Improved handling and preservation of freshwater giant prawn, Macrobrachium rosenbergii for producing safe and wholesome products

Studies were undertaken to evaluate the quality changes in freshwater giant prawn, Macrobrachium rosenbergii during various storage conditions of handling and preservation and producing safe and quality products. The samples kept in ice immediately after catch with head-on and head-less condition were found to be acceptable for 6 days and 7 days, respectively. Delaying of icing considerably shortened the shelf-life. The pH value increased from 6.36 to 8.0 after 10 days in ice. The initial average TVB-N value of sample increased from below 10 mg/100 g to 25 mg/100 g with the lapse of storage period. The Ca++ ATPase activity in presence of 0.1M KCl slightly decreased at the end of 10 days of ice storage. Immediately after harvest, initial aerobic plate count (APC) was 2.88x10^6 CFU/g which gradually increased to 1.12x10^8 CFU/g after 6 days in ice storage and showed early signs of spoilage. Initial bacterial genera in the prawn iced at 0 hours were comprised of Coryneform (22.21 %), Bacillus (7.40%), Micrococcus (11.11 %), Achromobacter (48.14%), Flavobacterium/Cytophaga (7.40%), Pseudomonas (3.70%) and Aeromonas (3.70%). During ice storage Coryneforms and Bacillus were always dominating along with less prominent ones - Micrococcus, Achromobacter and Flavobacterium. Studies were conducted on the stability of myofibrillar protein of M. rosenbergii under different storage and pH conditions. The influence of a wide range of pH on the remaining Ca++ ATPase activity of M. rosenbergii muscle myofibrils after storage at -20°C for 2 days, at 0°C for 2 days and at 35°C for 30 minutes demonstrated that ATPase activities were lower in acidic and alkaline pH regions and the activity remained relatively high. Mg++ ATPase activities both in presence and absence of Ca++ remained high at neutral pH compared to those of acidic and alkaline region. The solubility of myofibrillar protein decreased gradually both in acidic and alkaline pH regions. The study also examined the bacteriological quality of freshly harvested M. rosenbergii, pond sediment and pond water from four commercial freshwater prawn farms at Fulpur and Tarakanda upazilas in the district of Mymensingh. The study included aerobic plate count (APC), total coliform count, detection, isolation and identification of suspected public health hazard bacteria and their seasonal variation, salt tolerance test, antibiotic sensitivity test of the isolates and washing effect of chlorinated water on the bacterial load in the prawn samples. APC in sediment soil and water of the farm and gill and hepatopancreas of freshly harvested prawns varied considerably among the farms and between summer and winter season. The range of coliform count in water, gill and hepatopancreas ranged between 6 - 2.8x10^2 CFU/ml, 1.2x10^2 - 3.32x10^2 CFU/g and 1.43x10^2 - 3.89 x10^3 CFU/g, respectively. No coliform was detected in pond sediment sample. Suspected health hazard bacteria isolated and identified from pond sediment, water, gill and hepatopancreas included Streptococcus, Bacillus, Escherichia coli, Klebsialla, Salmonella, Staphylococcus, Pseudomonas and Aeromonas. Bacillus, Salmonella and Staphyloccus [sic], and were found to be highly salt tolerant and capable of growing at 10% NaCl. The antibiotic discs with different concentration of antibiotics were used for the sensitivity test. The organisms were found to be most sensitive against Tetracyclin and Gentamycin.

Impacto do pH final na maciez do músculo Longissimus lumborum de animais zebuínos: mudanças estruturais de proteínas da carne crua e cozida durante a maturação

O objetivo do presente trabalho foi determinar a força de cisalhamento Warner-Bratzler do músculo Longissimus lumborum de animais zebuínos machos inteiros (Bos indicus) durante o período de maturação, nas faixas de pH final (pHf 48 horas post mortem) normal (pH entre 5,5 e 5,8) e anormal (pH entre 5,81 e 6,19) e temperaturas internas de cozimento. Concomitante com a avaliação de força de cisalhamento, foram avaliadas também a degradação da desmina e troponina T, o comprimento do sarcômero, o teor de colágeno total e solúvel, as temperaturas máximas de desnaturação das proteínas e a morfologia geral de agregação das fibras do músculo no cozimento. A degradação da desmina e troponina T foi maior no pHf normal, aparecendo produtos de degradação a partir do dia 7 nessa faixa de pHf. Não houve diferenças nos valores de comprimento do sarcômero, descartando-se assim, a contribuição desse parâmetro sobre a temperatura máxima de desnaturação (Tmáx) das proteínas, determinada utilizando calorímetro exploratório diferencial (DSC). Similarmente, não foram encontradas diferenças para os teores de colágeno total e solúvel, e os valores de colágeno total foram baixos, sugerindo que sua contribuição na segunda transição térmica e nos valores de força de cisalhamento foi mínima. As Tmáx1 e Tmáx2, correspondentes à desnaturação da meromiosina leve e pesada, respectivamente, foram menores no pHf normal, mas o efeito foi maior para a Tmáx2. A Tmáx3 da actina e titina aumentou até 14 dias post mortem na faixa de pHf normal, e posteriormente diminuiu significativamente após 21 dias, sugerindo possível degradação dessas proteínas nesse período de dias. Não foram encontradas diferenças nos valores de Tmáx no pHf anormal, em todos os dias post mortem, o que sugere a contribuição de um possível mecanismo de proteção que estabiliza as miofibrilas no aquecimento. Houve maior agregação das fibras do músculo no pHf normal nas temperaturas internas de cozimento de 65 e 80°C, provavelmente devido à maior desnaturação térmica das miofibrilas. Os valores de força de cisalhamento foram maiores com o aumento da temperatura interna de cozimento, devido ao aumento da desnaturação térmica das miofibrilas do músculo. Independente da temperatura interna de cozimento, os valores de força de cisalhamento foram altos em quase todos os dias post mortem para ambas as faixas de pHf, o que sugere a necessidade de utilizar métodos físicos ou químicos para aumentar a maciez do músculo Longissimus lumborum de animais zebuínos.

Determination of binding constants by affinity chromatography

This review summarizes developments in the use of affinity chromatography to characterize biospecific interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes. By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitatiative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities. In addition, the link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with muscle myofibrils. an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment. (C) 2004 Elsevier B.V. All rights reserved.

The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs. (C) 2013 Elsevier Inc. All rights reserved.

Is titin a 'winding filament'? A new twist on muscle contraction.

Recent studies have demonstrated a role for the elastic protein titin in active muscle, but the mechanisms by which titin plays this role remain to be elucidated. In active muscle, Ca(2+)-binding has been shown to increase titin stiffness, but the observed increase is too small to explain the increased stiffness of parallel elastic elements upon muscle activation. We propose a 'winding filament' mechanism for titin's role in active muscle. First, we hypothesize that Ca(2+)-dependent binding of titin's N2A region to thin filaments increases titin stiffness by preventing low-force straightening of proximal immunoglobulin domains that occurs during passive stretch. This mechanism explains the difference in length dependence of force between skeletal myofibrils and cardiac myocytes. Second, we hypothesize that cross-bridges serve not only as motors that pull thin filaments towards the M-line, but also as rotors that wind titin on the thin filaments, storing elastic potential energy in PEVK during force development and active stretch. Energy stored during force development can be recovered during active shortening. The winding filament hypothesis accounts for force enhancement during stretch and force depression during shortening, and provides testable predictions that will encourage new directions for research on mechanisms of muscle contraction.

High pressure/thermal treatment effects on the texture of beef muscle

The effects of high pressure (to 800 MPa) applied at different temperatures (20-70 degreesC) for 20 min on beef post-rigor longissimus dorsi texture were studied. Texture profile analysis showed that when heated at ambient pressure there was the expected increase in hardness with increasing temperature and when pressure was applied at room temperature there was again the expected increase in hardness with increasing pressure. Similar results to those found at ambient temperature were found when pressure was applied at 40 degreesC. However, at higher temperatures, 60 and 70 degreesC it was found that pressures of 200 MPa caused large and significant decreases in hardness. The results found for hardness were mirrored by those for gumminess and chewiness. To further understand the changes in texture observed, intact beef longissimus dorsi samples and extracted myofibrils were both subjected to differential scanning calorimetry after being subjected to the same pressure/temperature regimes. As expected collagen was reasonably inert to pressure and only at temperatures of 60-70 degreesC was it denatured/unfolded. However, myosin was relatively easily unfolded by both pressure and temperature and when pressure denatured a new and modified structure was formed of low thermal stability. Although this new structure had low thermal stability at ambient pressure it still formed in both the meat and myofibrils when pressure was applied at 60 degreesC. It seems unlikely that structurally induced changes can be a major cause of the significant loss of hardness observed when beef is treated at high temperature (60-70 degreesC) and 200 MPa and it is suggested that accelerated proteolysis under these conditions is the major cause. (C) 2004 Elsevier Ltd. All rights reserved.

Ultrastructural analysis of the low level laser therapy effects on the lesioned anterior tibial muscle in the Gerbil

Low level laser therapy (LLLT) is known for its positive results but studies on the biological and biomodulator characteristics of the effects produced in the skeletal muscle are Still lacking. In this Study the effects of two laser dosages, 5 or 10 J/cm(2), on the lesioned tibial muscle were compared. Gerbils previously lesioned by 100 g load impact were divided into three groups: GI (n = 5) controls, lesion non-irradiated; GII (n = 5), lesion irradiated with 5 J/Cm(2) and GIII (n = 5), lesion irradiated with 10 J/cm(2), and treated for 7 consecutive days with a laser He-Ne (lambda = 633 rim). After intracardiac perfusion, the muscles were dissected and reduced to small fragments, post-fixed in 1% osmium tetroxide, dehydrated in increasing alcohol concentrations, treated with propylene oxide and embedded in Spurr resin at 60 degrees C. Ultrafine Cuts examined on a transmission electron microscope (Jeol 1010) revealed in the control GI group a large number of altered Muscle fibers with degenerating mitochondria, intercellular substance containing degenerating cell fragments and budding blood capillaries with Underdeveloped endothelial cells. However, groups GII and GIII showed muscle fibers with few altered myofibrils, regularly contoured mitochondria, ample intermembrane spaces and dilated mitochondrial crests. The clean intercellular Substance showed numerous collagen fibers and capillaries with multiple abluminal processes, intraluminal protrusions and several pinocytic vesicles in endothelial cells. it was concluded that laser dosages of 5 or 10 J/cm(2) delivered by laser He-Ne (lambda = 633 rim) during 7 consecutive days increase mitochondrial activity in muscular fibers, activate fibroblasts and macrophages and stimulate angiogenesis, thus suggesting effectivity of laser therapy tinder these experimental conditions. (C) 2009 Elsevier Ltd. All rights reserved.

Structural characteristics of the tendinous cord-papillary muscle junction in healthy and hypertensive rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Histochemical study of muscle fiber types in Synbranchus marmoratus Bloch, 1795

The myotomal muscle of Synbranchus marmoratus was investigated using histochemical and immunohistochemical reactions. This musculature is composed of a superficial red compartment, uniformly distributed around the trunk circumferentially and also in the lateral line. The red compartment fibers are small in diameter and have an oxidative metabolism, a high rate of glycogen and a negative reaction to alkaline and acid myofibrillar ATPase (mATPase). The white muscle forms the bulk of the muscle mass. Its fibers are large in diameter and have a glycolytic metabolism, a negative reaction to glycogen, a strong reaction to alkaline mATPase and a negative reaction to acid mATPase. Between these two compartments there is an intermediate layer of fibers presenting a mosaic metabolism pattern with a high rate of glycogen. These fibers stained moderately for alkaline and acid m-ATPase. Several clusters of red muscles were observed inside the white muscle. Each cluster is composed of three fiber types, with a predominance of red and intermediate fibers. Reactivity to anti-MHC BA-D5 was positive only in the intermediate fibers. Reactivity to anti-MHC SC-71 was negative in all fiber types.

Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na+ channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.

Morphological aspects of muscle regeneration in the Nile tilapia (Oreochromis niloticus)

The aim of our study was to analyze the morphological events in the skeletal muscle of the Nile tilapia (Oreochromis niloticus) after a traumatic lesion. Thirty-two fish were used, on which a small longitudinal incision was made in the muscle. The fish were sacrificed after 7, 14, 21, and 42 days and muscle samples were collected from the lesion and processed for morphological analysis. Muscle regeneration in the tilapia occurred gradually through the analyzed period, possibly due to the proliferation and differentiation of myosatellite cells, which were more morphologically evident 7 and 14 days after lesion.