Postmortem Process Research Articles

A new SNP in the 3′UTR region of the bovine calpain small subunit (CAPNS1) gene

Calpains are a ubiquitous cytoplasmic cysteine protease, the activity of which is absolutely dependent on calcium. This proteolytic system degrades myofibrillar protein under post-mortem conditions and appears to be the primary enzyme in the postmortem tenderization process. In the present study a new single nucleotide polymorphism was found in the bovine CAPNS1 gene exon 11 coding for the 3'UTR. Transition C --> T at position 6536 was detected and identified using PCR-SSCP and DNA sequencing techniques, and then analysed with PCR-RFLP using MboII nuclease. The genotype frequencies and alleles distribution were studied in 190 bulls including, Charolaise, Hereford, Limousine, Simmental, Polish Red and Fresian breeds.

New Single Nucleotide Polymorphisms in the µ-Calpain Gene in Spanish Maternal Beef Breeds

Calpains play an important role in the postmortem tenderization process of meat and several SNP in the μ-calpain gene (CAPN1) have been reported to be associated with tenderness in beef cattle. Our objectives were to identify the previously reported CAPN1 331G>C SNP and to detect new polymorphisms in this gene in Spanish maternal beef breeds. A fragment (exon 8 to 10) of the bovine CAPN1 gene was sequenced and genotyped in a sample of the main Spanish maternal beef breeds including Retinta, Morucha, and Avileña Negra-Ibérica. These breeds are characterized for their high meat quality, their adaptation to adverse environmental conditions, and their good maternal aptitude. This adaptation makes it possible to rear these breeds in the south and west of Spain, where drought and feed shortages occur frequently. Six SNP in the μ-calpain gene were found, five of which (CAPN1 80C>T, 302C>G, 310G>A, 445C>T, 524A>C) have not been reported previously. Sequences obtained for these five newly found SNP were submitted to GenBank (Accessions EU386166 to EU386183).

Sarcomere length influences postmortem proteolysis of excised bovine semitendinosus muscle

The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.

EFFECTS OF LOW VOLTAGE ELECTRICAL STIMULATION AND CHILLING METHODS ON QUALITY TRAITS OF PORK M. LONGISSIMUS LUMBORUM

ABSTRACT The effects of low voltage electrical stimulation and chilling methods on the quality aspects of pork carcasses were evaluated in this study. A total of 16 pigs (Long white × Huainan) were randomly allocated immediately prior to slaughter to two groups: low voltage electrically stimulated (LVES or ES) or nonstimulated (NS). Carcasses were split before cooling. Each side of the carcass was then randomly allocated to either conventional chilling or blast chilling. Results showed that LVES increased the rate of muscle pH decline in the early time postmortem and accelerated the onset of rigor. But LVES with conventional chilling had disadvantageous effects on water‐holding capacity (WHC), Warner‐Bratalor shear forces and lightness of meat color. However, LVES combined with blast chilling can avoid this disadvantage. And blast chilling achieved low internal muscle temperature faster than with conventional chilling and delayed the rate of muscle pH decline. Furthermore, blast chilling increased WHC of pork without risk of cold toughing. The disadvantageous effect of LVES on pork quality can be equalized by the advantageous effect of blast chilling. Therefore, it is desirable for commercial pig abattoirs to apply blast chilling, or low voltage electrical stimulation combined with blast chilling for rapid processing without compromising meat quality.PRACTICAL APPLICATIONSThere are many pork quality characteristics (i.e., appearance, texture and flavors as well as tenderness, juiciness and other subjective characteristics) that attract consumer attention. Among these, the water‐holding capacity of the pork, color and tenderness are considered the most important quality characteristics. Postmortem treatments involved with the postmortem biochemical process influence pork qualities. Therefore, investigations into the effects of electrical stimulation of pig carcasses, chilling methods, suspension and their combinations on pork qualities should be prerequisites for improving pork qualities.

Pink Teeth in a Series of Bodies Recovered From a Single Shipwreck

Pink teeth have most often been observed in victims of drowning but have also been reported in subjects who died suddenly and unnaturally. There is general agreement that there is no obvious connection between the occurrence of pink teeth and the cause of death, but the condition of the surroundings (especially humidity) must certainly play an important role in the development of the pink-tooth phenomenon. The frequency and distribution of postmortem pink coloration of the teeth have been studied among a representative sample of 52 cadavers. All the bodies were victims of a single shipwreck that occurred on March 13, 1997, in the middle of the Otranto Canal (Mediterranean Sea). The bodies were recovered from the seawater after approximately 7 months. A distinct pink coloration of the teeth was found in only 18 cadavers (13 females and 5 males) of ages ranging between 13 and 60 years. The phenomenon was more pronounced in younger individuals due to age-related changes of the root canal, less penetrable by the pigment responsible for the postmortem pink staining. By histochemical methods and autofluorescence, hemoglobin and its derivatives have been identified as the most likely pigments responsible for this postmortem process that can be considered analogous to postmortem lividity. These data are consistent with previous reports on pink teeth, indicating that the diffusion of the blood in the pulp into the dentinal tubules causes the red discoloration of the teeth. Based on the results, the pigmentation is more prominent on the teeth with single roots rather than in the posterior teeth with multiple roots.

Expression profile of Bag 1 in the postmortem brain

Bag 1 is a protein intimately involved in signaling pathways that regulate cell survival. Here we examined the expression profile of Bag 1 in the brain to consider issues associated with the sampling of anti-apoptotic proteins in a rat model of the human postmortem process. Following a 4 h postmortem interval, we analyzed the hippocampus of rats maintained at 24 or 4 °C storage temperatures using immunocytochemical and Western blotting techniques. Remarkably, postmortem tissue (up to 4 h) showed a significant and prominent up-regulation of Bag 1 in CA1 and CA3 subfields of the hippocampal formation. Over-expression of Bag 1, however, could only be traced down to a storage temperature of 24 °C. These data suggest that storage temperatures, but not postmortem intervals, significantly affect the expression profile and cellular stability of Bag 1 proteins.

Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.

Molecular identification of Stearibia nigriceps: An example of the usefulness of Cytochrome b gene for the identification of entomofauna species

In Forensics, the study of entomological evidence is focused on the accurate calculation of the variables “manner and place of death” and “postmortem interval (PMI)”. Carrion-feeding fly species morphological differentiation is often a difficult task. DNA-related methodologies, those based on mitochondrial DNA (mtDNA) in particular, are a powerful alternative to achieve species identification. For this reason, we have used the Cytochrome b (Cyt-b) gene to identify the necrophagous fly species Stearibia nigriceps (Meigen, 1826) (Diptera; Piophilidae) involved in the postmortem process.

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.

GLYCOGEN, GLUCOSE AND GLUCOSE‐6‐PHOSPHATE CONTENT IN FRESH AND COOKED MEAT AND MEAT EXUDATE FROM CARRIERS AND NONCARRIERS OF THE RN– ALLELE

ABSTRACT The objective of this study was to investigate the content of glycogen, glucose and glucose‐6‐phosphate (G‐6‐P) in fresh and cooked meat and meat exudate (purge and cooking loss) from carriers (RN–/rn+) and noncarriers (rn+/rn+) of the RN– allele. Loins (M. longissimus dorsi) from 13 commercial crossbred female pigs were used. The total content of glycogen, glucose and G‐6‐P was significantly higher in RN–carriers compared with noncarriers both in fresh and cooked muscle, as well as in fresh and cooked meat exudate. Also the sum of glucose and G‐6‐P was higher in all fractions for the RN– carriers compared with noncarriers. Loins from the RN– carriers had lower ultimate pH and higher drip, purge and cooking loss compared with loins from noncarriers. We conclude that after the postmortem process in RN– carriers there is muscle glycogen left which is subsequently released with meat exudate during storage and cooking, lowering the glycogen level in the cooked meat.

SSU rDNA analysis of Kudoa rosenbuschi (Myxosporea) from the Argentinean hake Merluccius hubbsi

The cloning and sequencing of the small subunit (SSU) ribosomal DNA gene from Kudoa rosenbuschi (myxosporean species associated with post-mortem myoliquefaction process in the Argentinean hake Merluccius hubbsi) is reported. The SSU rDNA was found to contain 1740 bp with a single polymorphic site with either a C or T at position 221. The sequence data obtained in this study and those known sequences of Kudoa species deposited in the GenBank were all analyzed to construct a phylogenetic tree. Nucleotide sequences showed the highest degree of identity with K. funduli, followed by K. miniauriculata, K. clupeidae and K. dianae. Phylogenetic analysis placed K. rosenbuschi in the same branch of K. clupeidae and K. funduli, and showed it to be closely related to K. dianae, K. paniformis and K. miniauriculata.

Effect of supplemental vitamin D3 concentration on concentrations of calcium, phosphorus, and magnesium relative to protein in subcellular components of the longissimus and the distribution of calcium within longissimus muscle of beef steers.

The effect of supplementing diets with various levels of vitamin D3 to provide 0, 0.5, 1, and 5 million IU/(steer x d) for 8 d before slaughter on the mineral content and localization of Ca in LM and muscle fragments was studied during the postmortem aging process. Twelve feedlot steers of three biological types were given access to the four levels of vitamin D for 8 d before slaughter. Differential centrifugation techniques were used to determine the concentrations of minerals relative to protein in different muscle fragments on d 3 and 21 postmortem. Electron microscopy visualization of bound Ca indicated that vitamin D3 mobilized Ca from the sarcoplasmic reticulum and transverse tubule system into the myofibrils. Bound Ca was concentrated near the Z-line at the A-band/I-band juncture within the sarcomere. Supplementing steers with 1 and 5 million IU/(steer x d) of vitamin D3 increased (P < 0.05) Ca, P, and Mg concentrations per unit of protein in the cytosol. Soluble cytosolic Ca concentrations were greater (P < 0.05) on d 21 than on d 3 postmortem only when steers were supplemented with 5 million IU/d. Concentrations of Ca, P, and Mg in isolated tissues were increased (P < 0.05) in nuclei and myofibrilar proteins by supplementing steers with 1 and 5 million IU/ (steer x d) of vitamin D3. All supplemental vitamin D3 treatments also increased (P < 0.001) Mg concentrations in the cytosol, regardless of aging treatment, and increased Mg concentrations (P < 0.04) within the mitochondria at d 3 postmortem. Thus, supplementation of feedlot steers with vitamin D3 at levels of 0.5 to 5 million IU/(steer x d) increased Ca concentrations within respiring muscle, resulting in increased bound tissue Ca concentrations. When the respiring muscle was converted to meat, the increased bound tissue Ca resulting from vitamin D3 treatment released Ca concentrations into the cytosol during aging (P < 0.05). Results of this study indicate that vitamin D3 supplementation increased total cytosolic Ca, P, and Mg concentrations in meat.

Molecular identification of arthropods by cytochrome b analysis

This work has been developed to improve the analysis of cadaveric entomofauna. Taking into account that the cytochrome b is widely use to identify vertebrates, we have used it for the identification of some necrophagous species involved in the postmortem process.

Analysis of colored teeth from Precolumbian Tlatelolco: postmortem transformation or intravitam processes?

The etiological basis of the abnormal coloration of archaeological teeth has been an unsolved question for a long time. Differences in the appearance of some archaeological teeth from Precolumbian adult and infant skeletons, detected by external optical inspection, led us to study this problem. A blue stain is visible in a few of the deciduous erupted teeth, and a brown color in various unerupted teeth in the collection, while brown spots appear on some permanent teeth. Several processes or factors that may occur during one's life, others around the time of death, and still others resulting from postmortem alterations have been reported as potential causes of abnormal tooth coloration.A sample of 35 colored teeth and two soil layers taken from Tlatelolco were analyzed by particle-induced X-ray emission (PIXE) as well as selective dissolution techniques. Concentrations of total and extractable elements in enamel and soil layers (Cg1-Cg2) were obtained. This paper describes the occurrence and implications of a substantial secondary concentration of Zinc (Zn), manganese (Mn), strontium (Sr), and iron (Fe) in the deciduous erupted and nonerupted teeth as compared to that in the adult teeth. Our interpretation is that, in this archaeological context, the brown spots and blue stains on the teeth are due to differences in tooth enamel porosity and to a postmortem biogeochemical process. The alterations involve cumulization and diagenesis of iron, manganese, and organic matter solutions that were eluviated from the soil and are not the result of antemortem or perimortem conditions such as trauma or disease.

POST-MORTEM ORGAN DONATION AND GRIEF: A STUDY OF CONSENT, REFUSAL AND WELL-BEING IN BEREAVEMENT

Concern about the grief processes of organ donors' families are reported by medical staff as a reason not to ask for organ donation. Objectives of the current study were to examine the relation between consenting to a post-mortem organ donation procedure and subsequent process of grief in the bereaved. A cross-sectional survey was conducted in a representative time-sample of 95 bereaved who lost a first-degree family member on intensive care wards in 27 Dutch hospitals. In 36 cases an organ donation procedure took place, in 23 cases consent was refused, and in 36 cases no request for organ donation was made to the bereaved.The authors found there were no differences in levels of depression and problems with detachment from the deceased between bereaved (first-degree family members) who participated in an organ donation procedure, those who refused consent, and families who were not approached for post-mortem organ donation. No differences were found in levels of main outcome measures between three donation conditions. However, dissatisfaction with hospital care was associated with depressive and grief symptoms.The results indicate that consenting to organ donation in itself neither hinders nor furthers the grief process.

Significance of the Recent Extraction to the Postmortem Dental Identification: A Case Study

This case report illustrates the significance of the partially healed extraction site to the postmortem dental identification process. It presents the remains of two individuals with documented extractions--one, 6 1/2 months prior to death, the second, only three weeks prior to death. These photographs, compiled with others, can be used as a guideline for the forensic scientist who seldom has the opportunity to study skeletonized remains.

Staphylococcal cell wall: morphogenesis and fatal variations in the presence of penicillin.

The primary goal of this review is to provide a compilation of the complex architectural features of staphylococcal cell walls and of some of their unusual morphogenetic traits including the utilization of murosomes and two different mechanisms of cell separation. Knowledge of these electron microscopic findings may serve as a prerequisite for a better understanding of the sophisticated events which lead to penicillin-induced death. For more than 50 years there have been controversial disputes about the mechanisms by which penicillin kills bacteria. Many hypotheses have tried to explain this fatal event biochemically and mainly via bacteriolysis. However, indications that penicillin-induced death of staphylococci results from overall biochemical defects or from a fatal attack of bacterial cell walls by bacteriolytic murein hydrolases were not been found. Rather, penicillin, claimed to trigger the activity of murein hydrolases, impaired autolytic wall enzymes of staphylococci. Electron microscopic investigations have meanwhile shown that penicillin-mediated induction of seemingly minute cross wall mistakes is the very reason for this killing. Such "morphogenetic death" taking place at predictable cross wall sites and at a predictable time is based on the initiation of normal cell separations in those staphylococci in which the completion of cross walls had been prevented by local penicillin-mediated impairment of the distribution of newly synthesized peptidoglycan; this death occurs because the high internal pressure of the protoplast abruptly kills such cells via ejection of some cytoplasm during attempted cell separation. An analogous fatal onset of cell partition is considered to take place without involvement of a detectable quantity of autolytic wall enzymes ("mechanical cell separation"). The most prominent feature of penicillin, the disintegration of bacterial cells via bacteriolysis, is shown to represent only a postmortem process resulting from shrinkage of dead cells and perturbation of the cytoplasmic membrane. Several schematic drawings have been included in this review to facilitate an understanding of the complex morphogenetic events.

Tenderness of normal and callipyge lamb aged fresh or after freezing ∗

The present experiments were conducted to determine if tenderness differed between normal (N) and callipyge (CLPG) lamb during post-mortem aging of: (1) longissimus muscle (LM), (2) semimembranosus muscle (SM), and (3) LM after freezing for 6 weeks. In the LM, CLPG chops were tougher ( p < 0.01) than N at every post-mortem age and after 24 h CaCl 2 marination of a day 1 chop. Post-mortem aging reduced ( p < 0.05) shear force of the LM in both N and CLPG; however, this reduction proceeded at a slower rate (−0.127 vs −0.341 kg d −1; p = 0.01) and for a longer time period (24 vs 6 days; p < 0.05) in CLPG than N. In the SM muscle, shear force values did not differ ( p > 0.05). due to phenotype, post-mortem age, or the two-way interaction. Freezing prior to aging accelerated the rate of post-mortem tenderization in CLPG (−0.130 vs −0.060 kg d −1; p = 0.004) while no changes ( p > 0.05) were observed for N. Phenotypic expression of the CLPG gene increased the toughness of the LM with no change in SM; however, freezing prior to aging accelerated the post-mortem tenderization process in CLPG LM.

The effect of post-mortem aging on meat flavor quality in Brangus beef. Correlation of treatments, sensory, instrumental and chemical descriptors

The flavor of muscle foods is dependent upon factors such as the animal's age, breed, sex, nutritional status and manner of cooking. Most important to the final flavor of the meat is relative age in the post-mortem aging process as it is during this time that many chemical flavor components are formed (Spanier et al., 1990). These components serve either directly as flavor components or as a pool of reactive flavors and intermediates that form many of the characteristic meat flavors after cooking. The post-mortem aging process is identified with enhancement of beef sensory quality due to enhanced tenderization. While this is true for beef texture, it is not true for the overall flavor of meat. Data is shown indicating that, during post-mortem aging, desirable flavors such as beefy, brothy, browned-caramel, and sweet decline while off-flavors such as bitter and sour increase.

Postmortem Metabolic and Morphologic Alterations of the Dog Brain Thalamus with Use of In Vivo 1H Magnetic Resonance Spectroscopy and Electron Microscopy

To investigate postmortem metabolic and morphologic alterations in the dog brain thalamus and to contribute diagnostic aids in medicolegal science. A homogeneous group of 17 dogs was used for in vivo 1H magnetic resonance (MR) spectroscopy and electron microscopic studies, which were performed under intravenous and intramuscular anesthesia before and after the dogs were killed. All 1H MR spectroscopy and electron microscopy examinations were performed on a 1.5 T MR imaging/MR spectroscopy system using a stimulated-echo acquisition mode pulse sequence and an electron microscope, respectively. The lactate resonance signal, which could be used as a criterion for tissue survival, appeared during the entire postmortem period. The lactate: creatine ratio significantly increased in the early postmortem metabolic process. The inositol: creatine ratio showed a mild gradual increase. However, the N-acetyl-aspartate: creatine and choline/creatine ratios generally were unchanged. The decomposition of proton metabolites was observed in the MR spectrum 48 hours postmortem. In the postmortem period, high power field (x10,000) electron microscopic results showed significant morphologic alterations of the thalamus, revealing nuclear pyknosis and perikaryal condensation, cytoplasmic vacuoles, clumping of nuclear chromatin, and destruction of cellular organelles and nuclear membrane. Results suggest that the combination of 1H MR spectroscopy and electron microscopy may simultaneously provide good quality metabolic and morphologic information of cerebral tissue in the field of thanatochronology.