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To save time and simplify the matching genetic information of bereaved family with the collected samples for identification of individuals in a mass-scale disaster and terrorism using molecular biological methods, we propose a method to initially screen samples according to different populations. We found a unique deletion (26822-3) in an allele that 62.0% of East Asian individuals had in intron 33 of type I collagen alpha 2 (COL1A2), while most Europeans and Africans did not have it. By making a heteroduplex complex, a visual discrimination was possible using 10% PAGE. A PCR and heteroduplex analysis could identify genetic polymorphism of COL1A2 intron 33 both clearly and accurately, even from decomposed samples or very small amounts of samples within a very short time period (approximately within 150min). As a result, genetic polymorphism of COL1A2 intron 33 is thus considered to be useful to quickly and easily screen East Asians from vast number of samples using a heteroduplex analysis. Consequently, we consider that an initial screening method for East Asians which use a heteroduplex analysis of genetic polymorphism of COL1A2 intron 33 can smoothly and effectively perform various identifications among East Asians when supplies are logistically poor after a mass-scale disaster or terrorism.

The lipid peroxide 4-hydroxynonenal (HNE) was measured in rat intestinal mucosa after lipopolysaccharide (LPS) injection (0.5 mg/kg, ip) by a highly sensitive time-resolved fluoroimmunoassay. HNE was increased, with a small peak at 20 min followed by a sustained elevation at 2–4 h, after injection of LPS. Immunohistochemistry demonstrated enhanced labeling with anti-IgA and anti-HNE antibodies in the plasma cells followed by diffusion of the labeled materials into the submucosal tissue after LPS injection. Immunoprecipitation with anti-IgA antibody and Western blotting with anti-HNE antibody showed that IgA is modified with HNE after LPS injection. The HNE (5 μM–5 mM) modification in vitro reduced the bactericidal activity of IgA and anti-Escherichia coli serum. The HNE modification in vitro also promoted polymerization of IgA as shown by nondenaturing gel electrophoresis. This is the first demonstration of the modification of IgA with HNE in an in vivo model of intestinal inflammation as well as in vitro effects of HNE on bactericidal activity and polymerization of IgA. These findings will help in understanding the involvement of oxidative stress in the IgA-mediated immune response exerted by plasma cells in early intestinal inflammation.

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.

A new method to measure two different drugs simultaneously by time-resolved fluoroimmunoassay (TR-FIA) has been developed. In the TR-FIA reported here, psychopharmaceuticals [chlorpromazine (CPZ) and desipramine (DSP)] and methamphetamine (MA) contained in serum are assayed by a combined use of a new europium (Eu) chelate and a samarium (Sm) chelate, as labels. The drug concentrations were determined by the competition between a labeled antigen with Eu(3+) or biotin and a sample antigen. A microtiter plate coated with a mixture of rabbit IgGs (anti-MA and anti-CPZ or anti-MA and anti-DSP) was used. In the assay of MA and CPZ, Eu(3+) labeled MA-bovine serum albumin conjugate (MA-BSA) and biotinylated CPZ-BSA were added to the well with their non-labeled standard solutions or samples. MA was assayed by measuring the fluorescence intensity of Eu(3+) at 615 nm. After incubation of the Sm(3+) labeled streptavidin, CPZ was assayed by measuring the fluorescence of Sm(3+) at 643 nm. In the assay of MA and DSP, Eu(3+) labeled DSP-BSA and biotinylated MA-BSA were used. In our dual-assay, the minimum detection limits of these drugs were 1ng/ml for MA, 10 ng/ml for CZP and 10 ng/ml for DSP. Since the simultaneous detection of different drugs by TR-FIA is time and sample saving, the method can be employed in rapid and sensitive screening tests.

To analyze serum proteins adsorbed to a hemodialysis membrane, immunostaining with colloidal gold and thermal-lens microscopy were employed. A total of 14 types of hemodialysis membranes were tested. The cross sections of the membranes were stained with fluorescent antibodies. Observations of these specimens using a laser microscope revealed that serum proteins, such as albumin and immuno globulins, tended to adsorb into the inner wall of these membranes. The degree of adsorption of serum proteins varied according to various conditions, such as what materials the membranes were made of or what proteins were adsorbed. For a better quantitative analysis of these proteins, the membranes were stained with immunogold and were observed by a thermal lens microscope. Colloidal gold coated with various anti-human serum proteins was used for the staining, and the quantity of colloidal gold was measured with a thermal-lens microscope. The quantity of colloidal gold, coated with anti-human sera antibody, gradiently decreased from the inner to the outer wall of the membrane. Probably, proteins with larger molecules were adsorbed to the inner parts, and those with smaller molecules were adsorbed to the outer parts of the membrane wall. High-resolution thermal-lens microscopy makes possible the sensitive detection of localized proteins adsorbed to a membrane without extracting it. Thus, the analysis of adsorbed proteins should serve to determine the adaptability of medical appliances to the human body and should also be helpful to a retrospective analysis of substances like drugs which are conjugated to the serum proteins.

The morphology of erythroid cells changes dramatically during the course of their terminal differentiation. According to calculations made with cytospin preparations obtained from Syrian hamster yolk-sac-derived erythroid cells, the area of nuclei at day 10 of gestation ranges from 25 to 85 micron 2 and is reduced to 15-25 micron 2 on day 13 [K. Morioka and R. Minamikawa-Tachino, Dev. Growth Differ. 35, 569-582, 1993]. The DNA and protein contents of each nucleus also decrease during this period. Nonspecific fragmentation of DNA was detected by agarose gel electrophoresis in all samples obtained from day 10 to day 13 of gestation, while distinct ladders of DNA fragments were not detected. DNA fragmentation was also detected by an in situ DNA-end labeling (TUNEL) assay. As the terminal differentiation proceeded, gradual decreases in levels of both histone H1 and most nonhistone proteins were observed by SDS-polyacrylamide gel electrophoresis, while levels of core histones appeared to be constant. In particular, lamin B2 was almost completely lost from the nuclear matrix fraction on day 11. These results suggest that the terminal differentiation of erythroid cells and apoptosis might have common mechanisms. However, expansion of the cytoplasm during the terminal differentiation distinguishes these processes. In addition, in the erythroid terminal differentiation, nuclei never form lobules or become fragmented; no apoptotic bodies are formed, occurrence of the apoptosis-like cellular change is not sporadic but rather synchronous, and the process is slow, with at least several days being required for cell death. These characteristics are different from those of typical apoptosis. Thus, the terminal differentiation of nucleated embryonic erythroid cells exhibits both apoptotic and nonapoptotic features.

This case is one in which a baby was found dead, apparently drowned in a bathtub. To confirm the inhalation of bathwater, an extract taken from the baby's lungs was analyzed with high-performance liquid chromatography. Bathsalts contained in the bathwater were detected in the extract. Bathsalts are usually used in home bathtubs and, as a dye material, they contain fluorescein which is highly sensitive to detection. The presence of fluorescein in the lung tissue and in the other tissues greatly helps to confirm the bathwater drowning.

The quantitation of drug concentration in a cell using a new microscopic method, named laser-induced photo-thermal microscopy, was demonstrated. We used a paraformaldehyde-glutaraldehyde- fixed guinea pig′s kidney to which we administered tobramycin and assayed the quantity of colloidal gold immunologically bound to the tobramycin in the epithelial cell of one renal tubule. We could detect the locally concentrated tobramycin in the epithelial cell, as well as its local distribution quantitatively. The detection limit of the tobramycin concentration (10zmol ) was 10-times superior to that of confocal scanning laser microscopy. The colloidal gold which we used for immunochemical staining remained stable to laser-beam radiation and gave a low background. Therefore, it makes possible highly sensitive detection of a drug in fixed organs.

Caenorhabditis elegansUNC-18 protein, homologous to yeast Sec1p, is important in neurotransmitter release, because theunc-18mutation leads to severe paralysis and presynaptic acetylcholine (ACh) accumulation. To examine the functional conservation in mammals, we tried to isolateunc-18isoforms from mouse and human brain cDNA libraries and obtained two classes of isoforms—neural genes and ubiquitous genes. Neural genes were identical to Munc-18 (also known as n-Sec1 or rbSec1), identified in rat and bovine brains as a syntaxin-binding protein. According to “Munc-18” terminology, we call the neural genes Munc-18-1 and the ubiquitous genes Munc-18-3. These mammalian isoforms exhibit 58% (Munc-18-1) and 42–43% (Munc-18-3) amino acid sequence identity with UNC-18. Next, we constructed transgenicunc-18mutants to test biological activity of mouse Munc-18-1 and Munc-18-3 under the control ofC. elegans unc-18promoter. Munc-18-1 compensates for severe locomotion disability and cholinergic defects, e.g., abnormal sensitivities to cholinesterase inhibitors and cholinergic receptor agonists inunc-18mutants, but Munc-18-3 fails. These data suggest that Munc-18-1 andC. elegans unc-18may play positive roles in ACh release and that the molecular mechanism of neuronal regulated secretion has been partially conserved from nematodes to mammals.

Yolk-sac derived erythroblasts undergo semi-synchronous maturation and some of them enucleate in the peripheral blood of embryos. We have studied the assembly and distribution of actin, myosin, and spectrin during the enucleation of Syrian hamster embryonic erythroblasts. At day 11 of the gestation, that is just before the start of the enucleation, formation of a cytoskeletal structure consisted chiefly of particulate associations of F(filamentous)-actin was detected by the staining with rhodamine-labeled phalloidin. Stress-fiber-like structures were not observed in each differentiation stage after day 10. Distribution of myosin, actin, and spectrin was studied immunocytochemically to know the role of them in the enucleation of erythroid cells that starts at late day 11 or early day 12 in the gestation. The enucleation is preceded by the approach and the subsequent attachment of nucleus to the plasma membrane. At that time, actin and myosin are present in the cytoplasmic and cortical region of the cells. From the time when the extrusion of nucleus has started, condensation of actin and myosin was observed at the cell cortex area surrounding the extruding nucleus, and a contractile ring-like structure was infrequently observed. Spectrin was observed in the cortical region of the cells, and the change of the localization of spectrin was not observed throughout the terminal differentiation process (days 10-12) of the embryonic erythroid cells. The results show the possible involvement of a myosin-actin contractile system that appears around the extruding nucleus within the mechanism of erythroid enucleation.

The purpose of this study is to elucidate the effect of wheel training on oxidative stress maker levels in spontaneous hypertensive rats (SHR). 4-hydroxynonenal and 3-nitrotyrosine levels in the aorta of SHRs were allowed to run for 10 weeks from the age of 15 weeks were measured and compared with those of nonexercised SHRs. The 4-hydroxynonenal and 3-nitrotyrosine levels in the exercised group were significantly lower than those in the nonexercised group. The exercised group showed a significant increase of manganese-containing superoxide dismutase. Endurance exercise showed a possible suppressing effect on the arteriosclerosis development by reducing oxidative stress, even after emergence of hypertension.

Methamphetamine activates catecholaminergic neurons and releases noradrenalin from their nerve endings in the central nervous system. Many reports have described the distribution of methamphetamine in the central nervous system after systemic administration. However, there have been only a few studies on the effect of this drug on peripheral organs, and known dealing with the inner ear.The authors used immunohistochemical methods to investigate the effect of methamphetamine on the pheripheral auditory system. Methamphetamine was administered to guinea pigs via intraperitoneal injection. The distribution of methamphetamine in the cochlea was studied by light microscopy following its immunohistochemical staining, and the methamphetamine concentration in the serum was also measured.1. Histochemical examination revealed that methamphetamine was present in the hair cells of the organ of Corti, spiral ligament and spiral ganglion cells 30 minutes after administration.2. The drug concentration in the serum rose rapidly, and then dropped in a logarithmic way with time until it became nearly horinzontal after 6 hours.The results suggest that methamphetamine penetrated the blood-labyrinth barrier (BLB) and then reached the organ of Corti, although some methamphetamine took a route to the inner ear via the cerebrospinal fluid. The pharmacological effect of methamphetamine on the cochlea was excitation of the sensory cells, the afferent nerve fibers connecting to them, and the spiral ganglion cells.

モルモット1匹あたり10mg/kgのメタンフェタミンを腹腔内注射し, 同剤が聴覚系に及ぼす影響をプライエル反射域値測定器やABRを用いて測定した。 この結果, プライエル反射域値は薬剤投与後30分, 60分で有意に低下した。 一方, ABRについては, 薬剤投与後ABRのI, II, IV波潜時は240分まで, III波潜時は180分までに有意に短縮し, 波間潜時についてもほぼ同様の傾向を示した。 しかし, 振幅や域値については, 薬剤投与前後で変化がみられなかった。 以上より, メタンフェタミンは聴覚伝導路全搬において, 潜時を短縮させる作用をもつことが判明した。

The priorities for mass disaster victim identification are rapid investigation turn-around time and low cost. We describe a DNA typing procedure to selectively identify members of Asian populations by a real-time PCR method using polymorphisms of the α2 chain of the type I collagen gene ( COL1A2 ) and mitochondrial DNA (mtDNA). Among the 50 members of the Asian population included in the present study, 37 harbored a deleted allele in intron 33 of COL1A2 (26822–26823del) or the 10400C>T substitution mutation in mtDNA to give a probability of 0.740 for these SNPs in the Asian population.

The analysis of single nucleotide polymorphisms (SNPs) together with conventional short tandem repeat (STR) and mitochondrial DNA (mtDNA) typing provide a forensic genetic approach for the identification of pathological and autoptical specimens in cases where the average length of DNA fragments is shorter than 150 bp in highly degraded samples. We applied a forensic genetic approach to digesta accidentally left after a training autopsy. PCR products were not amplified from samples containing the STR loci or common sequences used for mtDNA typing. The application of SNPs and deletion polymorphisms provides an alternative approach for DNA typing analysis.

A total of 20 autopsy cases involving deaths due to penetrating cardiac injuries were analyzed pathologically: the causes of death of the victims who died before cardiorrhaphy were exsanguination or hemorrhagic shock in nine cases, pericardial tamponade in three, and a combination of the two in another three. Three of the remaining five cases of early postoperative death resulted from hemorrhagic shock, one resulted from pulmonary air embolism, and one resulted from a combination of hemorrhagic shock and coronary platelet embolism. We should pay more attention to air embolism and platelet embolism as causes of death of victims who have had penetrating cardiac injuries, whose exsanguinating hemorrhage and/or pericardial tamponade may be controlled by emergency-room thoracotomy and cardiorrhaphy. Language: en

We report a medico-legal autopsy of a 4-year-old girl without any history of allergies, who died following anaphylactic shock due to local anesthesia with lidocaine during dental treatment. The shock symptoms were overlooked due to a rubber-dam-sheet covering her face. The autopsy revealed laryngeal edema, secretory fluid filling the trachea, severe pulmonary congestion and edema. An immunohistochemical investigation showed numerous mast cells releasing histamine in lungs, and the fluids in the pulmonary alveoli and veins also showed positive staining. In addition, the plasma concentration of histamine in the heart blood showed a high value. From these findings, the cause of death was determined to be respiratory failure due to anaphylactic shock induced by lidocaine. In this case, her death was considered to be due to medical malpractice; adequate life support had not been performed, because anaphylactic shock was overlooked until the rubber-dam-sheet removed. In addition, due to two previous autopsy case reports that described infantile fatalities involved with the misusage of a rubber-dam-sheet for the patients under local anesthesia, we therefore recommend immediate improvements in rubber-dam-sheets and/or better applications of them in pediatric dentistry.

A rapid and simple homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium (Eu) to cyanine dye (Cy5) has been developed for the quantitation of methamphetamine. In this assay, Eu chelate was labeled to a conjugate of methamphetamine and bovine serum albumin (MA-BSA), as an energy donor, and Cy5 was labeled to anti-MA as an energy acceptor. The close proximity between the two labels in the immunocomplex permits energy transfer from the excited Eu(3+) donor. Therefore, by measuring the sensitized emission of Cy5 with the time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 min. By a competitive immunoassay, MA could be assayed in the range 0.1-1,000 ng/mL. The intra-assay variations were 5.4-14.8% at 5 different concentrations. When urine or serum samples were examined, the quenching of Eu fluorescence was observed, but the acceptor-to-donor ratio constantly depended upon the dilution of samples. Twenty urine samples were assayed, and the data showed a good correlation to those obtained by gas chromatography (r = 0.94). The homogeneous assay using Eu-Cy5 energy transfer is time-saving without any washing procedures and is suitable for screening drugs that are commonly abused.

Chymase, a serine protease, is stored mainly in secretory granules of human mast cells. Serum chymase concentration was examined in 8 autopsy cases with anaphylaxis as well as in 104 control cases without anaphylaxis. It was detected in all 8 cases with anaphylaxis (range 3-380 ng/ml, mean 89.8 ng/ml), while it was detected in only 2 of the 104 controls and was below a detectable level (

The influence of neonatal deafness on cochlear electrically evoked Fos expression in the auditory brainstem was examined. Newborn rats were deafened by systemic injection of kanamycin, 1 mg/g daily for 12 days. At 4, 5, 6 or 8 weeks of age, these animals received cochlear electrical stimulation with a basal monopolar electrode for 90 minutes. Age-matched untreated control animals received similar stimulation. Experimental and control animals were assessed for spiral ganglion cell densities and Fos immunoreactive staining in the central nucleus of the inferior colliculus. Spiral ganglion cell assessments showed significant decreases in spiral ganglion cell densities in deafened rats compared to age-matched controls, at 5 weeks of age in lower turns and 6 and 8 weeks in all turns. Cochlear electrical stimulation induced Fos immunoreactive staining in the nucleus of auditory brain stem neurons in treatment and control groups. A significantly greater number of Fos immunoreactive neurons was found in the contralateral central nucleus of inferior colliculus in 5, 6 and 8 week old deafened animals compared to age-matched controls. The increases were larger with a longer duration of deafness. These results suggest that there are changes in auditory processing as a consequence of neonatal deafness.

Quantitation of drug concentration in a cell: Laser-induced photo-thermal microscopy and laser microscope (LSM) were used for quantitation of drug concentration in a cell of the fixed tissue sections. The colloidal gold labeled with antibodies was used as an indicator for detection of drugs or other antigens. The detection limit (10 zmol) of the tobramycin distributed in a epithelial cell of one renal tubule was 10-times superior to that of scanning laser microscope. The colloidal gold remained stable to laser-beam radiation and gave a low background. Therefore, the use of colloidal gold and laser microscopy make possible highly sensitive detection of drug in the fixed tissues. Highly sensitive quantitation of drug: A simple and highly sensitive time-resolved fluoroimmumoassay using a fluorescent europium chelate (4, 4'-bis (1", 1", 1", 2", 2", 3", 3"-heptafluoro-4", 6"-hexanedion-6"-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu+3) was used for analysis of methamphetamine (MA). Two kinds of assay methods (one step assay and two step assay) using a microtiter plate coated with anti-MA were employed. The smallest measurable amounts of MA for the one-step and the two-step methods were 25 pg/assay and 25 fg/assay, respectively. Molecular biological approach to the forensic problem: a. The diagnosis of the death caused by drowning was possible by detection of the DNA fragments of aquatic plants from the organ of victims. The detection of the DNA fragments of chloroplast gene, ribose-bisphosphate carboxylase large subunit (rbcL), was performed using PCR method with the newly synthesized probes. b. Taq-Man probes were useful to perform the personal identification of many samples. Mitochondrial DNA analysis was also brought about good results for putrefactive samples. So, the combination of some examine methods was need for personal identification of multiple samples. DGGE method was also very useful and easy for multiple gene analysis for personal identification with SNPs.

The denaturant gradient gel electrophoresis (DGGE) method was used in order to simultaneously estimate the genotypes of different factors in a gel plate consisting of one sheet. A genotype analysis of the blood groups (MN, Duffy, Kidd type) and serotype (Gc system) was carried out. DNA samples were extracted from trace deposits which were transferred on adhesive film from a blood trace obtained from a car tire after a fatal car accident. The reference DNA was prepared from the victim's blood. The PCR amplification fragments were amplified from the gene which controlled each blood group. The primers were designed in order to analyze the genotypes with one to three base substitutions in the amplification product. The denaturant concentration limit of the gel for the DGGE method to detect each genotype of the blood groups (MN, Duffy, Kidd type and Gc system) and other conditions of electrophoresis were performed according to previously methods. The each genotype of the blood groups and the Gc system were all simultaneously distinguished in one plate.

An automatic and rapid DNA typing system was employed for personal identification, using fragmentary tissue samples from victims in an airplane accident. Two victims were crushed into small pieces, and 33 samples suspected to belong to them were recovered from under the sea. From each sample, 10 mg was used for testing. The parents' bloods of two presumptive victims were also examined. DNA extraction from samples was performed by the NaI method, and the obtained DNA samples were analyzed with the ABI PRISM system. Among 33 samples, 31 samples were identified to be human tissues, possibly from two victims. The other two samples seemed to be parts of marine animals. ABO blood group, STR polymorphism, and mitochondrial DNA polymorphism typing were possible in every examined human sample. Two victims' fragmentary tissues were identified by determining ABO genotype, STR type and mitochondrial DNA type. The system we employed enabled an accurate typing of many fragmentary samples in a short time, thus contributing to the fast and secure identification of many victims in such cases as big air accidents.

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Objective. To investigate the effects of the interleukin-6 (IL-6) family cytokines, such as IL-6, IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM), on the production of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) in human articular cartilage. Methods. Effects of IL-6 family cytokines and growth factors on TIMP-1 production were studied in human articular chondrocytes, grown as monolayer and cartilage explant culture. TIMP-1 protein levels in the cultured medium were measured by sandwich enzyme immunoassay. TIMP-1 messenger RNA levels in the cultured chondrocytes were analyzed by Northern blotting. Western blot analysis was performed to evaluate the release of matrix metalloproteinases (MMPs) in the cultured medium. Cell proliferation of chondrocytes was determined by 3H-thymidine uptake. Results. IL-6 family cytokines induced TIMP-1 expression in monolayer and explant culture, and the production of TIMP-1 was most stimulated by OSM. In contrast, OSM did not modulate the release of MMPs and cell proliferation. Conclusion. These results suggest that OSM may be characterized as one of the chondroprotective mediators in cartilage destruction.

The topographic localization of methamphetamine and phenobarbital in the rat retina was studied. It is interesting that the secondary and primary ganglionic cells were stained predominantly only in the case of methamphetamine while in phenobarbital these were not stained. The staining patterns of the inner plexiform layer (strong reaction in phenobarbital and weak reaction in methamphetamine), outer nuclear layer (weak reaction in phenobarbital and strong reaction in methamphetamine) and choroid (very strong reaction in phenobarbital and no reaction in methamphetamine) also showed some differences.

The myoglobin content of chicken myocardial cells was studied using indirect-immunoperoxidase histochemistry. While ordinary myocardial cells exhibited a homogeneous reaction pattern, the reactions for ventricular Purkinje fibers were remarkably heterogeneous. On the basis of the degree of staining, three types of cells, i.e., dark, intermediate, and clear, were distinguishable. In addition, the cytological heterogeneity of Purkinje cells was confirmed using conventional and immunological electron microscopy. The dark cells contained more myofibrils, mitochondria, and other organelles (e.g., ribosomes) than the clear cells.

Investigation of genomic polymorphisms detected by a minisatellite named tentatively "Myo", which is expected to correspond to the minisatellite in human myoglobin gene of Jeffreys et al., gives distinct and different aspects of chorionic villus and the decidual membrane in the same placenta. The chorionic villus, which is regarded as the extraembryonal tissue, represents the essential embryonal DNA fingerprint pattern, while the decidual membrane reveals the maternal one. A comparison between the DNA fingerprints from the chorion villus and from the blood sample of the suspected father provides the possibility of setting a paternity determination which can be achieved during the first trimester of a pregnancy.

Six hundred and eight red cell hemolysates were screened for glutamate-pyruvate transaminase (GPT) by means of isoelectric focusing. Two new variant phenotypes were detected, neither of which could be distinguished from GPT2–1 and GPT2 by conventional starch gel electrophoresis. The two types were considered to correspond to GPT2B-1 and GPT2A-2C reported previously in samples of European origin.

Posttraumatic acute cholecystitis is an often unrecognized and potentially fatal complication seen among patients hospitalized for trauma, and differs in etiology from cholecystitis which develops de novo. The cause, although not yet clearly defined, is believed to be related to bile stasis, ischemia, bacterial infection, sepsis, the activation of factor XII, and the Shwarzman reaction. A case is described in which a 53-year-old man with pelvic fractures developed acute acalculous cholecystitis and died of multiple organ failure 3 weeks following cholecystectomy. The histopathological findings are also reported; these are most likely attributed to the Shwarzman reaction or the activation of the factor XII pathways. There has been a tendency to regard posttraumatic acute acalculous cholecystitis as induced by trauma, and calculous as mere coincidence. We believe, however, that it is not calculous but histopathological findings that determine whether acute cholecystitis following trauma was more than coincidence or just mere coincidence. Although progress in clinical care has improved the chances of survival of severely traumatized patients, posttraumatic acute cholecystitis has been increasing in frequency. We cannot be careful enough in judging the relationship of this fatal complication to the initial trauma.

Snail agglutinin (anti-AEc, anti-A1 of cold agglutinin type) from the albumin gland of Euhadra callizona amaliae was purified by affinity chromatography (meconium-A aminoethyl cellulose). Its physicochemicl properties are as follows: S20, w: 5.3S. Isoelectric point: pH 3.6. Molecular weight: 8.9 × 104. Hexose content: 5.1%. The amino acid composition is that of an acidic protein. The agglutination is inhibited by GalNAc, GNAc, raffinose, and melibiose at low concentration. It does not agglutinate B- and O-RDE-treated human red cells. Some blood group A-active substances (secretor A saliva and hog gastric mucin A + H) have low potency to inhibit the agglutination. Anti-ABC has no ability to release the cell-fixed anti-Eorssman haemolysin and the cell-fixed blood group A-decomposing enzyme. The significance of the antigen distribution on the limited area of the erythrocyte and protein surface is also discussed. Precipitin reaction of this agglutinin with various blood group-active substances has shown that anti-AEc is directed to a narrow-range spectrum of determining antigenicity.

A method for the demonstration of the topographical distribution of phenobarbital at the cellular level in various tissues was established. Mice that had been exposed to various doses of phenobarbital by intraperitoneal injection were killed, and their tissues were fixed with 0.1 M phosphate buffer solution (pH 7.4) containing paraformaldehyde and glutaraldehyde. Thereafter, paraffin and frozen sections were made and stained by the indirect immunoperoxidase method using antisera obtained from commercial sources and used for the immunochemical assay of the blood level of phenobarbital in clinical medicine. A specific positive reaction was observed solely in testing the intoxicated tissues, and this reaction was inhibited when phenobarbital was added to the antisera. The minimal sensitivity of the positive reaction, which can be discerned by observing the stained slides macroscopically, was in the range of 10 mg/kg. Thus, the diagnosis of phenobarbital intoxication in the forensic autopsy can be made by immunohistochemistry. A positive reaction was found in various tissue cells, including nerve cells, myelin sheaths, glia cells, hepatocytes, cells of the alveolar and bronchial wall, epithelial cells of the distal part of the renal tubules, and so forth. Endothelial cells of the capillaries in all tissues gave a strong positive reaction. The immunocytochemical electron microscopy of the hepatocytes revealed that the positive reaction in the cytoplasm was located solely in the intraluminal space of the smooth endoplasmic reticulum. These results indicate some interesting aspects of the pharmacokinetics of phenobarbital in vivo. It is expected that the antisera, which are used widely for the assay of the blood concentration of various drugs (phenobarbital, amphetamines, morphine, and so forth), may be regarded as excellent reagents for immunocytochemistry. This clearly indicates that morphological evidence in toxicology, which had so far remained obscure, can be easily obtained by applying these antisera against various drugs.