Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation.

Autosomal recessive muscular dystrophy is genetically heterogeneous. One form of this disorder, limb-girdle muscular dystrophy type 2C (LGMD 2C), is prevalent in northern Africa and has been shown to be associated with a single mutation in the gene encoding the dystrophin-associated protein gamma-sarcoglycan. The previous mutation analysis of gamma-sarcoglycan required the availability of muscle biopsies. To establish a mutation assay for genomic DNA, the intron-exon structure of the gamma-sarcoglycan gene was determined, and primers were designed to amplify each of the exons encoding gamma-sarcoglycan. We studied a group of Brazilian muscular dystrophy patients for mutations in the gamma-sarcoglycan gene. These patients were selected on the basis of autosomal inheritance and/or the presence of normal dystrophin and/or deficiency of alpha-sarcoglycan immunostaining. Four of 19 patients surveyed had a single, homozygous mutation in the gamma-sarcoglycan gene. The mutation identified in these patients, all of African-Brazilian descent, is identical to that seen in the North African population, suggesting that even patients of remote African descent may carry this mutation. The phenotype in these patients varied considerably. Of four families with an identical mutation, three have a severe Duchenne-like muscular dystrophy. However, one family has much milder symptoms, suggesting that other loci may be present that modify the severity of the clinical course resulting from gamma-sarcoglycan gene mutations.     

High proportion of new mutations and possible anticipation in Brazilian facioscapulohumeral muscular dystrophy families.

A gene responsible for facioscapulohumeral muscular dystrophy (FSHD) has been localized at 4q35. Subsequently, it was found that probe p13E-11 detects a polymorphic EcoRI fragment, usually > 28 kb, in normal individuals, whereas in sporadic and familial FSHD cases, an EcoRI fragment, usually < 28 kb, was found. Although these findings have been amply confirmed, several aspects are as yet either controversial or unsolved. In the present investigation, 34 Brazilian FSHD families were studied at the clinical and the molecular level for the following purposes: to assess the frequency of new mutations and their effect on estimates of biological fitness, to characterize FSHD-associated EcoRI fragments detected with probe p13E-11 in familial--as compared with isolated--FSHD cases, and to assess whether anticipation occurs in multigenerational families. Results from our study suggest that new mutations are apparently frequent for FSHD and may account for at least one-third of the cases, that somatic mosaicism may not be rare, and that biological fitness appeared to be reduced in FSHD, ranging from 0.6 to 0.82 by different estimates, with no difference in sexes. Interestingly, the size of the new EcoRI fragment is apparently smaller in more severely affected isolated patients. Moreover, the age at onset of clinical signs, as well as the age at ascertainment, in patients from multigenerational families suggests that anticipation occurs for FSHD in the majority of the families.     

Vasoactive Intestinal Peptide Stimulates Chick Pineal Melatonin Production and Interacts with Other Stimulatory and Inhibitory Agents but Does Not Show α1-Adrenergic Potentiation

Vasoactive intestinal peptide (VIP) is known to mimic the effects of beta-adrenergic receptor stimulation in the rat pineal, including marked potentiation by alpha 1-adrenergic receptor stimulation, and to cause increased melatonin synthesis. In contrast, the chick pineal does not respond to beta-adrenergic stimulation, and melatonin synthesis is inhibited by norepinephrine via an alpha 2-adrenergic receptor. The present experiments show that chick pineal cells in primary culture do, however, respond to VIP with increased melatonin production. The effect of VIP was inhibited by addition of norepinephrine or of nitrendipine or by exposing the cells to "unexpected" white light. Stimulation by VIP was enhanced by addition of forskolin or Bay K 8644 but not by alpha 1-adrenergic receptor stimulations. Although stimulation by VIP appears similar in the chick pineal to that seen in the rat pineal and other systems, "dual-receptor regulation," at least with alpha 1-adrenergic receptors, appears to be absent.     

Photoendocrine Transduction in Cultured Chick Pineal Cells: IV. What Do Vitamin A Depletion and Retinaldehyde Addition Do to the Effects of Light on the Melatonin Rhythm?

Abstract: Light has at least two distinguishable effects on the circadian rhythm of melatonin output displayed by dispersed chick pineal cells in static culture: acute suppression of melatonin output and entrainment (phase shifts) of the underlying pacemaker. Previous results indicated that these two effects of light are mediated by different mechanistic pathways. The pathways for the acute and phase-shifting effects of light either branch from the same, single photopigment or differ from the outset, starting from separate photopigments. If a single rhodopsin-like photopigment mediates both effects of light, then vitamin A depletion and retinoid addition should affect both responses in parallel, although not proportionately. We therefore compared the effects of vitamin A depletion and retinoid addition on the acute and phase-shifting effects of light under several experimental conditions. When chick pineal cells were depleted of vitamin A, acute responses to light were markedly reduced. Addition of 11-cis-retinaldehyde specifically restored (and enhanced) the acute response. When allowed to free run in constant red light, depleted cells displayed a rhythm of melatonin output with the same period as that of control cells. In contrast to the acute effects, phase shifts in response to 2- or 4-h light pulses did not differ between depleted and control cells. Addition of retinaldehyde to depleted cells did not, by itself, reduce melatonin output or induce phase shifts. Retinaldehyde did increase the acute response to 4-h light pulses but not the ensuing phase shifts. Responses increased with duration of the light pulse: Both the acute effect and the phase shifts induced by 4-h light pulses were considerably larger than those induced by 2-h (or 1-h) light pulses. Addition of retinaldehyde to depleted cells increased the acute effect of 2-h (or 1-h) light pulses to at least that seen with 4-h light pulses but did not Increase the size of the ensuing phase shifts. These results strongly confirm previous dissociations of the mechanistic pathways mediating the acute and phase-shifting effects of light on chick pineal cells. They also support a role for rhodopsin-like photopigment in the acute, but not phase-shifting, response. They favor, but do not prove, the conclusion that separate photopigments mediate the acute and entraining effects of light.     

Regression of Albuminuria and Hypertension and Arrest of Severe Renal Injury by a Losartan-Hydrochlorothiazide Association in a Model of Very Advanced Nephropathy

Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization.     

Identification of ubiquinone-50 as the major methylated nonpolar lipid in human monocytes. Regulation of its biosynthesis via methionine-dependent pathways and relationship to superoxide production

Human blood monocytes incorporated the methyl group from methionine into their neutral lipids. The major methylated product was identified as ubiquinone-50 in monocytes, lymphocytes, and a variety of human tumor cell lines by several analytical procedures including TLC or high performance liquid chromatography and as ubiquinone-45 in a mouse tumor cell line. Up to three methyl groups were shown to be derived from methionine by mass spectrometry. The rate of synthesis of ubiquinone-50 by monocytes as assessed by measuring labeled methyl group incorporation was shown to be linear over a 3-h period. Degradation of ubiquinone proceeded slowly; 80% of the labeled compound persisted after 18 h. The dependence of ubiquinone-50 synthesis upon methionine concentration was established in monocytes, with an estimated apparent Km for methionine of about 20 microM. The tumor promoter, tetradecanoate phorbol acetate, a potent stimulator of superoxide anion (O2-) production in phagocytic cells, inhibited ubiquinone-50 synthesis at nanomolar concentrations in monocytes, but not in lymphocytes, under conditions where oxidation of methionine takes place. Degradation of the labeled ubiquinone was unaffected. Formylmethionylleucyl-phenylalanine, a chemoattractant peptide which stimulates O2- production in phagocytic cells, also inhibited ubiquinone-50 synthesis. The degree of inhibition by either stimulus was increased when the methionine concentration in the medium was low. These findings demonstrate that in human monocytes ubiquinone-50 biosynthesis is regulable and that methionine concentration modulates both its rate of synthesis and the inhibitory effects of two stimuli of O2- production.     

Combined Effect of AMPK/PPAR Agonists and Exercise Training in mdx Mice Functional Performance

The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR (AMPK agonist, 50 mg/Kg-1.day-1, ip) and GW 1516 (PPARδ agonist, 2.5 mg/Kg-1.day-1, gavage)], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression (AMPK, PPAR, myoglobin and SCD) and intracellular calcium handling (DHPR, SERCA and NCX) protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies.     

Mechanism of action of thymosin. I. Thymosin fraction 5 increases lymphokine production by mature murine T cells responding in a mixed lymphocyte reaction

The effect of thymosin on the murine thymocyte mixed lymphocyte response was studied. Thymosin fraction 5 (TF5) caused a two- to threefold enhancement of the proliferative response and production of IL 2 when murine thymocytes were cultured with alloantigenic stimulator cells. Production of a second lymphokine, CSF, was increased up to sevenfold. The target cell for thymosin was a mature T cell, because the PNA- subpopulation of thymocytes, as well as peripheral lymph node lymphocytes, responded to culture with TF5 and alloantigen by enhanced proliferation and lymphokine production. The active component of TF5 appears to be one or more as yet unidentified peptides, because neither of the well-characterized TF5 component peptides, alpha 1 or beta 4, were active. After incubation with TF5 in primary culture, cells remaining after 10 to 14 days were increased both in number and in secondary response to alloantigen, as measured by lymphokine production. These results suggest that TF5 contains one or more biologically active components which can modulate mature T cell activity and lymphokine production, and which provide the basis for understanding some of the previously reported diverse effects of thymosin.     

Translocation of Protein Kinase C in Anterior Pituitary Tumor Cells

Previous studies have shown that phorbol esters and lithium each stimulate the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary tumor cell line AtT20/D16-16. Pretreatment with either lithium or phorbol ester desensitizes the cells to subsequent stimulation by phorbol ester. An early consequence of phorbol ester action in other systems is the translocation of protein kinase C from cytosol to membranes. We have assayed protein kinase C activity in cytosol and membranes of AtT20 cells after treatment with phorbol dibutyrate, lithium, or other agents that stimulate secretion of ACTH in these cells. Phorbol dibutyrate clearly induced translocation of protein kinase C, but lithium treatment did not cause translocation itself, nor did pretreatment with lithium affect the translocation induced by phorbol dibutyrate. These results are consistent with a role for translocation of protein kinase C in the stimulatory and desensitizing effects of phorbol esters but fail to implicate translocation in the actions of lithium on AtT20 cells.     

Acylation of disc membrane rhodopsin may be nonenzymatic

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.