Proteolytic and physicochemical mechanisms involved in meat texture development.

Development in meat texture is a complex process originating very likely from a softening of the structural elements, especially myofibrils. This process probably involves two sets of mechanisms: 1) an enzymatic mechanism involving at least two of the three proteolytic systems so far identified and present in this tissue, namely lysosomal (cathepsins) and calcium dependent (calpains) proteinases; 2) a physicochemical mechanism based on the important post mortem rise in muscle osmotic pressure which could be twice as high as in live animals. Despite the large progress in muscle enzymology, the nature of the proteinases responsible for the post mortem proteolysis associated with the development in meat texture is still not clearly established. In the present review, data obtained from two different approaches attempting to answer this question were analysed. The first one was based on the identification of a set of structural and biochemical changes associated with meat texture development and to examine which proteolytic system or proteinase would be able to reproduce them when incubated with either myofibrils or muscle fibres as substrate. The second tentatively relates the rate and the extent of the changes in meat texture to the proteolytic equipment of the tissue. The first approach led to the conclusion that changes in muscle proteins and structure can be only explained by considering a synergistic action of both lysosomal and calcium-dependent proteinases. From the second, it was concluded that the process of meat texture development did not depend on the proteinase levels but was related to their initial potential efficiency assessed by measurement of the enzyme/inhibitor ratio. With respect to the physicochemical mechanisms, the post mortem rise in muscle osmotic pressure was shown to be responsible for some biochemical changes occurring in myofibrils. This was further substantiated by the fact that the greatest osmotic pressure values were observed in muscles exhibiting highest tenderising rate. On the other hand we provide evidence suggesting that the substrate, namely myofibrils, might constitute an important limiting step of the efficiency of both types of mechanism. Taken together, the findings presented emphasize that improvement of our knowledge in this field will greatly depend on the development of basic research on these different topics notably: 1) the mechanisms by which proteinases activities are regulated in living and post mortem muscles; and 2) the myofibrillar structure, especially in slow-twitch or type I muscles.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichia coli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.     

Non-enzymatic weakening of myofibrillar structures during conditioning of meat: calcium ions at 0.1 mM and their effect on meat tenderization.

The tenderness of meat is set by the properties of connective tissue and myofibrils. Skeletal muscle connective tissues become firm with chronological aging concomitantly with the increase in intermolecular non-reducing cross-links of collagen, and this process toughens meat, however, connective tissues hardly change during conditioning of meat. Therefore, the tenderization of meat during post mortem aging, or to put it more precisely, during post rigor aging, stems for the most part from changes in myofibril structures. My research derives its origin on findings of two kinds of post mortem changes in myofibril structures; i) fragmentation of myofibrils; and ii) restoration of rigor-shortened sarcomeres. These results were published in 1967 [1], and were, thereafter proved by many workers to be closely related to meat tenderization. I report in this paper the essential molecular mechanisms of these phenomena, and of structural changes in connectin or titin filaments. All of them are non-enzymatically induced by 0.1 mM calcium ion, which is the ultimate concentration of sarcoplasmic calcium ion in post mortem muscles.     

Anaplerotic Triheptanoin Diet Enhances Mitochondrial Substrate Use to Remodel the Metabolome and Improve Lifespan,Motor Function,and Sociability in MeCP2-Null Mice

Rett syndrome (RTT) is an autism spectrum disorder (ASD) caused by mutations in the X-linked MECP2 gene that encodes methyl-CpG binding protein 2 (MeCP2). Symptoms range in severity and include psychomotor disabilities, seizures, ataxia, and intellectual disability. Symptom onset is between 6-18 months of age, a critical period of brain development that is highly energy-dependent. Notably, patients with RTT have evidence of mitochondrial dysfunction, as well as abnormal levels of the adipokines leptin and adiponectin, suggesting overall metabolic imbalance. We hypothesized that one contributor to RTT symptoms is energy deficiency due to defective nutrient substrate utilization by the TCA cycle. This energy deficit would lead to a metabolic imbalance, but would be treatable by providing anaplerotic substrates to the TCA cycle to enhance energy production. We show that dietary therapy with triheptanoin significantly increased longevity and improved motor function and social interaction in male mice hemizygous for Mecp2 knockout. Anaplerotic therapy in Mecp2 knockout mice also improved indicators of impaired substrate utilization, decreased adiposity, increased glucose tolerance and insulin sensitivity, decreased serum leptin and insulin, and improved mitochondrial morphology in skeletal muscle. Untargeted metabolomics of liver and skeletal muscle revealed increases in levels of TCA cycle intermediates with triheptanoin diet, as well as normalizations of glucose and fatty acid biochemical pathways consistent with the improved metabolic phenotype in Mecp2 knockout mice on triheptanoin. These results suggest that an approach using dietary supplementation with anaplerotic substrate is effective in improving symptoms and metabolic health in RTT.     

Different rates of glycolysis affect glycolytic activities and protein properties in turkey breast muscle

Protein alterations of turkey breast muscles (Pectoralis major) were investigated at 20 min and 24 h post mortem. Specific activities, quantities and kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase A were also determined at 20 min post mortem. Based on the pH values at 20 min post mortem, two groups of samples were classified as rapid glycolysis group (RG; pH20 min = 5.80 ± 0.07, n = 20) and normal glycolysis group (NG; pH20 min = 6.21 ± 0.01, n = 20). RG had lower specific activities of GAPDH and aldolase A than NG while Vm and Km values of both enzymes were not different between groups. RG showed lower high ionic strength (HIS) and pellet protein extractabilities at 20 min post mortem. It also had lower low ionic strength (LIS) and HIS protein extractabilities at 24 h post mortem. Besides pellet protein, muscular protein extractabilities at 24 h post mortem were higher than at 20 min post mortem. From SDS-PAGE of samples at 24 h post mortem, RG exhibited lower band intensities at 45 and 200 kDa, which were further identified as actin and myosin heavy chain (MHC), respectively. Western blots revealed that relative amounts of actin and MHC at 20 min post mortem were not different between groups. However, RG muscles had less relative amount of actin at 24 h post mortem. It also indicated that amounts of actin and MHC increased with regard to post mortem time.     

Proteomic analysis of rat soleus and tibialis anterior muscle following immobilization

A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.     

Proteomic analysis of 3T3-L1 adipocyte mitochondria during differentiation and enlargement

The increase in adipose tissue mass arises in part from progressive lipid loading and triglyceride accumulation in adipocytes. Enlarged adipocytes produce the highest levels of pro-inflammatory molecules and reactive oxygen species (ROS). Since mitochondria are the site for major metabolic processes (e.g., TCA cycle) that govern the extent of triglyceride accumulation as well as the primary site of ROS generation, we quantitatively investigated changes in the adipocyte mitochondrial proteome during different stages of differentiation and enlargement. Mitochondrial proteins from 3T3-L1 adipocytes at different stages of lipid accumulation (days 0-18) were digested and labeled using the iTRAQ 8-plex kit. The labeled peptides were fractionated using a liquid phase isoelectric fractionation system (MSWIFT) to increase the depth of proteome coverage and analyzed using LC-MS/MS. A total of 631 proteins in the mitochondrial fraction, including endoplasmic reticulum-associated and golgi-related mitochondrial proteins, were identified and classified into 12 functional categories. A total of 123 proteins demonstrated a statistically significant change in expression in at least one of the time points over the course of the experiment. The identified proteins included enzymes and transporters involved in the TCA cycle, fatty acid oxidation, and ATP synthesis. Our results indicate that cultured adipocytes enter a state of metabolic-overdrive where increased flux through the TCA cycle and increased fatty acid oxidation occur simultaneously. The proteomic data also suggest that accumulation of reduced electron carriers and the resultant oxidative stress may be attractive targets for modulating adipocyte function in metabolic disorders.     

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.     

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.     

Tissue-specific changes in protein synthesis associated with seasonal metabolic depression and recovery in the north temperate labrid, Tautogolabrus adspersus

The tissue-specific changes in protein synthesis were tracked in relation to the seasonal metabolic depression in cunner (Tautogolabrus adsperus). In vivo protein synthesis rate and total RNA content were determined in liver, white muscle, brain, heart, and gill during periods of normal activity before metabolic depression, entrance into and during winter dormancy, and during the recovery period. The decrease in water temperature from 8 degrees C to 4 degrees C was accompanied by a 55% depression of protein synthesis in liver, brain, and heart and a 66% depression in gill. Protein synthesis in white muscle fell below detectable levels at this temperature. The depression of protein synthesis is an active process (Q(10) = 6-21 between 8 degrees C and 4 degrees C) that occurs in advance of the behavioral and physiological depression at the whole animal level. Protein synthesis was maintained at these depressed levels in white muscle, brain, heart, and gill until water temperature returned to 4 degrees C in the spring. Liver underwent a hyperactivation in the synthesis of proteins at 0 degrees C, which may be linked to antifreeze production. During the recovery period, a hyperactivation of protein synthesis occurred in white muscle, which is suggestive of compensatory growth, as well as in heart and liver, which is considered to be linked to increased activity and feeding. Seasonal changes in total RNA content demonstrate the depression of protein synthesis with decreasing temperature to be closely associated with translational capacity, but the stimulation of protein synthesis during recovery appears to be associated with increased translational efficiency.     

Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.     

Chianina beef tenderness investigated through integrated Omics

In the present study we performed an integrated proteomics, interactomics and metabolomics analysis of Longissimus dorsi tender and tough meat samples from Chianina beef cattle. Results were statistically handled as to obtain Pearson's correlation coefficients of the results from Omics investigation in relation to canonical tenderness-related parameters, including Warner Bratzler shear force, myofibrillar degradation (at 48 h and 10 days after slaughter), sarcomere length and total collagen content. As a result, we could observe that the tender meat group was characterized by higher levels of glycolytic enzymes, which were over-phosphorylated and produced accumulation of glycolytic intermediates. Oxidative stress promoted meat tenderness and elicited heat shock protein responses, which in turn triggered apoptosis-like cascades along with PARP fragmentation. Phosphorylation was found to be a key process in post mortem muscle conversion to meat, as it was shown not only to modulate glycolytic enzyme activities, but also mediate the stability of structural proteins at the Z-disk. On the other hand, phosphorylation of HSPs has been supposed to alter their functions through changing their affinity for target interactors. Analogies and breed-specific differences are highlighted throughout the text via a direct comparison of the present results against the ones obtained in a parallel study on Maremmana Longissimus dorsi. It emerges that, while the main cornerstones and the final outcome are maintained, post mortem metabolism in tender and tough meat yielding individuals is subtly modulated via specific higher levels of enzymes and amino acidic residue phosphorylation in a breed-specific fashion, and whether calcium homeostasis dysregulation was a key factor in Maremmana, higher early post mortem phosphocreatine levels in the Chianina tender group could favor a slower and prolonged glycolytic rate, prolonging the extent of the minimum hanging period necessary to obtain tender meat from this breed by a few days.     

Characteristics of sarcoplasmic reticulum from slowly glycolysing and from rapidly glycolysing pig skeletal muscle post mortem

The composition and function of fragmented sarcoplasmic reticulum from pig skeletal muscle was examined in the period immediately post mortem. Muscle was defined as being either slowly glycolysing or rapidly glycolysing on the basis of colour, pH and concentrations of glycogen and lactate. The microsomal fraction was separated on a discontinuous gradient of 35, 40 and 45% (w/v) sucrose into heavy and intermediate fractions which sedimented to the interfaces, and a light fraction which remained on the surface of the 35%-sucrose layer. The sarcoplasmic reticulum from rapidly glycolysing muscle had a lower buoyant density than had that from slowly glycolysing muscle. This was reflected in the consistent lack of material in the heavy fraction and a greater proportion in the light fraction. The latter material had significantly lower ratios (w/w) of protein to phospholipid (2.3:1 versus 3.8:1) and of protein to cholesterol (10.4:1 versus 15.6:1). There were no gross differences in phospholipid content or in fatty acid composition of individual phospholipid classes in the membranes from the two types of muscle. Analysis of membrane proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that ATPase (adenosine triphosphatase) was a major component of each fraction and that its contribution to the total protein content of the membrane was greater in rapidly glycolysing muscle, suggesting a loss of non-ATPase proteins. The two fractions of sarcoplasmic reticulum prepared from rapidly glycolysing muscle had approximately one-third the normal activities of Ca(2+) binding and Ca(2+) uptake in the presence of ATP and one-half the passive Ca(2+)-binding capacity in the absence of ATP of the fractions from slowly glycolysing muscle. However, the (Ca(2+)+Mg(2+))-stimulated ATPase activities were similar. Efflux from actively loaded vesicles, after the addition of EDTA, consisted of a rapid and a slow phase. Vesicles from rapidly glycolysing muscle lost 60% of associated Ca(2+) (approx. 0.10mumol of Ca(2+)/mg of protein) during the rapid phase, compared with 30% (approx. 0.17mumol of Ca(2+)/mg of protein) in those from slowly glycolysing muscle. The efflux rate during the slower phase was comparable in both types of vesicles. Analysis of the temperature-dependence of (Ca(2+)+Mg(2+))-stimulated ATPase activity revealed that a high-activation-energy process operating in the temperature range 31-45 degrees C in the intermediate and light fractions from slowly glycolysing muscle was not apparent in vesicles from rapidly glycolysing muscle. Conditions that result in the prolonged activation of glycogenolysis in pig muscle post mortem primarily affect the protein components of the sarcoplasmic-reticular membrane, giving rise to a loss of loosely associated proteins. The function of the membranes observed under these conditions does not appear to be due to enhanced permeability of the membrane to Ca(2+) and may be the result of a defect in the transport of Ca(2+) into the vesicles.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.     

Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteome

Proteome analysis in the central nervous system area represents a large and important challenge in drug discovery. One major problem is to obtain representative and well characterized tissues of high quality for analysis. We have used brain tissues from normal mice to study the effect of post mortem time (up to 32 h) and temperature (4 degrees C and room temperature) on protein expression patterns. A number of proteins were identified using mass spectrometry and potential markers were localized. One of the proteins identified, dihydropyrimidinase related protein-2 (DRP-2), occurs as multiple spots in two-dimensional electrophoresis gels. The ratio between the truncated form of DRP-2 (fDRP-2) and full length DRP-2 is suggested as an internal control that can be used as a biomarker of post mortem time and post mortem temperature between unrelated brain protein samples. Results of this study may be useful in future efforts to detect disease specific alterations in proteomic studies of human post mortem brain tissues.     

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS)

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.     

Post Mortem DNA Degradation of Human Tissue Experimentally Mummified in Salt

Mummified human tissues are of great interest in forensics and biomolecular archaeology. The aim of this study was to analyse post mortem DNA alterations in soft tissues in order to improve our knowledge of the patterns of DNA degradation that occur during salt mummification. In this study, the lower limb of a female human donor was amputated within 24 h post mortem and mummified using a process designed to simulate the salt dehydration phase of natural or artificial mummification. Skin and skeletal muscle were sampled at multiple time points over a period of 322 days and subjected to genetic analysis. Patterns of genomic fragmentation, miscoding lesions, and overall DNA degradation in both nuclear and mitochondrial DNA was assessed by different methods: gel electrophoresis, multiplex comparative autosomal STR length amplification, cloning and sequence analysis, and PCR amplification of different fragment sizes using a damage sensitive recombinant polymerase. The study outcome reveals a very good level of DNA preservation in salt mummified tissues over the course of the experiment, with an overall slower rate of DNA fragmentation in skin compared to muscle.