Molecular species of collagen in the intramuscular connective tissues of the marine crab, Scylla serrata

Type V like collagens are widely distributed in marine invertebrates, particularly crustaceans and molluscs. We have been investigating the nature of collagens in the muscular tissues of crustaceans. The presence of type V like homotrimeric collagen in prawn muscle was noted before. We report here a comparative analysis of collagens purified from the pepsin digest of abdominal and pereiopod muscle tissues of the crab, Scylla serrata. The major collagen in either muscle precipitated at 1.2 M NaCl at acid pH, suggestive of a type V like property. The homotrimeric collagen was then purified to near homogeneity by precipitation with 20% ammonium sulphate. Solubility characteristics and biochemical studies indicated the leg muscle collagens to be highly crosslinked and stabilised by more bound carbohydrates, as compared to the abdominal muscle collagen. Analysis of amino acid composition revealed a close similarity to known type V collagens and the leg muscle collagen was characterised by more lysine hydroxylation and slightly reduced glycine content. The leg muscle collagen had a higher denaturation temperature and intrinsic viscosity than the abdominal muscle collagen. Our results confirm the similarity of major crustacean muscle collagens to vertebrate type V collagen. Further, the relative complexity of leg muscle collagen, unlike the abdominal muscle collagen, correlates to the specific functional requirements, where the former is involved in locomotion and preying and the latter in normal growth and development.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Summary— Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

The composition and characteristics of skin and muscle collagens from a freshwater catfish grown in biologically treated tannery effuent water

The skin of catfish Gariepinus spp. reared in tannery effuent water (ETP) had an increased collagen content, decreased acid solubility and high carbohydrate association as compared with skin of normal fish. The skin collagen in either fish was composed of three distinct α chains with mobilities different from that of vertebrate type I α chains and two of these three chains were invariably present in the muscle collagen. The amino acid composition of ETP fish skin and muscle collagens were similar but were characterized by higher degrees of proline and lysine hydroxylation, indicating higher stability. Both the skin and muscle collagens had significantly high denaturation temperatures. Electron microscopy of in vitro reconstituted fibrils of skin and muscle collagens of ETP fish displayed defined periodicities of around 64 nm. This demonstrates that the polymorphism in skin collagen chains is not confined to marine fish. There was a close similarity in the composition of skin and muscle collagens and the ETP environment influences the solubility and stability of the skin collagen. It is hypothesized that the third chain may play a role in the anchorage of skin to muscle, as such chain composition is now evident in fish such as hake, cod and catfish where the musculature is strongly attached to the skin.     

Lipid profiles of Nematocarcinus gracilis a deep-sea shrimp from below the Arabian Sea oxygen minimum zone

Specimens of the deep-sea benthic shrimp Nematocarcinus gracilis were collected from 900 m to 1000 m in the Arabian Sea, close to where the permanent oxygen minimum zone meets the sea floor. Lipid profiles, encompassing total lipid, lipid class and fatty acid composition, were compared with previously reported crustacean lipid assays and provided an insight into the life history of the species. The major storage lipid in N. gracilis was triglyceride, supporting the supposition that this species exists in benthic regions. Neutral lipid levels were commensurate with N. gracilis being an opportunistic feeder. Fatty acid composition was typical of an organism with a diet based on an ultimately photosynthetic source of organic carbon, but also reflected the reduction in the availability of labile organic carbon (in the case of lipid, highly unsaturated fatty acids) in the deep sea.     

Lobster locomotor activity as a measure of GABAAreceptor modulation

Gamma‐aminobutyric acid (GABA) is a well‐known neurotransmitter. A crustacean behavioural assay was developed for the examination of the effects of a benzodiazepine agonist and an antagonist of GABA_A‐type receptors. Both adult and juvenile male and female lobsters, Homarus americanus, were treated with single (or follow‐up) empirically determined doses of the agonist, diazepam, one of its metabolites (desmethyldiazepam), the antagonist, N‐methyl‐ß‐carboline‐3‐carboxamide (MBC), or vehicle alone. The effects of the compounds were monitored in a submerged circular open‐field for differences in locomotor activity, which was significantly inhibited by diazepam. Treatment with MBC following injection of diazepam reversed the latter's effects on locomotion. These results suggest that benzodiazepines may affect crustacean GABA receptors in a fashion similar to the GABA_A type found in the vertebrates, and that they may be involved in the regulation of locomotor behaviour.     

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Proliferation of myofibroblasts in the stroma of renal oncocytoma

Objectives: Myofibroblasts are a vital component of stroma of many malignant neoplasms, but it is not yet established whether stromal myofibroblasts also exist in benign tumours such as oncocytoma of the kidney. Materials and methods: Histomorphological and immunohistochemical analysis of 16 renal oncocytomas diagnosed at Chang Gung Memorial Hospital, Taiwan, has been performed. Results: Renal oncocytomas were composed of oncocytes, large cells with granular eosinophilic cytoplasm, arranged mostly in sheets, in tubulocystic or combined pattern. Few oncocytes appeared to be undergoing proliferation or apoptosis. MIB‐1 and active caspase 3 indices were low, but higher in tumour than in surrounding non‐tumour parenchyma (MIB‐1: 0.93 ± 0.09 versus 0.46 ± 0.07, P < 0.001 and active caspase 3: 0.76 ± 0.08 versus 0.41 ± 0.09, P < 0.001). Wnt/β‐catenin signalling was not implicated in this neoplasm, as there was no loss of E‐cadherin membranous localization or expression of intranuclear β‐catenin in the cells. Clumps of oncocytes were stained with periodic acid Schiff and had collagen I‐, collagen III‐ and fibronectin‐positive, but desmin‐ and human caldesmon‐negative stromas. Importantly, α‐smooth muscle actin (SMA)‐immunostaining established the myofibroblastic nature of many of the stromal cells. Some of the myofibroblasts were also positive for MIB‐1, indicating a proliferative role for them in the stroma. Conclusions: Renal oncocytomas were composed of two independent compartments: benign oncocytes and pronounced fibrotic stroma, which consisted of proliferating myofibroblasts (SMA‐ and MIB‐1‐positive) which were associated with excessive deposition of extracellular matrix (periodic acid Schiff‐component, collagen I‐, collagen III‐ and fibronectin‐positive, and desmin‐ and human caldesmon‐negative).     

cDNA cloning of a mandibular organ inhibiting hormone from the spider crabLibinia emarginata

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crabLibinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks ofLibinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.     

Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study

Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi‐step methods (methods A–E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X‐100 and/or sodium deoxycholate at 4–37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4‐6‐diamidino‐2‐phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X‐100, and 1% Triton X‐100/0.2% sodium deoxycholate was nuclei‐free and produced a viscous solution that formed a structurally stable white jelly‐like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.     

Metabolism of Hydrocarbons in Microorganisms

The fatty acid composition of the lipid of yellowfin tuna (Thunnus albacares) caught in two different localities, Philippine Sea (a tropical zone) and the Pacific coast area of Japan (a temperate zone) is described. The total lipids of various organs (dorsal ordinary muscle, ventral ordinary muscle, dark muscle, liver, heart, pyloric cecum, and orbital region) and of the stomach contents were extracted, and the fatty acid comosition was analyzed by gas-liquid chromatography (GLC).

Docosahexaenoic acid (DHA; 22:6n-3) was the major unsaturated fatty acid in the lipid of all organs in the specimens examined from both localities, the mean DHA content accounting for more than 25% (mean ± S.D. of 26.9 ±5.7%) of the total fatty acids. This value is markedly different from the fatty acid profile of other fish species, because, in general, the fatty acid composition of other species is variable and the DHA content is less than 20% of total fatty acids.

Although the mean DHA content of the total fatty acids in the lipid of yellowfin tuna caught in the tropical and temperate zones was markedly higher than that in other fish species, there was a small difference between that in the northern samples (temperate waters, 30.5 ±6.1%) and the southern samples (tropical waters, 25.9 ± 5.2%). It is suggested that this difference may be due to environmental effects, e.g., the fatty acid composition of the lipids of prey organisms, because there is also a small difference between the mean DHA content of northern prey fish (22.7 ±6.1%) and that of southern prey fish (19.2 ±4.0%).     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

The hemocytes of an lsopod Ligia exotica Roux

The morphological features of the hemocytes of the crustacean Ligia exotica are similar to hemocytes of insects and millipedes. Jones system of hemocyte classification is extended to crustacean hemocytes. As in insects, seven classes of hemocytes, identified as prohemocytes, plasmatocytes, granular hemocytes, cystocytes, oenocytoids, spherule cells and adipohemocytes, occur. The prohemocytes can be subdivided into five categories that probably represent the precursor of major cell types. The structural and chemical features of other major cell classes are distinct and support the concept of Jones ('62) that these types might have different lineages and might not be capable of transforming into one another. Some of the prohemocytes, plasmatocytes and granular hemocytes are amoeboid. Cystocytes do not bring about any visible plasma coagulation similar to their counterpart in millipedes. Oneocytoids and adipohemocytes are rare. Plasmatocytes, cystocytes and oenocytoids occur in conglomerates, the significance of which is discussed. The cell types are compared with those of the hemocytes of other crustaceans. It is suggested that the nomenclature based on morphological characters is more suited for crustacean hemocytes than a nomenclature based on behavioural and physiological characters.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Evidence for peripheral inhibition in an arachnid muscle

Summary Blectrophysiological investigations on the promotor tibiae muscle in the tarantulaDugesiella hentzi showed for the first time the existence of peripheral inhibition in an arachnid. The observed effects of GABA and picrotoxin are similar to those in crustacean and insect muscle.This investigation was supported by grants No. Ra 113/4 to Prof. W. Rathmayer and SFB 138 of the Deutsche Forschungsgemeinschaft.I thank Professors W. Rathmayer and E. Florey and Mr. C. Kleinsteuber for their support during various stages of this work.     

Characterization of muscle epimysium, perimysium and endomysium collagens

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.     

Collagen VI myopathies: From the animal model to the clinical trial

Collagen VI is an ECM protein which forms a prominent microfibrillar network in the endomysium of skeletal muscle. Mutations in the genes coding for the three chains of collagen VI cause skeletal muscle diseases; the severe wasting Ullrich congenital muscular dystrophy (UCMD) normally present at birth, and the milder Bethlem myopathy (BM). The pathogenesis of both collagen VI myopathies was unknown until 2003. Our group, utilizing Col6a1 deficient mice, discovered a latent mitochondrial dysfunction that caused increased apoptosis in muscle cells. These effects could be reverted by incubating Col6a1 null muscle fibres with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability pore; more interestingly, the treatment of Col6a1 null mice with CoA rescued the muscle phenotype in vivo.These findings demonstrated an unexpected collagen VI/mitochondrial connection as the basis for the UCMD and BM pathogenesis and suggested a strategy for a possible pharmacological treatment of the diseases. This was assessed by demonstrating that muscle biopsies from patients with UCMD showed an abnormal mitochondrial depolarization and that treatment with CsA normalized the mitochondrial phenotype.In this study we report the results of an open pilot trial of four UCMD and one BM patients, representing a range of collagen VI deficiency and having mutations in three of the collagen VI genes. As determined in muscle biopsies prior to treatment, all patients displayed mitochondrial dysfunction and muscle fibres showed an increased frequency of apoptosis. When patients were treated for 1 month with a low daily dose of CsA, primary muscle cell cultures of biopsies obtained at the end of the treatment showed a decreased apoptosis and increased immunohistochemical signs of muscle fibre regeneration. These results confirm that the pathogenic mechanism found in Col6a1 deficient mice also plays a crucial role in hereditary muscle diseases in human, and suggest that targeted treatment of these mitochondrial defects in patients with UCMD and BM may be effective in preventing and/or reverting muscle alterations.It is also important to consider that desensitization of the permeability transition pore by CsA occurs independently of calcineurin inhibition; because a CsA derivative that has no immunosuppressive activity appears to be as effective as the parent molecule, long-term trials should be designed to prevent irreversible muscle damages in young patients affected by collagen VI myopathies, without exposing them to infective risks.     

Isolation of two AKH-related peptides from cicadas

Two peptides related to locust adipokinetic hormone and crustacean red pigment concentrating hormone were isolated by high performance liquid chromatography from the cicadas Cacama valavata and Diceroprocta semicincta. Both species have the same peptides. The structure of one of the peptides is pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-amide. The mass spectrum, amino acid composition, and amino acid sequence of the other peptide suggest that it is almost identical to the first peptide. However, the exact nature of the difference between the two peptides could not be determined. © 1995 Wiley-Liss, Inc.     

Isolation of skeletal muscle collagen

A new method for the isolation of a high yield of collagen from human skeletal muscle is described. This technique employs rapid stirring of the homogenate of skeletal muscle with a magnetic stirrer. Fibrous material entangled during rapid stirring was recovered by passing the homogenate through a sieve and then sequentially extracted with Hasselbach-Schneider solution and 0.6 m KI-0.06 m Na₂S₂O₃. The insoluble residue obtained after these extractions was shown to be highly purified collagen by amino acid analysis, and the recovery of collagen by this method was found to be more than 90% of the total collagen in skeletal muscle. The isolated collagen from 5 years of age was mostly composed of Type I collagen, and small amounts of Type III collagen and an unidentified collagenous protein migrating in a position near Type V collagen (αB chain) were also found on a sodium dodecyl sulfate-polyacrylamide gel.