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The ever-increasing presence of environmental toxicants and their disruptive effect on the reproductive systems of wildlife raises the question of possible damage to the human reproductive system. Using medicolegal data from over 20,000 Japanese men subjected to necropsy from 1948 to 1998, we investigated temporal changes in testis weight to find possible evidence of male reproductive disorders. We also carried out a histological examination of 747 testes collected from 1978 to 1998. Our detailed analyses of the development of testis weight over the past 50 years have revealed four clear phenomena: (i) the age at which testis weight reaches its maximum has decreased; (ii) peak weight showed a general increase until it started to decline in boys born after 1960; (iii) the decline-rate at which testis weight decreases after its peak has greatly accelerated; and (iv) the onset of increasing testis weight of boys has occurred at a progressively younger age. Our quantitative analyses of testis weight indicate the possibility of a subtle reproductive disorder in Japanese men, especially in those born after 1960. Together with the accelerated development and decline in testis weight during the past 50 years, the decline in peak weight might be indicative of a subtle interference of environmental toxicants with male reproductive organogenesis.

Allele frequencies for the eight STR loci Hum-CSF1P0, F13A01, F13B, FES/FPS, LPL, TH01, TPOX and VWA were investigated in Japanese and Chinese populations. No significant deviations from Hardy-Weinberg equilibrium could be found for all loci. In the Japanese population VWA, CSF1PO, TH01, FES/FPS and TPOX were found to be useful for forensic applications and in the Chinese population, VWA, CSF1PO, TH01 and TPOX were found to be useful. Allele distributions were similar between both populations except for FES/FPS.

We report a study of polymorphism for seven short tandem repeat (STR) loci in Japanese and Chinese populations. Among 104 to 134 individuals in the both population samples, eight alleles were revealed for locus PLA2, thirteen for D3S1359, eleven for FGA, eight for D8S315 (kw38), ten for D8S1132, five for CYP19, and seven for D3S2459. They correspondingly constituted 10 to 39 genotypes therein. For most of the STRs, there was only a single allele active as the most frequent one among the others, except locus D3S1359 in Chinese samples (two alleles, 206 bp and 210 bp, frequency = 0.273 each). Also, the population genotype configurations were locus specific, varying in the patterns of commonest genotypes on each locus, e.g., one pattern for loci CYP19, D3S1359, and D8S315, one and two for loci PLA2 and D3S2459, two for locus D8S1132, and one and four for locus FGA. The distributions of observed genotypes were in Hardy-Weinberg Equilibrium. Furthermore, the seven STRs were exhibited highly polymorphic and informative for the both populations, and the alleles could be easily separated in electrophoresis and correctly interpreted with side-to-side allelic ladders. Together, the results suggest that the tri- and tetra-meric STRs are useful genetic markers for forensic practice.

The state of DNA methylation in the c-fos gene was examined in human livers of different ages, cirrhosis and hepatocellular carcinoma. The degree of methylation in the intron 1 to exon 4 region increased with age, whereas all of the 10 cirrhosis samples revealed a decrease in methylation when compared to normal livers of similar ages. The 11 hepatocellular carcinomas showed varied alterations suggesting that the alteration of the c-fos gene methylation is related to aging as well as to early-step of hepatocarcinogenesis.

A mouse monoclonal antibody against an amniotic protein carrying ABH antigenic epitopes was established. BALB/c mice were immunized by an amniotic protein of molecular weight over 200 kDa, which had proved to be the carrier protein of ABH blood group epitopes by analysis with SDS-PAGE and immunoblotting. The antibody, ASP-1, was directed to the amniotic carrier protein without affecting the ABH blood group antigenicity, and did not cross-react with other body fluids which included blood, saliva, semen, urine or vaginal secretion. The immunoglobulin class of ASP-1 was IgG1 with a titer of 1 : 1,600. ASP-1 was used to detect the ABH blood group of amniotic fluid by the sandwich ELISA in which wells of plates were coated with ASP-1, and the ABH blood group of the captured protein was detected with mouse IgM anti-A and -B antibodies and enzyme conjugated anti-mouse IgM. The sandwich ELISA could successfully detect the blood group of amniotic fluid in mixed body fluids.

In order to ascertain a generality of the age-dependent decrease in DNA methylation level among different mammalian species, methylated cytosine contents in human liver and spleen DNA at different ages have been determined using high performance liquid chromatography (HPLC). Unexpectedly, the liver DNA revealed no appreciable decline with age while the spleen DNA showed a slight reduction. It indicates that a decrease of methylation level in genomic DNA is not a common denominator of age-related changes in mammals.

To investigate the role of oxygen-derived free radicals in the pathogenesis of tourniquet shock, the authors present an experimental animal model. Two groups of rats were fastened with rubber tubes on both thighs (1.5 kg/cm2) for 6 h under pentobarbital anaesthesia. One group was administered liposomal superoxide dismutase (L-SOD 30,000 U/kg body weight), and the other liposome as a control 3 h prior to tourniquet removal. No rats in the control group (n = 20) survived more than 24 h after reperfusion, whereas 55% of animals treated with L-SOD (n = 20) survived for 24 h or more, and two recovered completely (P less than 0.005). Blood samples were obtained from the abdominal aorta after laparotomy of anaesthetized rats of both groups at different time intervals. Changes in the hematocrit value and blood urea nitrogen during the early periods after reperfusion were attenuated by prior administration of L-SOD, and the total plasma SOD activity of the control animals decreased promptly and continuously throughout the experimental period. This experimental model was very useful to study the pathogenesis of tourniquet shock with respect to reproducibility, induction of the shock stages and mortality. It is thought that oxygen-free radicals are involved in the induction of tourniquet shock, and L-SOD was, to a certain extent, effective against reperfusion injury in the early stages of shock.

Age-associated changes in DNA methylation of the c-myc gene in liver were compared between humans and mice which have large differences in aging rate. Although the overall methylation profiles of the gene and their age-related changes were found to be similar, the rate of change was much slower in humans than in mice. It suggests that the age-associated alteration of c-myc gene methylation is closely related to biological aging rather than to chronological aging.

Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

Four kinds of DNA probes that recognize hypervariable regions (HVR) were studied for parentage testing and individual identification. Allele frequencies and their confidence intervals among unrelated Japanese individuals were obtained. Codominant segregation of the polymorphism was confirmed in family studies. Two a priori probabilities were calculated for each HVR locus: the exclusion probabilities for an alleged father/mother/child trio and for an alleged parent/child duo, and the probabilities of matching of genotypes of two unrelated individuals or two siblings. The ease of availability of the probes and their highly discriminating polymorphic patterns mean they could be very useful for forensic purposes.

Three kinds of variable number of tandem repeat DNA probes (VNTR: pYNZ22, pYNH24, and pYNZ2) showing hypervariable polymorphisms were studied. Allelic frequencies and their confidence intervals among Japanese individuals were obtained. Co-dominant segregation of the polymorphism was confirmed in family studies. Two a priori probabilities were calculated for each VNTR locus: exclusion probabilities for an alleged father/mother/child trio and for an alleged parent/child duo, and probabilities of matching of genotyped two unrelated individuals or two siblings. Availability as well as highly discriminating polymorphic pattern of VNTR loci makes it potentially very useful for forensic and human genetic purposes.

Two PCR amplified loci and 3 single locus DNA probes were applied in a paternity case in which a married woman became pregnant after being raped. DNA analysis were performed using samples from the woman, her husband and amniotic fluid cells taken during the 16th week of pregnancy. The combined probability of paternity for her husband was calculated as 0.999997107. The application of PCR analyses and single locus DNA probes were considered to be extremely informative in prenatal paternity testing.

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.

In an attempt to find a common DNA alteration occurring in human glioma, we examined DNA methylation in 34 gliomas of various pathological grades and compared them with those in normal cerebral subcortex DNA. The total methylated cytosine levels in the genome did not differ appreciably between the tumors and the normal tissues; however, the degree of DNA methylation in several proto-oncogenes and suppressor oncogenes showed some alterations. Among them, the c-fos gene demonstrated deviation from that of normal tissues in all cases examined, suggesting that the alteration of c-fos gene methylation plays a role in the early steps of human glioma development.

A 12-year-old boy with no previous serious medical history experienced abdominal discomfort and chest pains for 5 days and suddenly died. The autopsy revealed diffuse and extensive infiltration of eosinophils into the myocardium, with poorly formed granulomas and few fibrotic changes. The necrotic change was so extensive that Charcot-Leyden crystals formed. The other visceral organs had no specific pathologic changes except for mild lymphocytic infiltration with an increase in goblet cells in the bronchial areas and eosinocytosis in the blood vessels. An initial viral infection. seemed to have caused subsequent eosinophil activation due to an allergic condition. Eosinophilic myocarditis is a rare cause of sudden death in apparently healthy children. Cardiac toxicity of eosinophils is, however, well established and dominates the ultimate prognosis of patients with complicated eosinophilia.

Objective To investigate the mechanism of the formation of a tetragametic chimera with true hermaphroditism (46,XX/46,XY). Design Molecular biologic analyses. Setting Outpatient clinic and laboratories of a university hospital. Subjects A true hermaphrodite with 46,XX/46,XY and the parents. Main Outcome Analysis Restriction fragment length polymorphism (RFLP) of the pseudoautosomal region on sex chromosomes. Results Whereas a normal diploid individual showed two bands, the true hermaphrodite showed four bands in the RFLP analyses. Evaluation of the molecular weights of the bands revealed two of them to be of maternal origin and the other two to be of paternal origin. Conclusion The two cell lineages composing the true hermaphrodite are heterogeneous because those originated from the fertilization of two genetically different maternal haploid cells by two different spermatozoa.

The ever increasing presence of environmental toxicants and their disrupting effects on the reproductive systems of wildlife, raise the question of their possible damaging effects on the human reproductive system. Using medicolegal data from over 20,000 Japanese men subjected to necropsy from 1948 to 1998, we investigated temporal changes of testis-weight to find possible evidence of male reproductive disorders. We also made histological observations of 747 testes collected from 1978 to 1998. Our detailed analyses indicates that the development of testis-weight over the last fifty years has followed four marked phenomena: (a) the age at which testis-weight reaches its maximum has become younger, (b) the weight at peak had become generally higher, until a decline in peak-weight beginning after 1960 birth years, (c) the decline-rate at which testis-weight decreases after its peak has greatly accelerated, and (d) the onset of testis-weight of boys has started progressively younger. Our quantitative analyses of testis-weight indicate some possibility of a subtle reproductive disorder in Japanese men, especially those born after 1960. Together with the accelerated development and decline of testis-weight during the last fifty years, the decline in peak-weight might be indicative of a subtle interference of environmental toxicants with male reproductive organogenesis.

Seven DNA probes which recognize single-locus hypervariable region (HVR) were applied to a paternity test in which the putative father and his wife were deceased. Three legitimate children, an illegitimate child and her mother were available for analysis. The cumulative paternity index of the illegitimate child derived from 15 conventional blood group markers was 18.71 and from 7 DNA probes 92,572.08, that is, 4,948 times higher than the former. Thus the DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the child. The application of highly discriminating polymorphisms of DNA which recognize single HVR loci is considered to be extremely informative in cases of disputed parentage.

A paternity case involving a putative father, a child, and the mother was referred to our laboratory for testing. Parentage was not excluded with 23 kinds of standard blood group markers and HLA, but the putative father requested more-affirmative evidence of paternity. Seven kinds of DNA probes that recognize hypervariable loci were applied. On the basis of the allelic frequencies and their confidence intervals previously reported among unrelated Japanese individuals, as well as confirmed codominant segregation of the polymorphism, the exclusion probability and paternity index were calculated for this case. The cumulative paternity index from the seven DNA probes was 1.4 × 10(6), which was 316 times higher than that from the 23 standard blood group markers and HLA. Accordingly, DNA polymorphism is considered to be informative enough for paternity testing.

Seven kinds of hypervariable DNA probes, which recognize hypervariable loci, were applied for individual identification of human bloodstains. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and codominant segregation of the polymorphism was confirmed in family studies. One-month-old bloodstains from ten individuals were investigated in the present study. The probability of matching was calculated using the database among Japanese population. Cumulative probability of matching from 7 kinds of hypervariable DNA probes was 1.1 x 10(-11), which was 4.5 x 10(9) times higher than that from 6 kinds of common blood group markers such as ABO, MN, Rh-Hr, P1, Hp and PGM1. Accordingly, DNA polymorphism is considered to be informative enough for individual identification of bloodstains.

The stability of DNA in human bloodstains and various post mortem tissues has been investigated. High molecular weight (HMW) DNA was usually recovered from dried bloodstains, even those up to a few years old, but very rapid degradation was found to occur post mortem in the liver, pancreas, spleen and kidney. Other tissues such as the heart, thyroid and skeletal muscle were found to give a reasonable yield of HMW DNA during the first few days after death. The feasibility of using DNA extracted from forensic bloodstain specimens for the detection of DNA polymorphisms was explored using a human haptoglobin (Hp) α chain specific probe. Using HindIII and XbaI digests the Hp genotypes Hp2, Hp1F and Hp1S were distinguished by Southern blot analysis in DNA prepared from 1 cm2 bloodstains up to 15–18 months old.

IWASA, M., YOKOI, T. and SAGISAKA, K. An Interaction between Human Seminal Acid Phosphatase and Canavalia gladiata DC Lectin. Tohoku J. exp. Med., 1982, 137 (4), 415-421 - Interactions between human seminal acid phosphatase (AcP) and five kinds of lectins were studied. Seminal plasma was mixed with the lectins at various ratios. The mixtures were centrifuged and the supernatants were assayed for AcP activity. The activity was effectively reduced only with Canavalia gladiata DC (CG) lectin. CG lectin purified by Sephadex G-200 gel filtration was added to two fractions of AcP separated by Sephadex G-200 gel filtration. There was no difference in the rate of reduction of the two fractions. To isolate seminal AcP by affinity chromatography, CG lectin was coupled to agarose gel. AcP bound with the agarose gel could be eluted with α-methyl-D-mannoside. Specific activity of the purified AcP was elevated to as much as 54-fold. - lectin; seminal plasma; acid phosphatase

Genetic polymorphisms of human serum orosomucoid ORM1 and ORM2 gene loci were investigated in a Libyan population sample from Tripoli, n=110, using isoelectric focusing in ultrathin layer polyacrylamide gel containing either Triton X-100 orN-(2-acetamido)-2-aminoethanesulfonic acid (ACES) with subsequent immunofixation. In ORM1 locus, six common phenotypes ORM1 1-1, ORM1 2-1, ORM1 2-2, ORM1 2.1-1, ORM1 2.1-2 and ORM1 3-1 were identified, with allele frequencies as follows:ORM1*1=0.6500,ORM1*2=0.3091,ORM1*2.1=0.0318 andORM1*3=0.0091. In ORM2 locus, one common phenotype ORM2 1 and four new heterozygote types represent products of three new variant alleles tentatively designatedORM2* Tripoli 1, ORM2* Tripoli 2 andORM2*Tripoli 3 were observed. In addition, one subject in which no ORM2 was detectable, the phenotype ORM2 0-0 is suggested. The ORM2 allele frequencies wereORM2*1=0.9816,ORM2*Tripoli 1=0.0092,ORM2*Tripoli 2=0.0046 andORM2*Tripoli 3=0.0046.

A recombinant DNA probe (pHY10) hybridizing specifically to human DNA family DYZ1, 3,000 copies of which are present on the long arm of the Y chromosome, was used for probing human genome DNA digested with various restriction enzymes. To our surprise, the probe detected a hypervariable polymorphism of autosomal origin in human DNA when digested with TaqI. None of other 12 restriction enzymes revealed polymorphic patterns. Codominant segregation of the polymorphism was established in family studies. This probe has been widely used in the detection of the Y chromosome. Its ease of availability as well as highly discriminating polymorphic pattern makes it potentially very useful for forensic and human genetic purposes.

It has been known that the agglutinability of human red cells is changed or enhanced by treatments with proteolytic enzymes or neuraminidase. In this paper, the serological properties of agglutinogens developed by proteolytic enzymes (bromelin, ficin, papain, trypsin and pronase) and neuraminidase are investigated by using antisera to trypsin- and neuraminidase-treated red cells. The adsorptions of the antiserum to trypsinized red cells with the cells treated with each of the proteolytic enzymes showed that the agglutinogens uncovered by bromelin, ficin and papain were different from those by pronase and trypsin. It was demonstrated that pronase was the most effective enzyme to uncover the agglutinogen located on deeper site of red cell membrane. This was confirmed by the agglutination with the test cells treated twice with two kinds of the enzymes. The reactions of the antiserum to neuraminidase-treated red cells treated with six kinds of the enzymes indicated that the agglutinogens developed by neuraminidase resembled those by bromelin, ficin and papain more than to those by trypsin and pronase. Blood

AOKI, Y., FUNAYAMA, M. and SAGISAKA, K. Detection of AB Antigen in Blood Stain Using Enzyme-Linked Immunosorbent Assay. Tohoku J. exp. Med., 1987, 152 (3), 277-281-A new method for detection of AB antigen from blood stain using enzyme-linked immunosorbent assay (ELISA) is described. Flat-bottomed-wells of polystylen plate coated with human anti-B or rabbit anti-A were sensitized with AB antigen which was extracted from blood stain with 1% octyl-glucopyranoside in 0.1M phosphate buffer pH 8.0. Mouse monoclonal anti-A or anti-B, and peroxidase conjugated anti-mouse immunoglobulin were added to the wells, respectively. Subsequently, the substrate was dropped into the wells, and the absorbance of the solution was measured. By this method, we could distinguish AB group blood stain from the mixed stain of A and B group bloods. When rabbit antiserum was used as the first antibody, differentiation between these antigens was unsuccessful presumably because of non-specific adsorption.

SAGISAKA, K., YAMASHITA, H., IWASA, M. and YOKOI, T. Human Specific Antigens and Species Difference in Non-Hemoglobin Proteins of Red Cell Lysate. Tohoku J. exp. Med., 1981, 135 (2), 197-204- Non-hemoglobin proteins (NHPs) of 12 species of mammalian were effectively isolated from red cell lysates with CM-Sephadex chromatography. Polyacrylamide gel disc electrophoresis demonstrated that human and monkey NHPs had 13 and 11 bands, respectively, but guinea pig and swine two to three bands. Rabbit anti-human NHP reacted with monkey, bovine, canine and badger as well as human. Absorption of the anti-NHP with monkey NHP resulted in preparing human specific anti-NHP. The specific antiserum gave two bands locating near the origin in immunoelectrophoresis. Human specific antigens were prepared by affinity chromatography with the anti-NHP. The antigens were composed of three bands, two of which had slightly faster mobility than carbonic anhydrase and the other migrated fastly on disc electrophoresis. These bands were proved to have neither LDH nor esterase activity.

A antigens of red blood cells and body fluids such as saliva, semen and sweat could be serologically distinguished using rabbit or guinea pig immune anti-A. As for antisera specific for red blood cell A, A+ rabbits were intravenously immunized with A group red blood cells. The resulting antisera were absorbed with O and B red cells and with A. Se saliva. The absorbed anti-A reacted with A red cells (titer 1:32) and was not inhibited with A. Se saliva. Guinea pigs were intramuscularly injected with A. Se saliva. Crude antisera contained agglutinins to human red cells which were abolished by absorption with A red cells. After absorption with O. Se saliva, the antisera were proved to have agglutinin activity with A group saliva using latex coated with A. Se saliva. A antigens from blood or body fluid stains could be distinguished by the elution method with these anti-A sera.

To examine common antigens between human and monkey sera, sera from 19 kinds of monkey were investigated with crossed and rocket immunoelectrophoresis using antisera to human serum proteins. In the crossed immunoelectrophoresis, the number of precipitation lines, 40 in human and 8 in the lower primates, increased according to the order of phylogenetic classification, and the lines due to albumin and gamma-globulin were observed in all the monkey sera examined. Quantitative investigation was performed by rocket immunoelectrophoresis with 8 kinds of mono-specific anti-serum protein. Alpha 2-Macroglobulin, orosomucoid, transferrin, IgG and IgM were not observed in monkeys of Loricidae family. Relatively large amounts of orosomucoid, albumin, IgG and transferrin were detected in monkeys of Cebidae family. It was considered that the distribution of serum proteins possessing antigens common to human is not related with the order of phylogenetic classification.

IWASA, M., YOKOI, T. and SAGISAKA, K. Individual Difference of Seminal Plasma Detected with Crossed Immunoelectrophoresis. Tohoku J. exp. Med., 1983, 140 (2), 187-192 - Seminal plasma from 120 subjects was examined by crossed immunoelectrophoresis using anti-seminal plasma. The samples were divided into two groups depending on a precipitation line which migrated fast in the first dimension electrophoresis. This precipitation line showed an incidence of 48 percent and was proved to have no acid phosphatase, esterase or lactate dehydrogenase activity and was not stained with Schiff's reagent. Analysis with column chromatography, Sephadex G-200 and Sephadex G-50, revealed that molecular weight of the protein was about 10, 000 dolton. The difference was not identified with the other methods; polyacrylamide gel electrophoresis and agarose immunoelectrophoresis.

IWASA, M., KANEMITSU, K. and SAGISAKA, K. An Esterase Isozyme in Non-Hemoglobin Proteins of Cell Lysate Reacting with Mushroom Lectin. Tohoku J. exp. Med., 1982, 136 (1), 53-59-Of 20 kinds of plant lectins examined, agglutinin activity of only mushroom lectin was inhibited with non-hemoglobin proteins of the red cell lysate. Chromatography of non-hemoglobin proteins on DEAE-cellulose and on hydroxylapatite resulted in isolating a protein which gave a single precipitation line with esterase activity in immunoelectrophoresis. SDS-polyacrylamide gel disc electrophoresis demonstrated that the protein consisted of two subunits molecular weight of which was approximately 100, 000 each.

An improved method for detecting diatom in blood is reported. Blood of cadavers was obtained by cardiac puncture at inquest or from the left atrium directly at autopsy. The blood was hemolyzed by sodium dodecyl sulfate (SDS) and filtered through membrane filter (47 mm in diameter and 5 microns in pore size). When the blood was putrefied, two or three pieces of membranes filters were needed because of choking membrane pore. The membrane filters were digested with fuming nitric acid and diluted with distilled water. The diluted solution was filtered through membrane filter (25 mm in diameter) again. After drying the membrane filter was immersed in oil. Diatoms on the membrane filter were clearly observed microscopically.

To elucidate immunochemical properties of Rh-Hr antigens, influence of various solubilizers on the activity, and isolation of the antigens were investigated. As a very sensitive method to detect Rh-Hr antigens, the agglutination inhibition test was recommended in which diluted antisera (1:400-1:1,600), papainized red cells and microtitrate plates were used. Extraction of D antigen with Brij 35 and Triton X-100 was effective, but SDS was not successful. However, D activity of SDS solubilized stroma was recovered by adding reductants. The activity was slightly demonstrated at the preparation from D negative red cells. The influence of the reductants was discussed. SDS solubilized stroma was fractionated with gel filtrations and activated thiol Sepharose 4B. The final preparation with D activity was proved to correspond with Band 3 which was not stained with PAS reagent. Deoxycholic acid solubilized stroma was fractionated with gel filtrations and affinity chromatography, resulting in the preparation corresponding to Band 3 with potent Rh-Hr blood type activities. The activities were detected without reductants and the preparation from D negative red cells had no D activity. Rh-Hr blood type activities other than D were observed in the same fraction.

To differentiate species-specificity of blood stains, anti-alpha 2-macroglobulin was raised in rabbits against Canavalia lineata DC lectin-serum complex (LSC). Adsorption of anti-LSC with human lipoprotein resulted in antiserum specific for alpha 2-macroglobulin. It was confirmed by Ouchterlony test that the antiserum adsorbed successively with monkey serum or anti-LSC adsorbed directly with monkey serum reacted with only human serum but not with mammalian ones. Immunoelectrosyneresis and anti-LSC consumption test could identify species-specificity of blood stains kept for up to two years and for up to several years, respectively. It is indicated that anti-LSC is quite effective for differentiating species-specificity of blood stains.

Immune agglutinin or precipitin loses its reactivity with the corresponding antigen by heating or some chemicals. A. Se group saliva or M group blood stain was sensitized with human IgM anti-A or rabbit IgG anti-M serum, respectively. After being washed with saline, the stains were heated or treated with formalin or 2-mercaptoethanol (2-ME) of various concentrations. The immunoreactivity of the antibodies in the state of immunecomplex was examined by the mixed agglutination or elution test, indicating IgM and IgG antibodies in the state of immunecomplex were proof against heating up to 150 degrees C for 10 min and against formalin and 2-ME of high concentrations. It is considered that IgM and IgG acquire a new property of heat and chemical stability by formation of immunecomplex although they are substantially heat and chemical labile.

Amylase activities were detected significantly in saliva from human, macaques and rodents and slightly in the vegetable and fruit extracts. Dried stains on filter paper prepared from human and mammalian saliva, and the vegetable and fruit extracts were subjected to starch-iodine test and blue starch polymer-agar plate test. Both tests showed strong positive reactions with the macaque and rodent saliva stains as well as human, but the vegetable and fruit stains showed clear positive reactions only in the starch-iodine test. The results suggest that these tests are not specific for human saliva and that for screening test of saliva stains, blue starch polymer-agar plate test is more suitable than starch-iodine test. Rabbit antisera against alpha-amylase isolated from the human submaxillary glands were prepared. In double immunodiffusion test with the human and mammalian saliva, human and macaque saliva produced precipitation lines. Human saliva gave patterns of partial identity with saliva from Japanese monkey and crab-eating monkey and these two macaque saliva gave total identity with each other. Anti-alpha-amylase sera absorbed with Japanese monkey saliva reacted only with human saliva. This suggested that the anti-alpha-amylase sera absorbed with macaque saliva make it possible to identify human saliva stain.

An effective isolation of carbonic anhydrase C (CA-C) from red cell lysate was described. The lysate dialyzed against 0.0175 M phosphate buffer pH 6.3 was applied onto a CM-Sephadex column equilibrated with the same buffer. The elution was performed with the starting buffer and 0.1 M dibasic potassium phosphate containing 0.14 M NaCl. The hemoglobin fraction eluted with the second eluant was applied onto a DEAE-cellulose column and eluted with 0.2 M glycine containing 0.01 percent KCN, resulting in complete isolation of CA-C at high recovery rate. For the preparation in a large scale, the hemoglobin fraction prepared from CM-Sephadex semi-batch-type chromatography was treated with cold ethanol and chloroform. The purity of these preparations was confirmed by polyacrylamide gel disc electrophoresis and immunoelectrophoresis.

A recombinant DNA probe hybridizing specifically to human repeat DNA sequence (pHY10) of which about 3000 copies are present on the Y chromosome was used for sex determination of degraded DNA samples of blood stains. Human blood stains of male and female origin were readily differentiated with the pHY10 DNA probe. This radioactive technique enabled reliable and sensitive sex determination from blood or dried blood stains greater than 20 years old. Less than 1 microliter of blood or 1 piece of 0.5 cm length thread of blood stain from cotton fabric was sufficient for the test using dot blot hybridization. Compared with the radioactive labeling method, the photobiotin labeling method showed one thirtieth to one fiftieth lower sensitivity and presented some problems which are expected to be resolvable.

SAGISAKA, K., YAMASHITA, H., IWASA, M. and YOKOI, T. Serological Distinction of H Antigen between Saliva and Red Cell in Forensic Practice. Tohoku J. exp. Med., 1983, 141 (2), 211-215-Rabbit antisera were prepared by immunization with O red cells or O.Se saliva. Anti-O red cells (anti-Hr) were diluted with O.Se saliva and each of diluents was adopted for elution method on blood or body fluid stains. Anti-Hr gave positive reactions only with red cell stains. On the other hand, anti-O.Se (anti-Hs) absorbed with O red cells kept activity reacting with latex coated with O.Se saliva. Anti-Hs reacted with various body fluid stains but not with red cell stain. It is considered that anti-Hr and -Hs are useful tools for individualization of blood or body fluid stains in forensic practice.

Lea substance or immune complex which was prepared with anti-D and D-active Band 3 was trapped in autologous red cell ghost. The trapped immunogens were administered intravenously to rabbits or guinea pigs. Rabbits immunized with Lea substance loading ghost produced antisera of relatively low titers, but they contained specific incomplete anti-Lea antibody (titer 1:128) after absorption procedure, which were higher than that of antisera prepared by the ordinary method. Guinea pig antiserum to the immune complex contained specific incomplete anti-D (titer 1:128). Immunoglobulin class analyses revealed that the anti-Lea consisted mainly of IgM and the anti-D of IgG. It was considered that intravenous injection of the immunogen trapped in ghost is useful for preparing hemagglutinin of sufficient titers.

Carbon monoxide (CO), total hemoglobin (Hb) and carboxyhemoglobin (HbCO) in the blood and reddish discolored body cavity fluids of cadavers which had not been exposed to fire and CO were analyzed. In 13 cadavers found on land, the maximum saturation of HbCO in the blood was 3.6%, and was 10.1% in the body cavity fluids. There was only one case in which the HbCO saturations in the body cavity fluids were more than 10%. In seven drowned bodies found in fresh water, the highest HbCO saturation in the blood was 6.1%, and was 44.1% in the body cavity fluids. There were three cases in which the HbCO saturations in the body cavity fluids were more than 10%. In 12 drowned bodies found in sea water, the HbCO saturations in the blood were not more than 6.2%, and the maximum saturation of HbCO in the body cavity fluids was 83.7%. There were eight cases in which the HbCO saturations in the body cavity fluids were more than 10%. The results seem to indicate that the interpretation of HbCO saturation in the blood would not be affected significantly by the postmortem formation of CO, and that body cavity fluids should not be used for CO determination.

Rabbit antisera were raised for red cell stroma extracted with each of the following solutions; 5 mM phosphate buffer pH 8.0 (A antigen). 5 mM EDTA containing 5 mM 2-mercaptoethanol (B), 1/15 M phosphate buffered saline (C), 0.05% SDS (D), 50 mM Tris-HCl buffer pH 8.0 containing 1% SDS, 1 mM EDTA and 1% 2-mercaptoethanol (E) and 1% Triton X-100 (F). Crossed immunoelectrophoresis showed that E antigen produced relatively numerous precipitation lines, 7-9, against each of the antisera, suggesting that E solution was superior to the others for membrane solubilization. However, the Ouchterlony test demonstrated potent precipitin activity in anti-C, and the antiserum absorbed with monkey stroma which was extracted with F solution was proved to have strict human specificity. By adopting the antiserum to affinity chromatography, human specific antigens were isolated. The antigens gave four bands moving faster than Band 5 at SDS-polyacrylamide gel electrophoresis none of which was stained by Schiff's reagent. Using absorbed anti-C, human specificity of blood stains kept us to for two years could be identified.

H and A antigens expressed only in the RBC were quantitated using specific antisera, and the amount of the antigens was compared with that of total H and A antigens shared by the RBC and saliva. Total H antigen decreased in the order of O, A, B and AB blood groups. However, H antigen specific for the RBC distributed similarly among four blood groups. In addition, the amount of total A antigen in A group was more than that of AB group, but the difference in the amount of A antigen specific for the RBC was not significant between the two blood groups. B antigen specific for the RBC was not examined because of failure in preparing specific anti-B serum. In conclusion, the quantitative difference of the total H or A between A and AB groups might be due to the difference in the amount of antigen common to the RBC and saliva but not one specific for the RBC.

In the previous paper, the present authors reported on diminution in agglutinability of trypsin-treated M and MN red cells with anti-M IgG antibody. This paper deals with the time course of changes in agglutinability and quantitative differences of M agglutinogen of red cells. M agglutinogen of M red cells disappeared much faster than that of MN, and the lowest titer of anti-M IgG antibody to agglutinate M red cells was higher than that to agglutinate MN red cells. The significant difference of agglutination scores was observed in the frequency distribution in 100 cases of M and MN red cells each. Subgroups such as M1 and M2 in M agglutinogen of trypsin-treated red cells reacting with anti-M IgG antibody were unsettled.

The present study deals with the distribution of periodic-acid-Schiff (PAS) positive substances in the thymus of dd-strain mice. The PAS-positive substances were found in about 15 per cent of the thymus lymphocytes in normal cases, and all of these cells were small thymic lymphocytes. In formalin stress, the substances disappeared just before morphological changes of the cells began to appear. From a standpoint of the maturity of thymic lymphocytes investigated by means of tritiated thymidine, it was considered that the substances might develop in the cytoplasm in accordance with the growth of cells. Autoradiographs showed that labeled large thymic lymphocytes were transformed into PAS-positive labeled small ones with the lapse of time after the injection of isotope. Therefore, it is conceivable that thymic lymphocytes produce these substances in the immature stage of cells.

The agglutinability of the proteolytic enzyme treated M, N and MN red cells with any one of the anti-M and -N antisera, eluted antibodies and class antibodies was studied. The enzyme-treated 141 and N red cells lost their group specificity in agglutination with the absorbed antisera. However, it was noted that the enzyme-treated M red cells acquired N agglutinogen instead of losing their own M agglutinogen in agglutination with the eluted antibodies. The enzyme-treated N red cells maintained the group specificity. Agglutination of the enzyme-treated red cells with the class antibodies showed clearly that the changes in agglutinability of the red cells were caused by each of the class antibodies.

A modified mixed agglutination method is described for detecting AB antigen from body fluid stain. Saline extract of body fluid stain was added to A.Se saliva stain which was beforehand sensitized with anti-A. AB antigen reacting with the stain was detected with the usual mixed agglutination method using anti-B and B red cells.

Anti-M and -N antibodies are usually prepared from rabbit immunized with human O.M or O.N red cells. The crude antisera must be adsorbed with non-corresponding red cells to remove heteroagglutinin. To specify the crude antisera, we have employed dextran gel (DEAE-Sephadex A-50) as an immunoadsorbent, which was mixed with M or N group substance prepared by the phenol-saline method, and was fixed with formaldehyde. The substance-coupled gel was added to the corresponding crude antiserum and washed with cold saline. Then, the gel added with an equal volume of saline was heated at 55 degrees C for 10 min and applied on a funnel with slight evacuating. By this method, specific anti-M and -N antibodies (titer 1:16 to 1:32) were prepared, but the antibody was not eluted from the gel reacted with IgM fraction. The recovery rate and specificity of the eluted antibodies are discussed.