Dysferlin is a plasma membrane protein and is expressed early in human development

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.     

Modification of T-cell receptor Vbeta repertoire in response to allergen stimulation in peanut allergy

BACKGROUND: Peanut is one of the most common foods causing allergic reactions and is the most common cause of fatal and near-fatal food-related anaphylaxis. Little is known of the immunologic mechanisms that underlie peanut allergy. OBJECTIVES: In this study we examined clonality of the T-cell response (TCR) to peanut in MHC class II identical, peanut allergy-discordant sibling pairs. METHODS: Four sibling pairs were investigated. The TCR repertoire was analyzed before and after in vitro stimulation of PBMCs with crude peanut or PHA, as control for general/nonspecific reactivity. Eighteen TCR-Vbeta families were examined by flow cytometry. Where significant differences in incidence of particular TCR-Vbeta families were observed, PCR familyspecific cDNA amplification and gene scanning were performed. RESULTS: After stimulation with peanut, no selective expansion of any TCR-Vbeta subpopulation was observed with flow cytometry, in either the peanut-allergic or nonallergic siblings, with the exception of 1 peanut-allergic subject who demonstrated a significant increase of TCR-Vbeta11(+) cells (0.3%-5.9% of the total CD3(+) cells). However, gene scanning revealed predominant single-size PCR products for TCRBV11 in all peanut-allergic subjects after peanut stimulation. TCRBV11 polyclo-nality was observed in allergic and nonallergic subjects before peanut stimulation and in nonallergic subjects after peanut stimulation. In comparison, all subjects, before and after stimulation with peanut, showed polyclonality for TCRBV2. CONCLUSIONS: Our results argue for clonal or oligoclonal TCRs to crude peanut and indicate that changes in the TCRBV11 subpopulation are restricted to peanut-allergic subjects after stimulation with crude peanut allergen.     

Comparison of biochemical, morphologic, and chemical characteristics of Centers for Disease Control fermentative coryneform groups 1, 2, and A-4.

A total of 24 strains of fermentative coryneform like bacteria isolated from clinical specimens form two distinct groups which have been designated Centers for Disease Control (CDC) fermentative coryneform groups 1 (13 strains) and 2 (11 strains). The phenotypic characteristics of group 1 were similar to those of a previously described CDC group designated A-4, with the major differentiating characteristic being the inability to hydrolyze esculin. Major differences in cellular fatty acid composition between CDC groups 1 and A-4 were also observed. The branched-chain fatty acids 14-methylhexadecanoate and 12-methyltetradecanoate, which account for more than 80% of the total acids of group A-4, were not detected in cells of group 1 strains. Groups 1 and 2, which have similar cellular fatty acid compositions, can be differentiated on the basis of fermentation of xylose, mannitol, lactose, sucrose, and melibiose by group 1 but not by group 2. The sources of isolation of the strains of both groups varied. Only group 1 strains were associated with eye infections.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Experimental interstitial renal fibrosis in rats: nephritis induced by N-(3,5-dichlorophenyl)succinimide.

The nephrotoxic properties of the chemical N-(3,5-dichlorophenyl)-succinimide were investigated in rats with a view to establishing the usefulness of this chemically-induced nephritis as a model of chronic interstitial renal fibrosis. The compound was synthesized and given daily by gastric intubation as a suspension in arachis oil B.P. to male WAG-strain rats, for periods of up to 108 days. Polydipsia and polyuria resulted rapidly in all treated animals and persisted for the duration of the experiment. There was a progressive increase in the extent of proteinuria in all treated animals and, by the end of the experiment, there was an increase in the plasma levels of urea and creatinine. Short term treatment (up to 3 days) resulted in focal areas of necrosis of some proximal convoluted tubules. Treatment for 28 days resulted in patchy but severe tubular interstitial nephritis with which was associated a moderate interstitial fibrosis. By 108 days, the nephritis was more widespread and the interstitial fibrosis was severe. The activity of proline hydroxylase, a part of the intracellular sequence of collagen synthesis, showed progressive increase in the renal cortex throughout the experiment and there was an associated increase in the cortical hydroxyproline content, a measure of the amount of collagen present. Associated with this biochemical evidence of an active, chronic fibrosis, was an increased water content of the cortical tissue. The results indicate that this chemically-induced, tubular interstitial nephritis is indeed a good and reliable model of interstitial renal fibrosis.     

Smoking Cessation in the Chicago Coronary Prevention Evaluation Program

Quit-rates for cigarette smokers in a lifestyle intervention program aimed at reducing coronary risk were 24 percent for all participants and 34 percent for non-dropouts. Recidivism remained very low during participation in the program. Half of the smokers who quit did so after being in the program more than two years. These data suggest that while engaging in an effort to make other changes in lifestyle, many smokers can be helped to quit. Sustained antismoking efforts in the clinical practice of medicine can be expected to share these same positive aspects. While mass public health programs to eliminate smoking and prevent young people from taking up the habit are being developed, health practitioners can make a significant contribution by including vigorous efforts at smoking cessation as part of routine practice.     

Towards the understanding of muscle contraction and its regulation,A Symposium in honor of John Gergely,MD, PhD

Journal of Muscle Research and Cell Motility -     

Modification of T-cell receptor Vβ repertoire in response to allergen stimulation in peanut allergy

Background: Peanut is one of the most common foods causing allergic reactions and is the most common cause of fatal and near-fatal food-related anaphylaxis. Little is known of the immunologic mechanisms that underlie peanut allergy. Objectives: In this study we examined clonality of the T-cell response (TCR) to peanut in MHC class II identical, peanut allergy–discordant sibling pairs. Methods: Four sibling pairs were investigated. The TCR repertoire was analyzed before and after in vitro stimulation of PBMCs with crude peanut or PHA, as control for general/nonspecific reactivity. Eighteen TCR-Vβ families were examined by flow cytometry. Where significant differences in incidence of particular TCR-Vβ families were observed, PCR familyspecific cDNA amplification and gene scanning were performed. Results: After stimulation with peanut, no selective expansion of any TCR-Vβ subpopulation was observed with flow cytometry, in either the peanut-allergic or nonallergic siblings, with the exception of 1 peanut-allergic subject who demonstrated a significant increase of TCR-Vβ11⁺ cells (0.3%-5.9% of the total CD3⁺ cells). However, gene scanning revealed predominant single-size PCR products for TCRBV11 in all peanut-allergic subjects after peanut stimulation. TCRBV11 polyclo-nality was observed in allergic and nonallergic subjects before peanut stimulation and in nonallergic subjects after peanut stimulation. In comparison, all subjects, before and after stimulation with peanut, showed polyclonality for TCRBV2.Conclusions: Our results argue for clonal or oligoclonal TCRs to crude peanut and indicate that changes in the TCRBV11 subpopulation are restricted to peanut-allergic subjects after stimulation with crude peanut allergen. (J Allergy Clin Immunol 2001;107:1089-94.)     

Glycoprotein synthesis in cells infected with vaccinia virus. I. Non-virion glycoproteins

When vaccinia-virus infected HeLa cells were incubated in medium containing radioactively labeled glucosamine, the specific activity of the UDP-N-acetylhexosamine pool increased linearly at a rate similar to that of uninfected cells. Nevertheless, starting at 2 hr after infection, hexosamines were incorporated into glycoproteins at a progressively lower rate. Furthermore, from electrophoretic analysis it appeared as though synthesis of the major glycoproteins of uninfected cells was arrested and new glycoproteins were labeled. After equilibrium centrifugation the major vaccinia-induced glycoproteins did not sediment with infectious virus but a large portion was recovered from fractions rich in cell membranes and containing particulate material. Glycoproteins of similar electrophoretic mobilities were made in vaccinia-infected HeLa and chick cells. Both actinomycin D and cycloheximide inhibited the labeling of all virus-induced glycoproteins whereas rifamycin derivatives had a selective effect.