Contraction Bands Are Induced by Cardiac Massage Itself.

Pathological contraction bands (CB) are recognized as a type of necrosis pattern found in the myocardium. It is well known that CB are induced by cardiopulmonary resuscitation (CPR) with cardiac massage. However, it is not known whether the reperfusion or massage itself causes the formation of CB. We studied 10 cardiac tissues taken from forensic autopsy cases that had not received CPR. We excluded the cases where the cause of death was a cardiac event. After making sections for forensic research, we massaged the left ventricles for 10 minutes. We found CB in all cases with massage performed within 12 hours after death, which is the time frame for supravital reactions. We did not find CB in any cases where more than 24 hours had elapsed since the time of death. Contraction bands were not observed in any sections that were taken before massage. We suggested here that CB induced by CPR were caused by the cardiac massage itself, and that most CPR-induced CB are not contraction band necrosis but rather artifactual CB.

Classification of contraction bands using immunohistochemistry.

Pathological contraction bands (CBs) are a type of necrosis pattern found in the myocardium. The composition of CB is not well studied. This is because CBs have diverse forms and can be observed in various causes of death. In pathology fields, CBs are classified artifactual CB and CB necrosis. We have identified different forms of CB by examining the expression and distribution of complement component C9 (CCC9) and Sirtuin1 by immunohistochemistry in the myocardium of patients who died because of different causes of death. We used cardiac tissues with CB from 30 forensic autopsy cases in our department from the last 2 years. We excluded the cases that had very little CB. We found that our CB classification based on expression levels of both CCC9 and Sirtuin1 correlated well with the agonal situation, including high temperature, myocardial infarction, cardiopulmonary resuscitation, and hypothermia. On the basis of these results, we here advocate a classification scheme based on immunohistochemistry. Furthermore, we found that CB necrosis could be detected using immunostaining with CCC9. Using our classification scheme, it will be possible to more accurately research each type of CB and the causative mechanisms.

Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE.

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was approximately 300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m(-3)) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation ofmicroflora during operation of the biofilter The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene.

Factor H in porcine seminal plasma protects sperm against complement attack in genital tracts.

We found that factor H (FH) exists in porcine seminal plasma. Purified FH strongly inhibited serum alternative pathway complement activation against lipopolysaccharide. The molecular weight, pI, and heparin-binding activity of the purified protein were different from those of purified FH from porcine serum. The complement regulatory activity of seminal plasma FH was approximately 2-fold stronger than that of serum FH. Treatment of purified serum FH with sialidase and N-glycosidase F gave almost the same results as those of seminal plasma FH. The deletion of sialic acid from the carbohydrate chains of both FHs contributed to heparin-binding and complement regulatory activities. Results of reverse transcriptase-PCR, Western blot analysis, and immunohistochemistry showed that seminal plasma FH is mainly secreted from epithelial cells of the seminal vesicle in male genital tracts. FH was also detected in the outer acrosomal region of ejaculated sperm by immunofluorescence staining, and found that the purified FH from the sperm membrane has the same complement regulatory activity as that of seminal plasma FH. The ejaculated sperm possessing FH in the outer acrosomal region considerably evaded complement attack. We also found that there is strong complement activity in fluids from female genital tract ducts. These findings indicate that FH bound to the outer acrosomal region and soluble FH play important roles in protecting sperm against complement attack in male and female genital tracts.

Genus identification of toxic plant by real-time PCR.

Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells.

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.

Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning.

Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.

Determination of Both Fetus' and Mother's Blood Type from an Autopsy Case Immersed in Formalin for Over 50 Years.

A female fetus which had been immersed in formalin for more than 50 years was found in Japan. Because no liquid blood could be obtained, we tried to use immunohistochemistry (IHC) methods to tissue samples obtained at autopsy to identify both the fetal and mother's blood type. We detected B antigens on endothelial cells in paraffin sections of the fetal organs. Furthermore, we observed both anti-A- and anti-B-positive red blood cells in the intervillous space, which is indicative of the mother's blood type. To our knowledge, this is the first case report on determining the blood type of both the fetus and the mother from tissue immersed in formalin for such a long time. The results suggest that IHC is valuable for the determination of ABO blood type in circumstances of long postmortem duration and unfavorable storage conditions.

Clinical Phenotype and Segregation of Mitochondrial 3243A>G Mutation in 2 Pairs of Monozygotic Twins.

IMPORTANCE: The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. OBSERVATIONS: In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. CONCLUSIONS AND RELEVANCE: The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

[ABO blood group typing in forensic autopsies].

In forensic science and medicine the ABO system has been a major focus, since the record of this blood system is a very prevalent one and A, B and O(H) antigens on erythrocytes are also associated with other cells and tissues throughout the body and are known to be considerably stable to the such violent conditions as heating or drying. However the determination of the ABO grouping from the body often encounters the difficulty due to haemolytic erythrocytes, and putrefaction, mummification or skeletonization of the body during post-mortem interval. In this presentation I review the merit and demerits of the ABO blood-grouping methods utilized in my division at the forensic autopsies according to the haemagglutination, absorption-elution and histochemical techniques and ABO genotyping method. It is important for ABO grouping to know the distribution of the ABO antigen in the body. I would like to emphasize that the species identification prior to ABO grouping is an important procedure because forensic materials such as from saliva, urine and seminal fluid might be contaminated with the fluid from animals, and DNA extracted from vertebrate species might be amplified with the primer for ABO genotyping and the amplified PCR products might be hybridized to those from human.

Axonal transport of puromycin-sensitive aminopeptidase in rat sciatic nerves.

Axonal transport of puromycin-sensitive aminopeptidase (PSA), a putative neuropeptide degrading-enzyme which removes amino acid residues from the amino-terminal of neuropeptides, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double-ligation technique. The soluble fraction of each segment was partially purified by MonoQ column chromatography, and showed two peaks of aminopeptidase activity. One of the aminopeptidases was PSA. At 48 h after the ligations, a significant amount of the axonal transport of PSA activity was found in the proximal segment. Western blot analysis of the segments also showed that immunoreactive PSA in the proximal segment was 2.1-fold higher than that in the middle segment. Furthermore, the immunohistochemical analysis of the segments showed an increase of the immunoreactive PSA in the proximal segment in comparison with the enzyme in the distal segment, indicating that PSA is mainly transported by anterograde axonal flow. These results suggest that PSA plays a role in the metabolism of neuropeptides in nerve terminals or synaptic clefts.

Localization of sepiapterin reductase in the human brain.

Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR is essential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purified recombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein and SPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebral cortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPR antibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPR protein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I (GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistry revealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of the hypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPR were colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOS neuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4 biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4 biosynthesis.

Demonstration of puromycin-sensitive alanyl aminopeptidase in Alzheimer disease brain.

Puromycin-sensitive alanyl aminopeptidase (PSA, EC 3.4.11.14) is a member of the ubiquitous aminopeptidase family, which cleaves N-terminal amino acids from proteins. PSA is suggested to function as a trimming protease in the MHC class I pathway, which is activated in brains of Alzheimer disease (AD). We examined the immunohistochemical localization of PSA in brains of AD and control cases using a rabbit anti-PSA. In the control cases, the antiserum revealed staining in a few glial cells and blood vessels. In AD brain, however, intensely stained cells were found richly in the cerebral cortex. Double immunofluorescence studies confirmed that PSA-positive cells were reactive microglia. Such PSA-positive reactive microglia tended to locate in and around senile plaques and were sometimes observed to associate with neurons containing neurofibillary tangles. The present result indicates that reactive microglia express PSA-immunoreactive molecules, probably in association with the pathological conditions of AD.

Number of striatal D-neurons is reduced in autopsy brains of schizophrenics.

The human striatum, especially its ventral part, the nucleus accumbens, contains numerous neurons immunoreactive for aromatic L-amino acid decarboxylase (AADC, the second-step monoamine synthesizing enzyme, =DDC: dopa decarboxylase), but not for tyrosine hydroxylase (TH, the first-step catecholamine synthesizing enzyme) or tryptophan hydroxylase (TPH, the first-step serotonin synthesizing enzyme) (Neurosci Lett 232 (1997) 111-114). These AADC (+)/TH (-)/TPH (-) neurons are named as D-neurons (Jaeger CB, Ruggiero DA, Albert VR, Joh TH, Reis DJ. Immunocytochemical localization of aromatic-L-amino acid decarboxylase. In: Bjorklund A, Hokfelt T, editors. Classical transmission in the CNS, Part I, Handbook of chemical neuroanatomy, vol. 2. Amsterdam: Elsevier, 1984. pp. 387-418). The nucleus accumbens is one of the brain regions that is involved in the pathogenesis of schizophrenia. We examined the distribution of striatal D-neurons using AADC immunohistochemistry and postmortem brains obtained by legal and pathological autopsies (nine controls (27-75 years old) and nine schizophrenics (32-78 years old), postmortem interval to fixation (PMI): 2-30 h). Because the number of AADC-positive neurons per section had a tendency to reduce in the case with longer PMI, we analyzed specimens of five controls (27-64 years old) and six schizophrenics (51-78 years old) in which the PMI was less than 8 h. The number of AADC-positive neurons was reduced in the striatum of schizophrenics compared to that of controls. The reduction was significant in the nucleus accumbens (P<0.05, t-test). D-Neurons might be involved in the pathogenesis of schizophrenia. Further studies using sex-, age- and PMI-matched controls are essential.

Histochemical characteristic of perivascular space in the brain with an advanced edema.

Amorphorous and colorless spaces, Virchow-Robin spaces (VRS), were often found by HE stain around blood vessels in the edematous brain. Histochemical characteristic of the enlarged VRS caused by an advanced edema and detected by lectin stain using Griffonia simplicifolia I agglutinin in the brain stem, the occipital lobe and/or the cerebellum was examined by means of immunohistochemical method. After pretreatment with formic acid or proteinase K, formalin fixed-paraffin embedded tissue sections were incubated with antibodies (ABs) against plasma proteins such as amyloid P component, Ig G, albumin (Al), apolipoprotein E (Apo E), and lactotransferrin (Lf), and cellular proteins such as ubiquitin (Ubt), Tau-protein (Tau), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), CD68 (KP-1) and heparansulfate proteoglycan (HSG). The tissue sections were also incubated with antibodies against alanyl aminopeptidase-S (AAP-S) and alanyl aminopeptidase-N (AAP-N) without pretreatment. The VRS showed intensive reactivity with ABs against Amy P, AAP-S and AAP-N, moderate with ABs against Apo E and HSG, weak with ABs against Ig G, Al and Lf, feeble with ABs against Ubt, Tau and CD 68, and no with ABs against GFAP and MBP, respectively. Although the substances detected in VRS might be of blood plasma origin resulting from abnormalities in the blood-brain barrier, the mechanisms whereby the serum proteins and/or other substances are enriched in VRS remain incompletely understood.

Utilization of lectin-histochemistry in forensic neuropathology: lectin staining provides useful information for postmortem diagnosis in forensic neuropathology.

We have investigated the deposition of glycoconjugates in human brain tissue with or without brain disorders. In this review we describe the application of lectin-histochemistry techniques to forensic neuropathology. Lectin staining is able to reveal several kinds of carbohydrate-related depositions in addition to the conventional degenerative changes including senile plaques, neurofibrillary tangles and corpora amylacea. The senile plaques and neurofibrillary tangles were clearly stained by Con A, PSA and GSI lectins, the corpora amylacea which is relevant to repeated brain hypoxia and mitochondrial damage was also easily detected by these and many other kinds of lectins. Amorphous spaces were detected around blood vessels and independently from blood vessels by lectin staining in the white matter from patients with brain disorders or severe edema. The white matter lesions were not considered relevant for forensic pathology, until a large group of cerebral white matter lesions were detected in the elderly with increasing frequency by modern neuro-imaging methods. The spherical deposits were newly detected by lectin staining in the molecular layer of the dentate gyrus of the hippocampal formation chiefly from patients with schizophrenia or cognitive dysfunctions.

Postmortem lung weight in drownings: a comparison with acute asphyxiation and cardiac death.

There are several controversial findings and arguments about the lung weight as a marker of drowning. The aim of the present study was to examine the difference in the lung weight and the amount of pleural effusion between freshwater and saltwater drownings (n=70 and n=75, respectively), in comparison with asphyxiation (n=85) and acute cardiac death (n=82), for the diagnosis of drowning. In drowning cases, a gradual postmortem time-dependent decrease in the lung weight and a reciprocal increase in the pleural effusion suggested postmortem transudation from the lungs. The decrease in the total value of the combined lung weight and the amount of pleural effusion was marked in saltwater immersion after 3 days postmortem, suggesting a leakage of the effusion out of the thoracic cavity under an osmotic effect of the immersion medium. In cases within 3 days postmortem, when the combined lung weight and amount of pleural effusion were added to estimate possible combined lung weight at the time of death, there was a gross difference among the causes of death: the value was the largest in saltwater drowning, followed by freshwater drowning, acute cardiac death and asphyxiation. However, the value depended on the gender and age of the subjects, suggesting a relation to the individual physical constitution and survival time or vital activity. These factors should be taken into consideration in evaluation of the lung weight in the diagnosis of drownings.

Lung-heart weight ratio as a possible index of cardiopulmonary pathophysiology in drowning.

The aim of the present study was to investigate the lung-heart weight ratio in fresh- and saltwater drowning (n=67 and n=75, respectively) as a possible index of cardiopulmonary pathophysiology, in comparison with acute myocardial infarction/ischemia (AMI, n=75) and asphyxiation (n=85). In drowning cases, the total value of the combined lung weight and the amount of pleural effusion was regarded as a possible total lung weight. The median value of the combined/total lung weight was the highest in saltwater drowning, which was followed by freshwater drowning, AMI and asphyxiation, showing a tendency to be mildly increased depending on the heart weight. The lung-heart weight ratio was significantly higher in fresh-/saltwater drownings (3.944+/-1.538 and 4.825+/-2.242, respectively) than in asphyxiation (2.846+/-1.042) and AMI (2.641+/-0.916) (P<0.0001), showing a tendency to be higher in saltwater than freshwater drowning. However, the value depended on the gender and age of the subjects, and the difference between freshwater drowning and asphyxiation was insignificant in females. These results suggested that the lung-heart weight ratio may be an index for investigating the influence of aspirated immersion medium in drownings.

Clinical course of acute chemical lung injury caused by 3-chloropentafluoropene.

Perfluoroallyl chloride (PFAC), a fluorine-containing compound, has very severe toxicity, but this toxicity is not well characterised. We report a fatal case of acute chemical lung injury caused by the inhalation of PFAC. A 39-year-old man, working at a chemical factory, inhaled PFAC gas and died 16 days later of acute lung injury with severe pneumothorax. We present his clinical course together with thoracic CT findings, autopsy and analysis of PFAC in blood and urine samples with gas chromatograph-mass spectrometry. Previously, a fatal case of PFAC was reported in 1981 but PFAC was not identified in any of the patient's samples. In our patient, we identified PFAC in both blood and urine samples. Our toxicological analysis may be used as a reference to detect PFAC toxicity in the future. Our study should be helpful for diagnosing lung injury induced by a highly toxic gas, such as PFAC.

Dopamine neurons in the ventral tegmental area: an autopsy case of disorganized type of schizophrenia.

The mesolimbic dopamine (DA) system has been associated with the pathogenesis of schizophrenia. Here, we examined DA-containing neuronal structures of the ventral tegmental area (VTA) of an autopsy case of disorganized type of schizophrenia (75-year-old female), using tyrosine hydroxylase (TH) immunohistochemistry. A free floating method using 50-µm cryostat sections and three-dimensional imaging analyzer AxioVision were applied to observe the wide range structures of TH-immunoreactive (-ir) neurons. TH-ir neuronal cell bodies in the VTA of the present case had irregular shape and various size, and TH-ir neuronal processes had irregular thickness and straightened shape or curved shape having many corners, when compared to a control autopsy case with no detectable neurological and psychiatric diseases (64-year-old male). The mechanisms underlying the morphological characteristics of DA neurons of the brains with schizophrenia should be elucidated epigenetically as well as genetically.