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.

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.

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.

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.

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.

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.

Fatal hypernatremia due to drinking a large quantity of shoyu (Japanese soy sauce).

We report a case of fatal salt poisoning in a 55-year-old woman who suffered from depression and drank a large quantity of shoyu (Japanese soy sauce). She presented with the highest ever documented serum sodium level of 187 mmol/L. This was associated with symptoms of cerebral damage which developed within hours after drinking the soy sauce. She died as a result of massive pulmonary edema, despite intensive medical treatment. Viewing the results of clinical and postmortem investigations together, her death could clearly be attributed to drinking a large quantity of soy sauce.

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.

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.

A fatal case of potential chronic overdoses of prescribed and proprietary remedies.

A 33-year-old man was found in a state of cardiopulmonary arrest. He was transported to an emergency hospital but was pronounced dead. He had suffered from depression for about 8 years and had attempted suicide repeatedly. A search by the police found 645 empty Press Through Package (PTP) sheets. They had included neuroleptics, antidepressants, hypnotics, proprietary antitussives containing caffeine, proprietary cold remedies containing caffeine, and other unidentified drugs. An autopsy showed higher rectal temperature (38 degrees C), severe pulmonary edema (left: 681 g, right: 821 g), and a large amount of urine in the bladder (about 760 mL). Toxicological analyses using gas chromatography-mass spectrometry (GC/MS) and high performance liquid chromatography (HPLC) demonstrated that doses of clomipramine hydrochloride (a tricyclic antidepressant), chlorpromazine (a phenothiazine), and caffeine (a methylxanthine derivative) were within the toxic range (0.68, 0.64, and 34.24 [microg/mL], respectively). Histological examination showed centrilobular necrosis of the liver with small fat droplets. We concluded that he had died of pulmonary edema due to combined drug intoxication including proprietary antitussives and cold remedies. Furthermore, there was a strong possibility that he had habitually taken overdoses of those drugs. Herein, the risk of misuse of prescribed and proprietary drugs, especially for people with psychological problems, should be reemphasized.

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.

Aromatic L-amino acid decarboxylase-immunoreactive structures in human midbrain, pons, and medulla.

The objective of the present study was to determine with precision the localization of neurons and fibers immunoreactive (ir) for aromatic L-amino acid decarboxylase (AADC), the second-step enzyme responsible for conversion of L-dihydroxyphenylalanine (L-DOPA) to dopamine (DA) and 5-hydroxytryptophan (5-HTP) to serotonin (5-hydroxytryptamine: 5-HT) in the midbrain, pons, and medulla oblongata of the adult human brain. Intense AADC immunoreactivity was observed in a large number of presumptive 5-HT neuronal cell bodies distributed in all of the raphe nuclei, as well as in regions outside the raphe nuclei such as the ventral portions of the pons and medulla. Moderate to strong immunoreaction was observable in presumptive DA cells in the mesencephalic reticular formation, substantia nigra, and ventral tegmental area of Tsai, as well as in presumptive noradrenergic (NA) cells, which were aggregated in the locus coeruleus and dispersed in the subcoeruleus nuclei. In the medulla oblongata, immunoreaction of moderate intensity was distributed in the mid and ventrolateral portions of the intermediate reticular nucleus, which constitutes the oblique plate of A1/C1 presumptive adrenergic and/or NA neurons. The dorsal vagal AADC-ir neurons were fewer in number and stained more weakly than cells immunoreactive for tyrosine hydroxylase (TH). AADC immunoreactivity was not identified in an aggregate of TH-ir neurons lying in the gelatinous subnucleus of the solitary nucleus, a restricted region just rostroventral to the area postrema. Nonaminergic AADC-positive neurons (D neurons), which are abundant in the rat and cat midbrain, pons, and medulla, were hardly detectable in homologous regions in the human brain, although they were clearly distinguishable in the forebrain.

Measurement of force to obstruct the cervical arteries and distribution of tension exerted on a ligature in hanging.

We experienced suicidal hanging cases without a ligature in front of the neck. We conducted several anatomical autopsies and the magnetic resonance imaging (MRI), and studied the dynamics behind hanging by applying an apparatus to cadaver and creating a dummy. MRI revealed that the vertebral artery protrudes diagonally upward from the second vertebra to the first one. We also found that this area was not covered with any bony tissue. The average length between the first and second transverse processes was 2 cm. We measured the minimum required force to obstruct the blood stream in both carotid and vertebral arteries exerting 130 mmHg. The required force was 6 kg for the carotid artery and 7 kg for the vertebral artery. Compared to the reported cases, there was not a significant difference in the force to obstruct the carotid artery, however, that of the vertebral artery was lower than the force reported so far. With an experiment of pressure sensitive plastic-sheet, we learned the lateral sides of the neck are compressed more. The results obtained from this study show that the complete and fatal obstruction of the carotid and the vertebral arteries may easily occur when the ligature transects at the lower part of mandible angle.

Shedding of gACE from residual body membrane of rat sperm.

Germinal angiotensin I-converting enzyme (gACE) is expressed only in the testis and is uniquely present in developing spermatids and sperm. We previously purified soluble gACE from porcine seminal plasma, and reported that gACE was secreted from residual body on spermatozoa by the other peptidase(s), namely Sheddase. Using Nma/DNP substrate, it was observed that the shedding activity in testicular fluid was stronger than in the sperm membrane and epididymal fluid. The shedding activity was inhibited by AEBSF and antipain, and not by EDTA and E-64. Accordingly, it is thought that Sheddase is an endo-type of serine peptidase.

A study of monoamine neuronal systems of schizophrenic patients: using forensic autopsy brains.

Studies on autopsy brains of mental illnesses are essential for psychiatric research. Brain materials from forensic autopsy cases have been valuable research resource. We introduced some characteristic neuronal structures of schizophrenic post-mortem brains and normal controls detected using both forensic and pathological autopsy cases and immunohistochemical method. Dopamine-related neuronal structures in schizophrenic cases were investigated by using immunohistochemistry for tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and phenylethanolamine-N-methyltransferase (PNMT). At the level of the striatum, the number of AADC-positive, but TH-negative neurons (D-neurons) reduced in schizophrenics compared with controls. In the brainstem structures, we described some morphological findings characteristic to catecholaminergic neurons of the ventral tegmental area of schizophrenic cases and reduction of the number of medullary catecholaminergic neurons. These results might be in accordance with recent reports describing neural stem cell dysfunction in schizophrenic brains. The authors also introduced the first systematic psychiatric brain bank in Japan, the Fukushima Psychiatric Brain Bank, constructed in 1997, and mentioned the necessity for arrangement of brain bank systems to further promote the psychiatric brain research.

Assessment of cervical venous blood flow and the craniocervical venus valve using ultrasound sonography.

Severe congestion with petechiae and/or small hemorrhages over a wide area consisting of the subcutaneous and mucous level of the head and neck is observed in most cases of strangulation and positional asphyxial death. Congestion present above the strangulation mark is accepted as a vital reaction. Although the congestion results from a disturbance of cervical venous flow, only a few reports concerning a relationship between venous flow and posture were found in the literature. We assessed the physiological situation of cervical venous return and movement of the craniocervical venous valve for many types of valve with respect to posture using color-duplex ultrasound sonography (US). We measured the Doppler wave of cervical veins consisting of internal jugular vein (IJV), external jugular vein (EJV) and vertebral vein (VV) and the motion of IJV valves in three positions on a seat which could be manually moved through +90 degrees to -90. The diameter of IJVs and EJVs were thicker in a head-down position, moderate in a supine position and flat in a sitting position. The diameter of VVs was thick in a sitting position, moderate in supine position and unclear in an upside-down position because of difficulty in scanning owing to very slow velocity and deep lying position. The velocities were high in a sitting position, but very slow and intermittent in an upside-down position. The EJV which is markedly congested in some cardiac tamponade cases and drains mainly the facial superficial region is very important in legal medicine but has not often observed in previous reports. We confirmed that cervical venous return (IJV, EJV, VV) changes its route according to the posture. The motion of IJV valves was very complicated and varied in three positions, even after the Doppler wave and the motion of valve became stable. Asphyxia and strangulation should be studied taking the complex venous structure into consideration. We could not find any consistent explanation for venous valve motion and further studies on the valves are necessary to understand the venous reflux in some diseases in legal medicine.

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.

Evaluation of suicide by hanging : From the video recording.

Most evaluations of the pathophysiological processes of suicidal hangings are based on animal experiments and only a few have examined the hanging process. The little information available is mostly the result of animal experiments that have focused on tracheal obstructions. In the study presented here, a camcorder was set up by the victim to record the entire hanging process. According to the analysis from the recording, the complete asphyxial process lasted only 2 minutes and 43 seconds. Stage 1 (the prodromal stage) was especially short. Typical convulsions were not observed in this case. Body movements, such as clonic and tonic spasms, were not noticed in stage 2 (the dyspnea stage). Because the body did not touch the ground, the pressure on the neck by the ligature caused cerebral ischemia as a result of the obstruction of the neck vessels. Consequently, it can be hypothesized that sui cide by hanging may not be based on the effect of changed concentration in the blood of carbon dioxide and oxygen, but on the effect of cerebral ischemia.

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.