Ikuko Ushiyama

Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.

Changes in choice of method and lethality between last attempted and completed suicides: how did suicide attempters carry out their desire?

Some researchers have emphasized that, from the perspective of suicide prevention, research into the methods of suicide seemed to be particularly promising, as it has been shown repeatedly that restricting access to the prevailing method of suicide in a country will decrease suicide rates and that the lethality of the method used significantly correlated with the degree of intention to die. In this study, we examined changes in choice of method and the lethality score between the last attempted suicide (LAS) and completed suicide (CS) in 416 victims (male: 197, female: 219) to point out the tendency on their choice of method in LAS and CS. There was a significant difference in choice of suicide method between LAS and CS, and injury to themselves (33.7%) was the most common method of LAS, while hanging (37.5%) was the most common method of CS. The mean lethality score of CS method was significantly higher than that of LAS method in both sex groups, suggesting that at least one of the causes that drives suicide attempters to commit suicide finally may be the difference in the lethalities of LAS and CS. At the time of CS, suicidal victims tend to choose the same method as that of LAS again. These findings suggest that although suicide attempters tend to choose the same method, they will use a more lethal method if they change the suicide method. Interestingly, moreover, there was no sex difference in the percentage of the mean lethality score at CS.

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

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

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

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

Lectin-histochemical detection of degenerative glycoconjugate deposits in human brain

Several lectins were used to study the localization of glycoconjugates in brain of elderly people and patients with Alzheimer type dementia (ATD) and Downs syndrome (DS). Five kinds of degenerated or deposited materials stained clearly by lectins specific to GalNAC, Gal, Fuc, and/or Man were recognized much in ATD and DS, less in elderly peoples, in addition to the binding of the lectins to neurons. (i) Round shape deposits called corpora amylacea (CA) which consisted of various sizes of round material, existed mainly on the surface of cerebral cortex and some in white matter of the brain. They were colored by Alcian blue (AB), Aldehyde fucsin (AF) and periodic acid shiff (PAS) and weakly by Hematoxylin (H), but not by Eosin (B). They showed clear reactivity with lectins specific to GalNAC, Gal, Fuc and Gal-GalNAC. (ii) Amorphous and variform amyloid deposits existed around blood vessels in the white matter were stained by thioflavin and lectins specific to GalNAC, Gal and Fuc, but not with Man specific lectins and PAS, AB, AF and HE. (iii) Another kind of amyloid deposits which showed a similar characteristic to the previous one and were recognized mainly in white matter and independent blood vessels. These deposits were stained by thioflavin but not by PAS, AB, AF and HE and showed good reactivity with lectins specific to GalNAC, Gal, Fuc, Gal-GalNAC, Gal-GIcNAc and Man. The reactivity with lectins specific to Gal, Fuc, and Man was seen in senile plaques (iv) and neurofibrillary tangles (v). Although at present we are unable to explain the origin of these deposits, it is clear from this study that the glycoconjugates form an integral part of the degeneration in the brain. The lectin staining with GS-I is useful in the forensic pathology to diagnose brain disorders at postmortem examination, since these lectin were able to detect five types of degeneration changes and/or deposits.

Phylogenetic analysis of picoplankton in Lake Biwa and application to legal medicine

Three strains of picoplankton designated as brown, green, and pink belonging to the Synechococcus genus in cyanobacteria (approximately 1 μm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria such as Proteobacteria and Bacillus by microscopy. In this study, we attempted to use the polymerase chain reaction (PCR) analysis of the 16S ribosomal DNA (rDNA) from picoplankton for the diagnosis of death by drowning. A segment of 16S rDNA was sequenced in order to investigate their phylogenetic relationships and to design the specific primers. The PCR products from three picoplanktons were compared with those from five other cyanobacteria, Melosira (diatom), Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The efficiency of PCR was increased more than hundredfold by isolating the picoplankton from human lung tissue. The specific PCR products of the picoplankton were obtained from a formalin‐fixed drowning body (lung and liver) that was found in a downstream river and Lake Biwa. The PCR analysis of the picoplanktion 16S rDNA is considered useful for the diagnosis of death by drowning.

Tissue-specific expression of Le(Y) antigen in high endothelial venules of human lymphoid tissues.

In this study, we demonstrated that the anti-LeYantibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The LeYexpression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the LeYexpression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of LeY-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of LeYwas observed in vessels of Payers patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-LeYantibody increased after enzyme digestion with α-N-acetylgalactosaminidase or α-galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs.

Intrathymic lymph nodes in humans

An unusual lymph node exists in the centre of the human thymus. This lymph node, which we call an intrathymic lymph node (ITLN), possesses some interesting morphological characteristics. In ontogeny, this node seems to appear at the latter half of fetal period. The function of the ITLN is still unknown, but it is assumed that it may play a different role in the immune system than other peripheral lymph nodes by its characteristics.

Species Identification by Analysis of the Genes for ABO Blood Group

The species identification is of a great importance in the forensic field. The immunological techniques have been utilized to distinguish human from other animal samples, such as blood stain, saliva or seminal fluid, however a keen method for identification of the origin of a sample has not been established. We have previously reported that ABH(O) blood group substances are detected in various species and that the lectin- and immuno-histochemistry can be available for species identification from tissue particles (Nishi 1992). It is considered that some animals possess the genes homologous to those for human ABO blood group. We have also reported the determination of ABO genotypes with DNAs extracted from fresh blood or formalin-fixed, paraffin-embedded tissues by PCR amplification and restriction enzymes digestion (Yamada 1994), depending on the polymorphism of the gene encoding blood group A-transferase. Here we describe that the DNA analysis of the genes for ABO blood group might be a tool of species identification.

ABH-Related Antigens Participate in the Spermatogenesis of Cats and Rats

The ABH antigens were expressed in the secretory cells of many mammals(1), including humans. Although the allelic cDNA of ABO blood group had been cloned and sequenced(2), no clear explanation of the biological significance of the ABH and related antigens has been proposed. In this study we examined the distribution of ABH related antigens in the urogenital organs from cats and rats in order to pro mote better understanding on the biological significance of the antigens, using monoclonal antibodies (MoAbs) against the ABH and related antigens, and lectins. The results obtained from the present study suggested that the ABH and related antigens may play certain roles in the processes of development of spermatogenic system. In view of forensic practice, the importance of species identification prior to ABO blood grouping from seminal, saliva and urinal stains are stressed on the basis of the present results.