Effect of temporary freezing on postmortem protein degradation patterns.

BACKGROUND: A precise determination of time since death plays a major role in forensic routine. Currently available techniques for estimating the postmortem interval (PMI) are restricted to specific time periods or cannot be applied for individual case-specific reasons. During recent years, it has been repeatedly demonstrated that Western blot analysis of postmortem muscle protein degradation can substantially contribute to overcome these limitations in cases with different background. Enabling to delimit time points at which certain marker proteins undergo distinct degradation events, the method has become a reasonable new tool for PMI delimitation under various forensic scenarios. However, additional research is yet required to improve our understanding of protein decomposition and how it is affected by intrinsic and extrinsic factors. Since there are temperature limits for proteolysis, and investigators are confronted with frozen corpses, investigation of the effects of freezing and thawing on postmortem protein decomposition in the muscle tissue is an important objective to firmly establish the new method. It is also important because freezing is often the only practical means to intermittently preserve tissue samples from both true cases and animal model research. METHODS: Sets of dismembered pig hind limbs, either freshly detached non-frozen, or thawed after 4 months of freeze-storage (n = 6 each), were left to decompose under controlled conditions at 30 °C for 7 days and 10 days, respectively. Samples of the M. biceps femoris were regularly collected at predefined time points. All samples were processed via SDS-PAGE and Western blotting to identify the degradation patterns of previously characterized muscle proteins. RESULTS: Western blots show that the proteins degrade predictably over time in precise patterns that are largely unaffected by the freeze-and-thaw process. Investigated proteins showed complete degradation of the native protein band, partly giving rise to degradation products present in distinct time phases of the decomposition process. CONCLUSION: This study provides substantial new information from a porcine model to assess the degree of bias that freezing and thawing induces on postmortem degradation of skeletal muscle proteins. Results support that a freeze-thaw cycle with prolonged storage in frozen state has no significant impact on the decomposition behavior. This will help to equip the protein degradation-based method for PMI determination with a robust applicability in the normal forensic setting.

Morphological changes and protein degradation during the decomposition process of pig cadavers placed outdoors or in tents-a pilot study.

The delimitation of the postmortem interval (PMI) is of utmost importance in forensic science. It is especially difficult to determine the PMI in advanced decomposition stages and/or when dead bodies are found under uncommon circumstances, such as tents, or other (semi-) enclosed environments. In such cases, especially when insect access is restricted, morphological assessment of body decomposition is one of the remaining approaches for delimitation of the PMI. However, as this method allows only vague statements/indications about the PMI, it is required to develop new and more reliable methods. One of the most important candidates is the biochemical analysis of protein degradation. In this regard, it has been demonstrated that specific skeletal muscle protein degradation patterns characterize certain time points postmortem and thus can be used as markers for PMI estimation. In order to test this method in different micro-environments, a pilot study using ten pig carcasses was conducted in summer in Northern Germany. The cadavers were openly placed outside (freely accessible for insects), as well as enclosed in tents nearby, and left to decompose to investigate decomposition processes over a time course of 10 days. Muscle samples of the M. biceps femoris were collected on a regular basis and processed via SDS-PAGE and degradation patterns of selected proteins identified by Western blotting. In addition, morphological changes of the cadavers during decomposition were assessed using the total body score (TBS). Results showed that postmortem protein degradation patterns are largely consistent between treatment groups (open field versus tents) despite major morphological differences in the decomposition rate. This field study provides evidence that muscle protein degradation is mostly unaffected by different levels of exposure, making it a sufficient candidate for PMI delimitation under various circumstances.

The dual role of zonula occludens (ZO) proteins.

ZO (zonula occludens) proteins are scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of intercellular junctions. In addition, they provide a link between the integral membrane proteins and the filamentous cytoskeleton. ZO proteins belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. Besides their structural function at cell-cell contacts, ZO proteins appear to participate in the regulation of cell growth and proliferation. Detailed molecular studies have shown that ZO proteins exhibit conserved functional nuclear localization and nuclear export motifs within their amino acid sequence. Further, ZO proteins interact with dual residency proteins localizing to the plasma membrane and the nucleus. Although the nuclear targeting of ZO proteins has well been described, many questions concerning the biological significance of this process have remained open. This review focuses on the dual role of ZO proteins, being indispensable structural components at the junctional site and functioning in signal transduction pathways related to gene expression and cell behavior.

Evolution of myogenesis in fish: a sturgeon view of the mechanisms of muscle development.

Patterns of initial muscle formation are well documented for teleost fish. Here, attention is focused upon sturgeons, which arose close to the base of the actinopterygian radiation and whose early development has remained largely unresearched. We demonstrate that some features of muscle development are common to both groups of fish, the most important being the origin and form of migration of adaxial cells to establish the superficial slow fibre layer. This, together with information on initial innervation and capillarisation, strongly suggests a common basis for muscle developmental mechanisms among fish. An important feature that is different between sturgeons and teleosts is that sturgeons lack any cellular dorsal-ventral separation of the myotome that involves the insertion of muscle pioneer (MP)-like cells at the site of the future horizontal septum. This, and information from other fish and from sarcopterygians, permits the supposition that such MP-defined dorsal-ventral separation is a teleost apomorphism. These and other findings are discussed in relation to their significance for the evolution of fish muscle developmental patterns.

Tonic fibres in axial muscle of cyprinid fish larvae: their definition, possible origins and functional importance.

Teleost fish are known to develop small populations of muscle fibres that are assumed to be tonic in nature although their contractile properties and many other characteristics remain unknown. Here we attempt to resolve some of the ambiguity and confusions surrounding the definition and functional role of tonic fibres in teleosts and provide new information on their ontogeny. We investigate the differentiation of tonic muscle fibres in three species of cyprinid fish using electron microscopy, histochemistry, immunohistochemistry and in situ hybridization. The fine structure of the fibres defined as tonic in the larvae used in this study complies with patterns known from studies in teleost adults. This allows formal definition of tonic fibres in cyprinid larvae. The tonic fibres may be recognized by a variety of features: (1) by their characteristic position along the medial confines of the red muscle insertion at the horizontal septum, (2) their fine structure, including solid clusters of irregularly cleaved myofibrils, thick and wavy Z-lines, and T-tubules at the A-band/I-band transitions, (3) their histochemical features, specifically weak but obvious staining for mATPase after alkaline preincubation, and lack of SDH activity in the more advanced larval stages, (4) their unique immunological properties, being the only fibre type in the myotome that reacts with a serum against chicken tonic myosin (anti- T2). Expression of tonic characters usually begins within a few fibres in the dorsal domain of the superficial red muscle insertion at the horizontal septum and hence involves a high degree of dorso-ventral polarity. The present evidence indicates that tonic fibres arise from separate myogenic stem cells rather than by transdifferentiation from existing red fibres. First appearance of tonic fibres during ontogeny correlates closely with the onset of free swimming and exogeneous feeding. We use this fact to argue that tonic fibres are probably a prerequisite for efficient locomotory control during prey capture, shoaling and predator avoidance.

Effects of rotator cuff ruptures on the cellular and intracellular composition of the human supraspinatus muscle.

Ruptures of the rotator cuff tendons of the human shoulder are a common incidence and lead to functional impairment of the four muscles connected to the cuff, entailing profound changes of their cellular tissue composition. Most importantly, such tendon tears lead to atrophy, fatty degeneration and fibrosis of the corresponding muscles. The muscle most commonly affected with such changes is the M. supraspinatus. The present study uses biopsy samples from the supraspinatus muscle of 12 elderly patients and 6 controls to examine the rupture-induced muscle change at both the cellular and the intracellular (ultrastructural) levels. Amounts of fatty tissue, connective tissue and muscle were assessed by light microscopy-based morphometry and stereology. Stereology of electron micrographs was employed to determine volume densities of muscle fibre mitochondria, myofibrils and intracellular lipid. Results demonstrate that the supraspinatus muscles of patients with a massive rupture contain significantly higher amounts not only of fatty tissue but also of intracellular lipid than those of control subjects. These patients further exhibit a major decrease in relative amounts of myofibrils, thus confirming that change of intracellular composition is a major component of the observed muscle degeneration. The results contribute to establish the true spectrum of supraspinatus muscle damage in humans induced by tendon rupture.

Lateral fast muscle fibers originate from the posterior lip of the teleost dermomyotome.

The predominant source of myogenic cells in vertebrates is the dermomyotome (DM). In teleost fish, recent research has provided a useful but limited picture of how myogenic precursors originate from the DM and how they develop into muscle fibers. Here, we combine detailed morphological analysis with examination of molecular markers in trout to describe the cellular mechanisms by which the lateral fast muscle growth zone is created during second phase myogenesis. Results suggest that this occurs by lateral-to-medial immigration of myogenic cells de-epithelializing from the posterior DM lip. These cells then appear to stop proliferation and migrate anteriorly to finally differentiate into muscle fibres. This seems to be a continuation of the rotational cell movement that creates the teleost DM during early somite development. These findings suggest an evolutionary conserved role of the posterior DM lip in amniotes and fish.

MyoD and Myogenin expression during myogenic phases in brown trout: a precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth.

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow-growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast-growing salmonid with exceptionally large embryos. Results confirm the three-phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second- and third-phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies.

Phases of myogenic cell activation and possible role of dermomyotome cells in teleost muscle formation.

Present knowledge indicates that fibre recruitment (hyperplasia) in developing teleost fish occurs in three distinct phases. However, the origin and relationship of the myogenic precursors activated during the different phases remains unclear. Here, we address this issue using molecular techniques on embryos and larvae of pearlfish, a large cyprinid species. Results provide comprehensive molecular characterisation of cell recruitment over the three phases of myogenesis, identifying muscle types as they arise. Specifically, we show that the myogenic cells arising during 2nd phase myogenesis are clearly different from the myogenic cells arising during the 3rd phase and that the dermomyotome is a major source of myogenic cells driving 2nd phase hyperplasia. These findings are discussed in relation to their implications for the generality of vertebrate developmental patterns.

Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish.

The somitic compartment that gives rise to trunk muscle and dermis in amniotes is an epithelial sheet on the external surface of the somite, and is known as the dermomyotome. However, despite its central role in the development of the trunk and limbs, the evolutionary history of the dermomyotome and its role in nonamniotes is poorly understood. We have tested whether a tissue with the morphological and molecular characteristics of a dermomyotome exists in nonamniotes. We show that representatives of the agnathans and of all major clades of gnathostomes each have a layer of cells on the surface of the somite, external to the embryonic myotome. These external cells do not show any signs of terminal myogenic or dermogenic differentiation. Moreover, in the embryos of bony fishes as diverse as sturgeons (Chondrostei) and zebrafish (Teleostei) this layer of cells expresses the pax3 and pax7 genes that mark myogenic precursors. Some of the pax7-expressing cells also express the differentiation-promoting myogenic regulatory factor Myogenin and appear to enter into the myotome. We therefore suggest that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates. The identification of a dermomyotome in fish makes it possible to apply the powerful cellular and genetic approaches available in zebrafish to the understanding of this key developmental structure.