A lesson from history? Worsening mortality and the rise of the Nazi Party in 1930s Germany.

OBJECTIVES: The aim of the study was to test the hypothesis that worsening mortality rates in the early 1930s were associated with increasing votes for the Nazi Party. STUDY DESIGN: The study consist of panel data with fixed effects. METHODS: We used district- and city-level regression models of Nazi vote shares on changes in all-cause mortality rates in 866 districts and 214 cities during federal elections from 1930 to 1933, adjusting for election and district/city-level fixed effects and sociodemographic factors. As a falsification test, we used a subset of deaths less susceptible to sociopolitical factors. RESULTS: Historical downward trends in mortality rates reversed in the early 1930s in Germany. At the district/city level, these increases were positively associated with a rising Nazi vote share. Each increase of 10 deaths per 1000 population was associated with a 6.51-percentage-point increase in Nazi vote share (95% confidence interval = 1.17-11.8). The strongest associations were with deaths due to infectious and communicable diseases, suicides, and alcohol-related deaths. Worsening mortality had no association with votes for the Communist Party or for other contemporary political parties. Greater welfare payments were associated with smaller increases in both mortality and Nazi vote share, and adjusting for welfare generosity mitigated the association by approximately one-third. CONCLUSIONS: Worsening mortality rates were positively associated with the rise of the Nazi Party in 1930s Germany. Social security mitigated the association between mortality and Nazi vote share. Our findings add to the growing evidence that population health declines can be a 'canary in the coal mine' for the health of democracies.

Awareness, knowledge, and perceptions of infertility, fertility assessment, and assisted reproductive technologies in the era of oocyte freezing among female and male university students.

PURPOSE: The aims of our study were to analyze university student's knowledge and attitude towards parenthood, female fertility, fertility assessment, and oocyte freezing and to explore associations between these aspects and the participant's sex or degree program they were registered for. METHODS: The study was designed as an online-based cross-sectional survey. A total of 1144 participants answered 27 questions. The data were analyzed using descriptive statistics. Linear regression models were employed to explore associations between sex or university program and attitude towards parenthood, fertility assessment, and oocyte freezing. RESULTS: Female students and students of non-medical degree programs were more likely to plan to have children earlier than male students or students of medical degree programs. Female sex or medical degree program was associated with an overall better knowledge about women's fertility. The better the participant's knowledge about fertility, the more likely the students would consider assisted reproductive technology (ART) treatments as an option to become pregnant when ovarian reserve is low. The majority of students knew the principal of oocyte freezing but would not consider using it. However, in the case of a low ovarian reserve, oocyte freezing would be accepted as an option. CONCLUSIONS: Students planned to have children at an age when women's fertility is already declining. Gaps in knowledge about female fertility and the potential of ART were more pronounced in male students and students of non-medical degree programs suggesting an increase of fertility awareness is necessary in these groups to prevent them from infertility and unwanted childlessness.

[Sealing of the hepatic resection area using hemostat devices does not improve results of adequate surgery]. / Versiegelung der Leberresektionsfläche mit Gewebsklebematerialien bewirkt keine weitere Verbesserung adäquater Chirurgie.

PURPOSE: In hepatic resections, there has been a high quality demand. The aim of this systematic clinical, prospective, unblinded unicenter observational study with two arms in an unselected patient cohort was to investigate whether hemostat device can significantly improve outcome in resective liver surgery, in particular, in high risk patients. METHODS: All consecutive patients (mean age, 60.5 [range, 17 - 96] years) who underwent hepatic resection (ntotal = 770) were prospectively documented in a computer-based registry at a university hospital (tertiary center) over a time period of 10 years and retrospectively evaluated specifically with regard to the use (-/+; in daily practice and intraoperative decision-making) of hemostat device (Tissucol(®), n = 59/Tachocomb(®), n = 202/combination, n = 55) indicated (among others) by drainage volume, inflammatory parameters and rate of specific complications (nvalidated = 541 [100 %]). RESULTS: Most frequently, (a-)/typical segmental resections were used: n = 192/90 (3-segment resection, only n = 38). 1) For the assignment of patients to the two different groups (-/+ hemostat device), weight loss and type of resection were found as significant factors (trend: ASA, cirrhosis), for the amount of drainage volume, ASA, sex, Karnofsky Performance Scale and also type of resections using independent distributed statistical tests (such as χ(2), U test [Mann/Whitney]; H test [Kruskal-Willis]; correlation coefficient by Spearman) - no impact: smoking, diabetes, BMI, ethanol. 2) Not taking into account these parameters, the use of hemostat device was characterized by an increased drainage volume (negative control < Tissucol = Tachocomb < combination). 3) Using multifactorial analysis of variance, it was found even under correction by the factors with significant impact elucidated in the single test that the application of hemostat device onto the hepatic resection area resulted unexpectedly rather in an increase than a decrease of the drainage volume but 4) under accompanying more pronounced increase of the white blood cell count (leucocytosis). 5) General and specific complications such as postoperative bleeding, biliary fistula and subhepatic abscess were not further lowered in a significant manner using hemostat device. CONCLUSION: Adequate surgery in the operative management of hepatic resection area cannot further be improved or optimized using hemostat device. In this context, drainage volume may not be considered a sufficient rather an orienting parameter. However, there is an inflammatory response detectable most likely indicated by a(n un-)specific effusion and increase of white blood cell count, which can be interpreted as a) being characteristic for the problematic group of patients, in whom hemostat device was decided to be useful and was finally used in daily prectice, or b) reactive inflammation to foreign material.

Utilization of virus φCh1 elements to establish a shuttle vector system for Halo(alkali)philic Archaea via transformation of Natrialba magadii.

In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus Ch1. The shuttle vector pRo-5, based on the repH-containing region of Ch1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The Ch1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of Ch1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus Ch1 in their extreme environments.

Characterization of T cell repertoire changes in acute Kawasaki disease.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease.

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA.

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.

Mitochondrial DNA deletions and the aging heart.

Mitochondrial DNA (mtDNA) mutations appear to be associated with a wide spectrum of human disorders and proposed to be a potential contributor of aging. However, in an age-dependent increase of the common 4977 bp deletion of human mtDNA still many unanswered questions remain. Comparing mtDNA copy levels in different tissues revealed that cardiac muscle had the highest, while the cortex cerebelli showed the lowest copy number of mtDNA in every donor. Intriguingly, mtDNA copy number showed no changes during aging. In heart tissue, the amount of 4977 bp mtDNA deletion increased in an age-dependent manner showing significant differences at the age of 40 years and older (p<0.005). In vitro studies analyzing human normal cells transfected with telomerase (BJ-T) revealed that oxidative stress (OS)--a well accepted promoter of aging--induced 4977 bp deletion and point mutations as demonstrated by real-time PCR and DHPLC analysis. Interestingly, OS induced apoptosis only in transformed human fibroblasts by activation of the intrinsic (mitochondrial-mediated) signalling pathway as indicated by morphological damage of mitochondria, DNA laddering and increase of the Bax/Bcl-2 ratio. In conclusion, in heart tissue, the amount of the 4977 bp deletion increased in an age-dependent manner and it was more detectable after the 4th decade of life, although there was some scatter in the data. Since, apoptosis was induced by the mitochondria-mediated pathway only in transformed cells, the role for apoptosis in normal tissue of the aging heart remains unclear.

Low hepatic lipase activity is a novel risk factor for coronary artery disease.

BACKGROUND: The crucial function of hepatic lipase (HL) in lipid metabolism has been well established, but the relationship between HL activity and coronary artery disease (CAD) is disputed. METHODS AND RESULTS: We measured HL activity in the postheparin plasma of 200 consecutive men undergoing elective coronary angiography and determined the degree of CAD with the extent score, which has been shown to be better correlated with known risk factors than other measures of CAD extent. We found a significant inverse correlation between HL activity and the extent of CAD (r=-0.19, P<0.01). This association was mainly due to patients with HDL levels >0.96 mmol/L (n=94, r=-0.30, P<0.005). HL activity was lower in 173 patients with CAD than in 40 controls with normal angiograms (286+/-106 versus 338+/-108 nmol. mL(-1). min(-1), P<0.01). To correct for potential confounding factors, we performed multivariate analyses that confirmed the independent association of HL activity with CAD extent. In addition, the presence of the T allele at position -514 in the HL promoter, which leads to a reduced HL promoter activity, was associated with lower HL activity (r=0.30, P<0.001) and higher CAD extent (42.2+/-20.8 versus 35.3+/-23.6 [extent score], P<0.05). In patients with heterozygous familial hypercholesterolemia, calcified lesions in ECG-gated spiral computed tomography were higher in patients with low HL activity (6.3+/-6.8 versus 1.5+/-3.1, P=0.01). CONCLUSIONS: Our data show that low HL activity is associated with CAD. Therefore, HL might be useful for CAD risk estimation and might be a target for pharmacological intervention.

Antidiabetic action of somatostatin--assessed by the artificial pancreas.

By means of a glucose-controlled insulin- and glucose-infusion system (GCIGIS) we examined the effect of somatostatin on insulin and glucose requirements following meals or oral glucose loads in juvenile diabetics. In six of seven patients the insulin requirement with somatostatin was remarkably reduced to between 38 per cent and 79 per cent of that of otherwise identical control experiments. No reduction could be found in the seventh case, fed only 575 kcal. In all cases we observed an increase in dextrose demanded from the GCIGIS ranging between 28 per cent and 192 per cent of the control amounts. In addition, a lowering and smoothing of postprandial blood glucose curves caused by somatostatin application was a general finding. It seems to us most likely that the well-known suppression of the secretion of growth hormone and glucagon, both insulin antagonists, is responsible for the antidiabetic action of somatostatin.

Effects of current therapy of Kawasaki disease on eicosanoid metabolism.

Coronary artery inflammation in Kawasaki disease is accompanied by thrombocytosis and platelet activation. It was hypothesized that abnormal metabolism of bioactive eicosanoids could result from or contribute to these events. Circulating plasma thromboxane B2, 6-keto-prostaglandin F1 alpha and prostaglandin E were measured by double antibody radioimmunoassay in patients with Kawasaki disease before and after aspirin alone or aspirin and intravenous gamma globulin therapy. Plasma prostaglandin E concentrations were normal in all patient groups. Pretreatment thromboxane B2 was elevated compared with age-matched controls, fell moderately with high-dose aspirin (60 to 100 mg/kg/day) and marginally increased with low-dose aspirin (3 to 5 mg/kg/day) 6 to 8 weeks after treatment. Plasma 6-keto-prostaglandin F1 alpha was not detected in 12 of 16 patients before therapy and remained low in all but 1 patients by 6 to 8 weeks. Thromboxane B2 correlated weakly with serum salicylate concentration but had no relation to platelet mass. The results in these patients with Kawasaki disease indicate only partial thromboxane suppression and depressed prostacyclin generation regardless of therapy. This balance favors coronary vasoconstriction and platelet aggregation capable of potentiating myocardial ischemia or infarction. The results justify consideration of higher or more frequent aspirin doses for longer duration and thromboxane receptor blockade in this disease.

Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus.

Mitochondria are considered a key element in the process of organismic aging, because of their fundamental role in cellular energy generation. In the course of oxidative phosphorylation, harmful free radicals are continuously produced damaging the mitochondrial (mt) genome. One of the consequences is the occurrence of large-scale deletions in mtDNA molecules. The 4977 bp common deletion accumulates exponentially with age, in a mosaic pattern, especially in postmitotic tissues. In order to investigate whether certain cell characteristics underlie this pattern of distribution, and to look for possible age-related changes, two cell types in the caudate nucleus of the human brain from five young and five senescent subjects were analysed by single-cell PCR.MAP2-positive neurons and GFAP-positive astrocytes were isolated by micromanipulation. For each of the 10 cases, at least 30 cells of each type were collected and subjected to PCR individually. Screening for the presence of the common deletion yielded no significant differences in relative distribution, neither between astrocytes and neurons, nor between healthy young and old humans. Our results imply that the age-dependent increase of the common deletion cannot come about by an increase of independent deletion events in a greater proportion of cells, and that mitotic rate is not a major cellular risk factor for deletion accumulation in the caudate nucleus.

Detection of the 4977 bp deletion of mitochondrial DNA in different human blood cells.

As recently reported, it is possible to detect and quantify the amount of the deleted human mitochondrial DNA (mtDNA) in whole blood, platelets and peripheral blood mononuclear cells using real-time PCR. The aim of this study was to identify the cell types in human blood carrying the 4977 bp deleted mtDNA and their accumulation with regard to donor age. Whole blood from 10 healthy donors (five individuals aged from 19 to 22 years, five aged from 57 to 61 years) was separated in various cell populations such as granulocytes, B cells/monocytes and T cells. Purity of the cell isolates was determined by flow cytometry. Total DNA was extracted and 250 ng DNA of each cell type was subjected to PCR using fluorescent-labelled primer pairs. The specific PCR product of the 4977 bp deletion was quantified using an automated detection system. The accumulation of the 4977 bp deletion was more pronounced in T lymphocytes and granulocytes in comparison to B lymphocytes/monocytes. The amount of the 4977 bp deletion in whole blood varied from 0 to 0.00018%, in T lymphocytes from 0.00009 to 0.00160%, in granulocytes from 0 to 0.00162% and in the B lymphocyte/monocyte fraction from 0 to 0.00025%. The higher amount of the deletion in T lymphocytes may be due to a subset of lymphocytes with a longer lifespan thus facilitating the accumulation of mitochondrial damage. The higher amount in granulocytes could have the explanation in the higher release of free radicals for prevention of infectious diseases, because free radicals are supposed to damage the macromolecules of this cell type. The 10 donors displayed differences in the pattern of the accumulation with regard to the different cell types, but no age-dependent accumulation was observed. Differences of the accumulation pattern may be due to actual individual living behaviour or environmental factors.

Brain injury after gunshot wounding: morphometric analysis of cell destruction caused by temporary cavitation.

In addition to the primary destruction of brain tissue readily visible at autopsy (permanent track), gunshot wounding to the brain creates a pulsating temporary cavity due to radial expansion along the bullet's track. To determine the maximum extent of this temporary cavitation in brains of victims of gunshots from weapons with low muzzle velocity, we carried out morphometric studies on 20 cases of death from gunshot wounding to the head from bullets with a muzzle energy <500 J and a survival time of <90 min. The brains were fixed in formalin, examine macroscopically and microscopically, and subjected to morphometric analyses. Surrounding the permanent track, a narrow mantle-like zone of astrocyte destruction was found within an area of hemorrhagic extravasation. Axons near the permanent track had been broken into tiny fragments. The axonal damage lessened with increasing distance from the permanent track, although axons continued to be fragmented and to exhibit varicose changes and clumping until assuming their normal structure beyond 18 mm. Nerve cells were extremely shrunken close to the permanent track but gradually took on their normal shape with increasing distance. We also assessed the loss of glial fibrillary acid protein expression by astrocytes in the white matter, the extent of traumatic bleeding, and damage to axons and neurons as measured radially from the permanent track. Axonal and neuronal damage were found to extend about 18 mm radially from the permanent track, tapering gradually along the track from entry point to exit point. The destruction was probably produced by the temporary cavitation and accords with theoretical considerations and experimental observations.

Regional potassium distribution in the brain in forensic relevant types of intoxication preliminary morphometric evaluation using a histochemical method.

A histochemical-morphometric method was used to measure potassium (K+) levels in gray and white matter of rats following sublethal intoxication with 11 different neurotoxic compounds of high forensic significance. Six rats were each given a single substance applied intraperitoneally, the same dosage being given to two animals each. The animals were subsequently killed, the brains immediately frozen, and cryosections cut. K+ levels were evaluated morphometrically. A drop in K+ levels was used as the criterion for cytotoxic edema. Application of ethanol, atropine, carbromal, carbon monoxide, morphine or triethyltin led to a rise in K+ levels in the gray matter and a simultaneous decline in the white matter. By contrast, administration of amitriptyline, glycerin, potassium cyanide, parathion or phenobarbital initiated an increase in K+ levels in both gray and white matter. A cytotoxic edema could thus be reliably excluded in these intoxications. Although the study design allows no statistical analysis, these conclusions are supported by the marked differences in K+ levels in gray and white matter induced by the different toxicants.

Demonstration of the 4977 bp deletion in human mitochondrial DNA from intravital and postmortem blood.

The 4977 bp deletion in mitochondrial DNA (mtDNA) is known to accumulate with age in various human tissues. Findings regarding its accumulation in blood, however, have so far been contradictory. We investigated the levels of the 4977 bp deletion in mtDNA from 100 intravital and postmortem blood samples. Applying an improved version of a PCR plus silver staining of polyacrylamide gels, we could detect the 4977 bp deletion in blood of healthy individuals over 20 years of age. While the 4977 bp deletion in blood is subject to a certain age dependence, it appears to be influenced by additional factors. A Primer-Shift-Assay amplifying four different deletion-specific fragments showed that the smaller fragments were amplified with a higher amplification efficiency than the larger fragments. The deletion-specific 389 bp fragment was demonstrated in 73% of individuals over 80 years of age, but in only 46% of individuals between 21 and 30 years old whereas the largest 802 bp deletion-specific fragment was detectable in 38% of subjects over 80 years of age, and in only 15% of individuals under 30 years of age. Deletion-specific fragments were not detected in a single individual under 20 years old, nor in fetal blood. In this work, we demonstrate for the first time the detection of 4977 bp specific fragments in blood of healthy individuals without the necessity of using a nested PCR. The deletion is detectable in postmortal and intravital blood, so that the occurrence of the 4977 bp deletion seems to be a physiological and not only a postmortal process.

Traumatic induced total myelomalacia of the cervical spinal cord associated with a space-occupying subdural hematoma.

We report the case of a 20-year-old male driver who suffered from a trauma to the cervical vertebral column in a head-on collision with a tree. The injuries included subluxation of the 2nd and 3rd cervical vertebrae and fracture of the odontoid process of the axis with ventrally directed displacement of the proximal fragment and dorsally directed displacement of the distal fragment. Already at admission to hospital a space-occupying spinal subdural hematoma was diagnosed. Clinically, paraplegia was diagnosed with progressive loss of consciousness. Pneumonia led to death 40 days after the accident. Autopsy disclosed a total myelomalacia of the cervical spinal cord obviously resulting from an ischemia caused by a traumatic lesion of the dorsal truncus arteriosus spinalis as well as a compression by the spinal subdural hematoma.

Death from thermal effects and burns.

One hundred and fifteen unselected autopsy cases of death from thermal effects and/or fire between 1990 and 1999 were analyzed with regard to time of death, signs of vitality at the scene of the fire, the manner and cause of death, and the extent of soft tissue loss. The cases represented approximately 6% of all autopsy cases at the Institute of Legal Medicine responsible for a catchment area with approximately 700,000 inhabitants. In 23 victims suffering burn injuries, death occurred during initial medical care or clinical treatment. The causes of death were primary respiratory arrest due to smoke poisoning or delayed shock caused by thermal injuries to the skin. Death occurred at the scene of the fatal event in 85 cases: eight cases exhibited no thermal effects; the cause of death in one of these cases was polytrauma incurred in a leap from a height; in seven cases there was poisoning due to smoke inhalation. The remaining 77 cases were characterized by signs of intensive thermal and/or fire effects. Clear signs of vitality (carboxyhemoglobin (COHb) in blood, inhalation and/or swallowing of soot) were found in 84.7% of the victims dying at the site of the fatal event. Of the 13 victims showing no signs of vitality at the scene, a cause of death could be determined in only seven cases; death in the other six cases remains unexplained. Quantification of the soft tissue loss revealed a possible correlation with the temperature and time course of heat exposure.

Brain injury after survived gunshot to the head: reactive alterations at sites remote from the missile track.

Gunshot wounds to the brain usually lead to acute respiratory arrest or death after a brief survival period, even in cases involving only slight direct tissue damage. It can be assumed therefore that the damage extends beyond the zone of recognizable destruction and hemorrhages. To determine the true extent of the tissue injury resulting from gunshot wounds to the brain, we carried out microscopic investigations for reactive changes (emigration of leukocytes and macrophages, axonal expression of beta-amyloid precursor protein (beta-APP) in 10 cases of gunshot wound to the narrow channel of the brain with survival times >2h. Demonstration of leukocytes expressing naphthol AS-D chloroacetate esterase activity in the brain tissue at the border of the missile track established the vitality of the gunshot effect. The presence of macrophages (CD68-epitope) allowed demarcation of a 1-2mm wide necrotic zone around the permanent cavity. Within this zone and beyond, beta-APP showed an initial increase followed by a decline in the number of injured axons. Three types of beta-APP positive staining could be differentiated. In the immediate vicinity of the missile track beta-APP positive neurons were present at a distance of 2-4mm from the margin of the permanent cavity (type 1) as a result of primary injured neuronal tissue by the gunshot itself. At longer distances from the narrow channel and the permanent cavity single beta-APP positive axons or axon fragments and two additional types were found; type 2 shows a parallel, wave-like arrangement of the damaged fibers, which suggests that the injury was produced by mechanical acceleration of the brain tissue created by the energy the projectile expended within the brain; irregular aggregation of beta-APP positive axons or axon fragments within a local edema represents type 3, which may be attributed to secondary ischemia or edema.

Estimation of age at death based on quantitation of the 4977-bp deletion of human mitochondrial DNA in skeletal muscle.

The 4977-bp deletion in human mitochondrial DNA (mtDNA) is known to accumulate in various tissues with age. Since this deletion in mtDNA correlates closest with age in muscle tissue, iliopsoas muscle tissue was taken at autopsy from 50 persons aged 24-97 years to determine whether age at death can be estimated based on the amount of the 4977-bp deletion in skeletal muscle. Total DNA (nuclear and mtDNA) was extracted from 100 mg tissue and the 4977-bp deletion quantified using a kinetic polymerase chain reaction (PCR) followed by visualization of the products on silver stained polyacrylamide gels. The amount of the 4977-bp deletion of mtDNA ranged from 0.00049% to 0.14% depending on age, with a correlation coefficient of r = 0.83 (P = 0.0001). In forensic practice this method can aid in the estimation of age at death with a relatively wide confidence interval, thus enabling a discrimination between young and elderly persons in the identification of human remains based solely on skeletal muscle.