Response of the taste receptor cell to the umami-substance stimulus. An electron-microscopic study.

The chemosensory gustatory cells in taste buds form chemical synapses with intragemmal neurites. We investigated the ultrastructure of the guinea pig gustatory cells after stimulation with a mixture of monosodium L-glutamate and guanosine 5'-monophosphate. The gustatory cells responded to the stimulus. The dense-cored vesicles localized in the presynaptic regions discharged the contents into the synaptic cleft by means of exocytosis, which resulted in a marked decrease of their population. These findings strongly suggest that the transmitter or transmitters contained in the vesicles are released in response to the taste stimulation at the cell apex, to conduct the excitement of the cell to the nerves.

A comparison of coronary arteries from Japanese and NZ subjects.

This paper describes a comparison of the anterior descending branch of the left coronary artery from 198 Japanese subjects of ages less than 60 yrs, with 301 New Zealand individuals of similar ages. The object of the study was to determine whether there were structural differences present which could be partially responsible for the low incidence of atherosclerosis in Japanese as well as the known low blood lipid levels. It was found that the internal elastic lamina of Japanese coronary arteries was less well formed at birth than that of NZ subjects. Intimal thickening was greater in Japanese coronary arteries from birth to the end of the first decade, but increased less rapidly with age, and was only about half as great as that of NZ vessels in the older age groups. The thickened intima of Japanese arteries was more uniform round the circumference of the vessel, the luminal surface was better formed with more stainable elastin present subjacent to the endothelial cells, and there was less evidence of mural thrombosis. NZ arteries showed pronounced eccentricity of the intima, more extensive lipid deposits, a poorly defined luminal surface, and frequent evidence of mural thrombosis.

Direct cardiotoxic effects of cocaine and cocaethylene on isolated cardiomyocytes.

We investigated the cardiotoxic effects of cocaine and cocaethylene on the Ca2+ flux responsible for excitation-contraction coupling in isolated ventricular rat myocytes. We simultaneously measured intracellular Ca2+ transients and cell length in isolated cardiac myocytes loaded with a fluorescent Ca2+ indicator, indo-1, during electrical field stimulation at 1 Hz. The cell length was estimated by video dimension analysis. We also measured the activities of Ca2+ ATPase and Ca2+ release channels of cardiac sarcoplasmic reticulum membrane vesicles. Both cocaine and cocaethylene produced significant decreases in both peak intracellular Ca2+ and the cell-contraction rate in a dose-dependent manner. The K0.5 for the reduction of peak intracellular Ca2+ was 157.5 microM for cocaine, but 90.0 microM for cocaethylene. Both cocaethylene and cocaine inhibited neither Ca2+ ATPase nor Ca2+ release channel activity. These results demonstrate that cocaethylene has a more potent direct negative inotropic action on cardiomyocytes, without preventing Ca2+ flux through the cardiac sarcoplasmic reticulum membrane.

Structure of the kidney in the crab-eating frog, Rana cancrivora.

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.

Postmortem changes in drug-metabolizing enzymes of rat liver microsome.

Postmortem changes in the drug-metabolizing enzymes in rat liver microsome were studied. Parameters investigated were: microsomal protein, NADPH-cytochrome P-450 reductase activity, NADH-cytochrome b5 reductase activity, cytochrome b5 content, cytochrome P-450 content, aminopyrine N-demethylase activity, aniline p-hydroxylase activity, p-nitroanisole O-demethylase activity, uridine diphosphate (UDP)-glucuronyl transferase activity and glutathione S-transferase activity. Nearly all the parameters based on microsomal protein decreased during autolysis and the time-dependent decrement ratios of the parameters changed by various amounts. Cytochrome b5 content decreased more rapidly than that of other components. By 36 h post mortem, levels of cytochrome b5 were not detectable. By 48 h post mortem, NADPH-cytochrome P-450 reductase activity decreased to 91%, NADH-cytochrome b5 reductase activity decreased to 94%, and cytochrome P-450 content decreased to 92% of relative activities. By 48 h post mortem, aminopyrine N-demethylase activity decreased to 87%, aniline p-hydroxylase activity decreased to 98% and p-nitroanisole O-demethylase activity decreased to 75% of relative activities. The activity of p-nitroanisole O-demethylase appeared to be more stable than that of aminopyrine N-demethylase or aniline p-hydroxylase. These results demonstrate that there are multiple forms of isozymes of the cytochrome P-450-linked monooxygenase system. Hepatic transferases showed decrease patterns different to those of monooxygenases, so UDP-glucuronyl transferase activity of approximately 32% of relative activity was detected at 48 h post mortem. Thus, UDP-glucuronyl transferase activity appeared to be more stable than the cytochrome P-450-linked monooxygenases. These results show that these activities and components would be useful as markers of postmortem time. The causes of the variety of instability of these enzyme systems are discussed.

Myocardial lesions induced after trauma and treatment.

In order to clarify the effect of trauma and treatment as stresses on myocardia, we examined histological changes of myocardia in victims who received various kinds of traumata and treatments. We also undertook a histochemical study for calmodulin, which we found useful in the diagnosis of early ischemia. Those who died shortly after stab wounds, traffic accident or head injuries, showed mild cardiac lesions such as contraction bands or fragmentation and mild diffusion of calmodulin, a marker for necrosis. A case with hemorrhagic shock after a traffic accident, involving intense resuscitation for 2 h, showed severe cardiac lesions such as contraction bands, hydropic change and subendocardial hemorrhage along with severe diffusion of calmodulin. In most of the instant death cases after falls, severe contraction band necrosis and severe calmodulin diffusion were observed. Myocardia of victims, who died several days after head injuries or traffic accidents, generally demonstrated distinct diffusion of calmodulin as compared to the mild and non-specific lesions detected by hematoxylin-eosin (H&E) staining. In cases of long-term survival in a state of brain death, calmodulin staining was very low, which was not always associated with the severity of the lesions on H&E staining. In cases with intensive or extended treatment, it appeared to be difficult to determine the cause-effect relationship between trauma and cardiac lesions or to distinguish the lesions due to extrinsic factors from those of disease. In some cases, calmodulin intensely stained the areas with hydropic appearance or hypereosinophilia, which may be related to calcium overload.

Forensic analysis of 10 barbiturates in human biological samples using a new reversed-phase chromatographic column packed with 2-micrometre porous microspherical silica-gel.

A high-performance liquid chromatographic method has been developed for the forensic analysis of 10 frequently used barbiturates (BARs) (allobarbital, amobarbital, barbital, cyclobarbital, hexobarbital, metharbital, pentobarbital, phenobarbital, secobarbital and thiopental) using a recently developed reversed-phase column packed with 2-micron particles. The results show that the new ODS column packing gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

Cardiac lesions and their reversibility after long term administration of methamphetamine.

In order to clarify the effect of methamphetamine (MA) on myocardium, histological, immunohistochemical and electron microscopic changes in the myocardium of rats were examined following daily intraperitoneal administration of MA at a dose of 1 mg per kg body weight for 4, 8, and 12 weeks before sacrifice. Normal saline (NS) was similarly injected for the same period before sacrifice to constitute a control group. Light microscopic changes found in the myocardium of the MA-treated group included atrophy, hypertrophy, patchy cellular infiltration, eosinophilic degeneration and disarray, edema myolysis, fibrosis, and the appearance of vacuoles. Ultrastructurally, nuclei and normal mitochondria had various shapes and there were dilated T tubules and sarcoplasmic reticulum, the accumulation of glycogen granules and fat droplets. Intra- and extra-cellular edema and intramyocytic vacuoles were often found. Withdrawal of MA at the twelfth week in another group of rats evidenced gradual recovery of the myocardial changes, commencing at 3 weeks after withdrawal. Optimism is therefore generated about the possibility of the affected hearts in MA-abuse patients returning towards the normal state if they give up the drug.

Determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) in human serum by wide-bore capillary gas chromatography with nitrogen-phosphorus detection.

A sensitive method for simultaneous determination of ester-type local anesthetic drugs (procaine, tetracaine, and T-caine) has been developed using wide-bore capillary gas chromatography with nitrogen-phosphorus detection (GC-NPD). The extraction procedure, the experimental conditions for heptafluorobutyryl (HFB) derivative formation, and the percentage of the ester-type local anesthetic drugs from the human serum are described. The HFB derivatives of ester-type local anesthetic drugs showed sensitivity of approximately 2-3 fold higher than that without derivatization. The detection limits of HFB derivatives of the ester-type local anesthetic drugs were approximately 60-70 pg on column. Recoveries from the human serum were 85-94%. This method could be used to determine concentrations as low as 24-28 ng/mliters of the ester-type local anesthetic drugs.

An electrocution death of an infant who had received an electric shock from an uncovered oval shaped lamp switch in his mouth while in a hospital.

A male infant aged one year and nine months was found dead on a bed after admission to hospital with suspected pneumonia. The patient apparently put an uncovered oval shaped lamp switch (pendant switch) into his mouth and died of electric shock after contacting the exposed wires of the switch (100 V, 60 Hz alternating current). There were extensive first- to fourth-degree burns on the inner surface of the both lips. Because the histological findings were consistent with electric burns and the burns showed vital reactions, electric shock was judged to be the cause of death. The pendant switch is normally a very convenient piece of bedside equipment for inpatients. However, when the patient is an infant who naturally puts all the objects into the mouth, such a switch should be placed out of reach, and it should be certain that the cap is not loose.

Development and application of immunoassay for paraquat: radioimmunoassay.

A sensitive radioimmunoassay for paraquat is reported. Anti-paraquat antisera were produced by repeated immunization in rabbits with 1-methyl, 1'-hexanoic acid-4,4'-bipyridinium (MHBP) coupled to bovine serum albumin (BSA). Less than 0.5 ng of paraquat dichloride was detectable by this assay system. These antisera were strongly cross-reactive with the bipyridyl ring and methyl group in either the 1- or 1'-position of paraquat. The determination of paraquat in tissues of paraquat-poisoned cadavers was also carried out.

Detection of D1S80 (pMCT118) locus polymorphism using semi-nested polymerase chain reaction in skeletal remains.

We evaluated the usefulness of a semi-nested polymerase chain reaction (PCR) method for detecting D1S80 (pMCT118) locus polymorphisms of DNA extracted from old skeletal remains. The semi-nested PCR has been applied to the amplification of D1S80 nucleic acid sequences. For amplification of the locus D1S80, a pair of oligonucleotide primers have been used widely as described by Kasai et al. We have designed another set of primers for semi-nested PCR. This method resulted in D1S80-VNTR detection from low-titered DNA isolated from old skeletal remains. The first and second step PCR achieved amplification from as little as 10 ng and 10 pg of template DNA, respectively. Specificity and sensitivity of the amplification products was markedly improved by semi-nested PCR. In DNA extracted from biological samples, this method took about 5 hours to amplify the target DNA and 3 hours for electrophoretic separation. We demonstrated that this semi-nested PCR method was superior in sensitivity to conventional 1-step standard amplification for VNTR typing of the D1S80 locus.

Rapid and clear detection of ABO genotypes by simultaneous PCR-RFLP method.

We reported a new approach of ABO genotyping by a polymerase chain reaction and restriction fragment length polymorphism method. Instead of amplifying the loci containing the positions of nucleotides 258 and 700 of cDNA of the A transferase separately, we successfully amplified these 2 loci together in one reaction mixture using 2 sets of primers. The amplified DNA products were digested at the same time with restriction enzymes Kpn I and Alu I. The digested DNA products were then separated by electrophoresis on polyacrylamide gel. In addition, we evaluated the influence of various amplification parameters (concentration of template DNA, primers, Taq DNA polymerase, MgCl2, and number of cycles). In particular, high Mg2+ concentration (3.5 mM) made effective amplification of this locus without producing any unspecific band. By using that optimized condition for PCR, together with a simultaneous approach, our study proved to be time saving, more economic, and convenient in interpreting the results.

Mixed cavernous angioma and glioma (angioglioma) in the hypothalamus--case report.

A 54-year-old female presented with a huge mixed cavernous angioma and astrocytoma in the hypothalamus manifesting as headache, visual field defect, gait disturbance, and convulsion. Radiological studies revealed a huge suprasellar tumor encasing all the major cerebral vessels. Craniotomy disclosed a hemorrhagic tumor poorly demarcated from the surrounding brain which was partially removed. Histological examination of the operative specimen revealed cavernous angioma with low grade glioma in the periphery. The residual tumor responded to radiation therapy remarkably well. An autopsy conducted 3 years later revealed a small hypothalamic astrocytoma with abundant vasculature.

Sarcomatoid renal cell carcinoma. An immunohistochemical and ultrastructural study.

A case of sarcomatoid renal cell carcinoma is reported with light, immunohistochemical and electron microscopic findings. The tumour consisted of typical clear cells of renal cell carcinoma and spindle cells compatible with malignant fibrous histiocytoma (MFH). Although an epithelial membrane antigen was demonstrated in the clear cells, this was not detected in the MFH-like spindle cells. In contrast, the spindle cells expressed vimentin that was not identified in the clear cells. Electron microscopy reveal epithelial features in the spindle cells. Comparisons were made with previous sarcomatoid renal cell carcinoma in the literature. Thus, ultrastructural study was vital in diagnosis of sarcomatoid renal cell carcinoma.

[Medical education reviewed by legal consultation of civil affairs].

I pointed out the following medical problems based on my own research and investigations of civil proceedings relating to accidents. 1. Pay little attention to the circumstances of the accident 2. Too much emphasis on laboratory investigations for medical diagnoses 3. Unsuitable medication or medical examination 4. Money-making medical care and treatment 5. Poor mental care 6. Unsuitable or incorrect descriptions of the medical conditions of the subject 7. Lack of moral sense 8. Lack of ability to guide the co-medical staff Pre and post graduate medical education must be improved and as forensic pathologists we have to participate in finding a solution for these problems. Now is the time to reform the medical system and the cost of medical examination, treatment and consultation.

Sudden death case of single coronary artery with special reference to the effect of ischemia on actomyosin.

A normal single left coronary artery was found in a high school boy who died suddenly after Rugby football practice. He had an infarct lesion in the posterior wall where there was no perfusion because of the absence of the right coronary artery. There was no significant lesion in the anterior wall except for contraction band necrosis. Myoglobin diffusion paralleled the extent of necrosis. The case was useful in evaluating the effects of ischemia on a human subject because variations due to personality and postmortem changes were not involved. Actomyosin ATPase activity was not different among the various infarct and non-infarct regions examined except in one infarct region where it was higher.

[Two autopsy cases of sudden death from dilated cardiomyopathy: calmodulin staining as an index of myocardial injury].

Thirty-four years old man and fourty-one years old man were suddenly died after exercise and drinking, respectively. Autopsy and microscopic examinations showed severe cardiac dilatation and fibrosis in both cases. Myocardial degeneration was observed only in the former case. Decreased (diffusion) or increased staining for calmodulin was observed in association with contraction band necrosis. The decrease and increase in calmodulin staining is thought to represent necrosis and calcium-overload, respectively. Both cases were diagnosed to have died from dilated cardiomyopathy without particular histories and causes.

Sex identification of old skeletal remains by DNA analysis: the report of three cases.

We analyzed the DNA in the remains of three skeletons affected by severe post-mortem modification to identify the sex of the cases. We analyzed DNA extracted from bone, bone marrow and a tooth by Southern blot hybridization with Y chromosome specific probe and PCR to amplify the sex chromosome specific sequence. The first case had been preserved underground in a deep grave for two months, the second case was found in a forest after more than five years, and the last case had been kept in a styrofoam box for few years. The detective sensitivity of Southern hybridization improved to nearly the same level as PCR with the aid of computed image processing. Although PCR was of obvious benefit when analyzing small amounts of biological evidence, amplification was impossible in case the target sequence became lost or changed. As a result, we identify the sexes of the three cases by Southern blot hybridization or PCR using bone, bone marrow and tooth DNA. The old skeletal remains were suitable for sex identification in spite of severe post-mortem change, due to preservation in a condition protected from external influences.

[Sudden death of alcohol withdrawal syndrome--report of a case].

A 50-year-old driver was arrested in a drunken stupor while he was driving a track. He suddenly died after five days of the arrest in a jail. As time went by, various symptoms of alcohol withdrawal appeared. He was in the state of delirium treatments for about a day before he died. Gross and microscopical examination revealed fibrosis and fatty degeneration of liver and heart lesions representing chronic and acute ischemia. We discussed the cause and the mechanism of the death in the case and reviewed those in the previous observations.