Myotonic dystrophy type 2 (DM2) : Diagnostic methods and molecular pathology

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are the most common forms of muscular dystrophy affecting adults. They are autosomal dominant diseases caused by microsatellite tri- or tetranucleotide repeat expansion mutations in transcribed but not translated gene regions. The mutant RNA accumulates in nuclei disturbing the expression of several genes. The more recently identified DM2 disease is less well known, yet more than 300 patients have been confirmed in Finland thus far, and the true number is believed to be much higher. DM1 and DM2 share some features in general clinical presentation and molecular pathology, yet they show distinctive differences, including disease severity and differential muscle and fiber type involvement. However, the molecular differences underlying DM1 and DM2 muscle pathology are not well understood. Although the primary tissue affected is muscle, both DMs show a multisystemic phenotype due to wide expression of the mutation-carrying genes. DM2 is particularly intriguing, as it shows an incredibly wide spectrum of clinical manifestations. For this reason, it constitutes a real diagnostic challenge. The core symptoms in DM2 include proximal muscle weakness, muscle pain, myotonia, cataracts, cardiac conduction defects and endocrinological disturbations; however, none of these is mandatory for the disease. Myalgic pains may be the most disabling symptom for decades, sometimes leading to incapacity for work. In addition, DM2 may cause major socio-economical consequences for the patient, if not diagnosed, due to misunderstanding and false stigmatization. In this thesis work, we have (I) improved DM2 differential diagnostics based on muscle biopsy, and (II) described abnormalities in mRNA and protein expression in DM1 and DM2 patient skeletal muscles, showing partial differences between the two diseases, which may contribute to muscle pathology in these diseases. This is the first description of histopathological differences between DM1 and DM2, which can be used in differential diagnostics. Two novel high-resolution applications of in situ -hybridization have been described, which can be used for direct visualization of the DM2 mutation in muscle biopsy sections, or mutation size determination on extended DNA-fibers. By measuring protein and mRNA expression in the samples, differential changes in expression patterns affecting contractile proteins, other structural proteins and calcium handling proteins in DM2 compared to DM1 were found. The dysregulation at mRNA level was caused by altered transciption and abnormal splicing. The findings reported here indicate that the extent of aberrant splicing is higher in DM2 compared to DM1. In addition, the described abnormalities to some extent correlate to the differences in fiber type involvement in the two disorders.

Magnetic resonance imaging and ultrasonography in brachial plexus birth injury

Brachial plexus birth injury (BPBI) is caused by stretching, tearing or avulsion of the C5-C8 or Th1 nerve roots during delivery. Foetal-maternal disproportion is the main reason for BPBI. The goal of this study was to find out the incidence of posterior subluxation of the humeral head during first year of life in BPBI and optimal timing of the ultrasonographic screening of the glenohumeral joint. The glenohumeral congruity and posterior subluxation of the humeral head associated to muscle atrophy were assessed and surgical treatment of the shoulder girdle as well as muscle changes in elbow flexion contracture were evaluated. The prospective, population based part of the study included all neonates born in Helsinki area during years 2003-2006. Patients with BPBI sent to the Hospital for Children and Adolescents because of decreased external rotation, internal rotation contracture or deformation of the glenohumeral joint as well as patients with elbow flexion contracture were also included in this prospective study. The incidence of BPBI was calculated to be 3.1/1000 newborns in Helsinki area. About 80% of the patients with BPBI recover totally during the follow-up within the first year of life. Permanent plexus injury at the age of one year was noted in 20% of the patients (0.64/1000 newborns). Muscle imbalance resulted in sonographically detected posterior subluxation in one third of the patients with permanent BPBI. If muscle imbalance and posterior subluxation are left untreated bony deformities will develop. All patients with internal rotation contracture of the glenohumeral joint presented muscle atrophy of the rotator cuff muscles. Especially subscapular and infraspinous muscles were affected. A correlation was found particularly between greatest thickness of subscapular muscle and subluxation of the humeral head, degree of glenoid retroversion, as well as amount of internal rotation contracture. Supinator muscle atrophy was evident among all the studied patients with elbow flexion contracture. Brachial muscle pathology seemed to be an important factor for elbow flexion contracture in BPBI. Residual dysfunction of the upper extremity may require operative treatment such as tendon lengthening, tendon transfers, relocation of the humeral head or osteotomy of the humerus. Relocation of the humeral head improved the glenohumeral congruency among patients under 5 years of age. Functional improvement without remodeling of the glenohumeral joint was achieved by other reconstructive procedures. In conclusion: Shoulder screening by US should be done to all patients with permanent BPBI at the age of 3 and 6 months. Especially atrophy of the subscapular muscle correlates with glenohumeral deformity and posterior subluxation of the humeral head, which has not been reported in previous studies. Permanent muscle changes are the main reason for diminished range of motion of the elbow and forearm. Relocation of the humeral head, when needed, should be performed under the age of 5 years.

Inverse relationship of the dehydrogenase and ADP-ribosylation activities in sodium-nitroprusside-treated glyceraldehyde-3-phosphate dehydrogenase is coincidental

Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) with sodium nitroprusside (SNP) decreased its activity in concentration- and time-dependent fashion in the presence of a thiol compounds, with DTT being more effective than GSH. Both forward and backward reactions were effected. Coinciding with this, HgCl2-sensitive labelling of the protein by [32P]NAD+ also increased, indicating the stimulation of ADP-ribosylation. Treatment with SNP of GAPD samples from rabbit muscle, sheep brain and yeast inactivated the dehydrogenase activity of the three, but only the mammalian proteins showed ADP-ribosylation activity. The SNP-modified protein of rabbit muscle GAPD, freed from the reagent by Sephadex filtration showed a concentration-dependent restoration of the dehydrogenase activity on preincubation with DTT and GSH. Such thiol-treated preparations also gave increased ADP-ribosylation activity with DTT, and to a lesser extent with GSH. The SNP-modified protein was unable to catalyze this activity with the native yeast enzyme and native and heat-inactivated muscle enzyme. It was possible to generate the ADP-ribosylation activity in muscle GAPD, by an NO-independent mechanism, on dialysis in Tris buffer under aerobic conditions , and on incubating with NADPH, but not NADH, in muscle and brain, but not yeast, enzymes. The results suggest that the inverse relationship of the dehydrogenase and ADP-ribosylation activities is coincidental but not correlated

The analysis of dieldrin residues in the brain, liver and muscles of the African catfish Clarias gariepinus (Burchell 1822) chronically exposed to dieldrin

Mature adult Clarias gariepinus were obtained at the ABRU hatchery in Sonning (UK), where they had beenbred and reared for several years. These were exposed to two concentrations of dieldrin in water (2.4 mu g super(-1) and 4.0 mu g super(-1). The residue analysis of diedrin in three tissues exposed for on moth at two concentrations was carried out. These were subjected to GLC analytical process. The results indicated significantly (P<0.05) higher residues in liver than in muscle and brain. The results also showed that residue levels were dependant on exposure concentration

Relation between TMAOase activity and content of formaldehyde in fillet minces and bellyflap minces from gadoid fishes

Minced fish is a significant component of a number of frozen fishery products like fish fingers, cakes and patties. Predominately minced fish is produced from gadoid species (Alaska pollack, cod, saithe, hake and others) possessing the enzyme trimethylamine oxide demethylase (TMAOase, E.C. 4.1.2.32) (Rehbein and Schreiber 1984). TMAOase catalyses the degradation of trimethylamine oxide (TMAO) to formaldehyde (FA) and dimethylamine (DMA), preferentially during frozen storage of products (Hultin 1992). In most gadoid species light muscle contains only low activity of TMAOase, the activity of red muscle and bellyflaps being somewhat higher. In contrast, the TMAOase activity in blood, kidney and other tissues, residues of which may contaminate minced fish flesh, may be higher for several orders of magnitude (Rehbein and Schreiber 1984).

A rapid and simple method for the determination of Formaldehyde in Fishery Products

The content of free formaldehyde (FA) in minced fish muscle was measured by the following procedure: A mixture of minced fish muscle and water was deproteinized by means of the Carrez reagent. The FA content of the filtrate was determined reflectometrically using the Reflectoquant test strips and the RQflex. The results agreed well with the colorimetrically (Nash test) measured FA content. Der Gehalt an freiem Formaldehyd (FA)in Fischerzeugnissen wurde mit folgender Methode bestimmt: Zerkleinertes Fischfleisch wurde mit Wasser homogenisiert und mit Carrez-Reagenz enteiweißt. Der Formaldehydgehalt des Filtrates wurde reflektometrisch unter Verwendung von Reflectoquant-Teststätbchen und des RQflex ermittelt. Die Ergebnisse stimmten gut mit kolorimetrisch (Nash Test) gemessenen FA-Gehalten überein.

Surface mucous as a source of genomic DNA from Atlantic billfishes (Istiophoridae) and swordfish (Xiphiidae)

Procedures for sampling genomic DNA from live billfishes involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples from Atlantic blue marlin (Makaira nigricans), white marlin (Tetrapturus albidus), sailfish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified polymorphic DNA analysis, and mitochondrial and nuclear locus sequencing. The nondestructive and less invasive characteristics of surface mucous collection may promote increased survival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens destined for release.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

Human muscle spindles act as forward sensory models.

Modern theories of motor control incorporate forward models that combine sensory information and motor commands to predict future sensory states. Such models circumvent unavoidable neural delays associated with on-line feedback control. Here we show that signals in human muscle spindle afferents during unconstrained wrist and finger movements predict future kinematic states of their parent muscle. Specifically, we show that the discharges of type Ia afferents are best correlated with the velocity of length changes in their parent muscles approximately 100-160 ms in the future and that their discharges vary depending on motor sequences in a way that cannot be explained by the state of their parent muscle alone. We therefore conclude that muscle spindles can act as "forward sensory models": they are affected both by the current state of their parent muscle and by efferent (fusimotor) control, and their discharges represent future kinematic states. If this conjecture is correct, then sensorimotor learning implies learning how to control not only the skeletal muscles but also the fusimotor system.

Some aspects of curing of sharks and rays

A simple method for eliminating urea almost completely from elasmobranch muscle consists in desalting the initially salted muscle in 5% brine. The initial salting removes about 58% of the urea, which occurs to the extent of 5.8 to 7.5% D.W.B. in the fresh muscle, and the desalting further reduces the urea content to negligible quantities (0.2%).

Quantitative and qualitative studies on the bacterial flora of fresh sardines

Plate counts at R T and 8 C on the skin with muscle and the gut contents of absolutely fresh sardines (Sardinella longiceps) caught off Cochin showed a seasonal variation when sampling was done over a period of 12 months. The counts of the gut contents ran parallel with those of the skin with muscle, but at a higher level of magnitude. Qualitatively, the analysis of 360 strains of bacteria isolated from the skin with muscle and 100 strains from the guts during a year's study revealed a very high preponderance of Gram negative rods, mainly of Achromobacter, Vibrio, and Pseudomonas groups. The percentage of Gram positive organism was very low or nil at times in the ocean fresh sardines.

Studies on isolation of salmonella from sea foods 1. Comparison of enrichment and selective media for recovery of salmonellae from fish

Three enrichment broths and six plating media were compared for efficiency of detection Salmonella in the presence of numbers of Coliforms (10super(5)/ml) and proteus (10super(3)/ml) from artificially inoculated fish samples. Recovery experiments Salmonella anatum, S. typhimurium and S. enteritidis indicated that the two enrichment broths Dulcitol Selinite (DSE) and Selinite Cystine (SC) were equally efficient. Further, the viability of Salmonella, inoculated into fish muscle and kept at 4°C for 48 hours, was found to be not affected by the low temperature storage. Selective plating media like Xylose Lysine Deoxycholate agar (XLD), Brilliant Green Sulphadiazine agar (BGS) and Brilliant Green agar (BG) were found to be superior in performance to Salmonella-Shigella agar: (SS) and Bismuth Sui phite agar (BiS).

Population diversity studies of pharaoh cuttlefish (Sepia pharaonis) in the Persian Gulf and the Gulf of Oman

The pharaoh Cuttlefish (Sepia Pharaonis) is the most abundant species in the persian gulf and oman sea. The stock patterns of this species was studied conserning biological, morphological and electrophoretical aspects. In addition to measuring the biological patterns; 21 quantitative and qualitative factors were measured or counted. There tissues namely muscle, eye and liver were also used for further polyacrylamide electrophoresis analysis (SDS-Page method). The densitograms of protein bands of each tissue were prepared by Gel-Scanner and also the amount of bands' areas were estimated. The results of LSD test showed that tentacle length (TL), Tentacle club length (TCL) and TCL were indicative factors which they showed significant difference between male TL and female specimen of Bushehr and Balouchestan regions. Regarding to length and weight frequencies data the results indicated that males are always bigger than females and also, the cuttlefishes of the gulf of oman are bigger than persian gulf's samples. There were found a significant sample correlation (95%) between different quantitative parameters and the most correlation (0.963) was found to be between TL and TCL; whereas the less correlation (0.384) was observed between GL and LA3. However the gill length factor illustrated the less correlation with the other factors. The results of Cluster analysis for both sexes showed that the cuttlefishes of both studied regions belongs to seperate stocks. The electrophoretic experiments of proteins showed that the protein bands of the sample tissue of muscle and eye from both sampling areas revealed significant differences; whereas the tissue of liver wasn't recognized as an indicative tissue for population studies. Taking into consideration the findings of the present study including: (1) difference in spawning season, (2) results of dendrograms, (3) observed Significant differences in one-way analysis of variance (ANOVA) for morphometric measurements, (4) differences in body length and weight, (5) ecological variations of the persian gulf and oman sea, (6) as well as the results of electrophoresis of proteins have indicatend that: "The Pharaoh cuttlefishes of Bushehr and Balouchestan waters belong to two seperate stocks. Also, it is believed that each region has propably two different populations and more studies are needed to approve this result.

Characterization of the porcine differentially expressed PDK4 gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.