTG90 clones, respectively). CTG90 cells exhibited a suppression of NGF-induced neuronal differentiation while empty-vector, CTG5 and CTG60 clones differentiated normally. CTG90 cells displayed normal activation of early differentiation markers, ERK1/2, but the up-regulation of the late marker MAP2 was dramatically reduced. Our neuronal cell system provides the first information of how the mutant MtPK 3'-UTR mRNA affects neuronal functions.     

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.