Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ~30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.     

Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes.

Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region. We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)). It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.     

Mutations in the N-terminal actin-binding domain of filamin C cause a distal myopathy

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.     

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.     

SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.     

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.