JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.     

Determination of IgG subclasses of immunoglobulin preparations for intravenous injection and the problems involved

The IgG subclasses of immunoglobulin for intravenous injection (IVIg) were investigated after various different types of treatment. The level of IgG2 was relatively low in preparations treated with pepsin, but by changing the clone producing anti-IgG2 antibody, the level approached that of normal human serum. Though undetectable in sulfonated immunoglobulin preparations, IgG3 was detected after reoxidation. When determining the IgG subclasses of IVIg, it must be kept in mind that the apparent value may subsequently become lower due to the depressed reaction of the antibody.     

Moving Mesh Method for Reconstructing Some Spread Sources in the Brain

The purpose of this paper is to propose a new algorithm for the analysis of biomagnetic field data obtained from magnetoencephalography (MEG) measurements. This new method overcomes two major problems faced by the current method of data analysis. The first problem is the need to determine the number of sites of brain activity before calculations can be performed. The second problem is inability of the analysis to provide any information regarding the volume of the brain activity. The new data analysis method, called the Moving Mesh Method (MMM), is capable of analyzing MEG data without the need to determine the number of sources beforehand. In addition, the MMM determines the location of brain activity as a three dimensional volume, instead of as a point source of activity. The MMM uses an iterative method of calculating the position of the sources to achieve greater accuracy, and a regularized g-inverse matrix to stabilize its solution. The feasibility of the MMM was examined by two methods. First, a computer simulation was used to confirm the MMM's capability to analyzing MEG data. In the second experiment, the MMM was applied to analyze somatosensory evoked fields obtained using a new imaging system (Shimadzu Biomagnetic Imaging System, Model-100). From the interpretation of the results, we have concluded that the MMM is a feasible method of biomagnetic data analysis.     

Vinyl polymerization by metal complexes, 14. Synthesis of 2-substituted and 2,4-disubstituted imidazole-copper(II) complexes as initiator systems for vinyl polymerization

A series of the copper(II) complexes of the type CuL₄X₂ (L: ligand, X: anion) was prepared with the following substituted imidazoles as ligands 2-ethyl- ( 1a ), 2-ethyl-4-methyl- ( 1b ), 2-phenyl- ( 1c ), 2-undecyl- ( 1d ), and 2-heptadecylimidazole ( 1e ). The structures of these complexes were confirmed by elemental analysis, reflection spectra of the crystals as well as IR-spectroscopy. Polymerization of acrylonitrile was tested in the presence of each imidazole-copper(II) complex in dimethyl sulfoxide solution.     

Chymase inhibitor ameliorates eosinophilia in mice infected with Nippostrongylus brasiliensis

BACKGROUND: Chymase is a chymotrypsin-like serine protease primarily stored in mast cells. Infection with helminth parasites is known to increase the level of mast cell chymase in the jejunum and serum in mice. The aim of the present study is to elucidate the role of chymase in helminth infection. METHODS: Chymase inhibitor SUN-C8257 was administered to mice infected with the nematode Nippostrongylus brasiliensis, and the number of eosinophils in the blood, serum IgE levels and fecal egg counts were determined. RESULTS: Administration of SUN-C8257 significantly inhibited blood eosinophilia in BALB/c mice infected with N. brasiliensis. The effect of SUN-C8257 was specific for eosinophils, in that it affected neither the number of total leukocytes nor serum IgE levels. SUN-C8257 did not alter the fecal egg counts in this model, showing that SUN-C8257 has no effect on infectivity and expulsion of the nematode. N. brasiliensis infection induced eosinophilia in mast cell-deficient mice (W/W(v)) as well as their littermates (+/+), and SUN-C8257 inhibited the eosinophilia in +/+ mice but not in W/W(v) mice. These results suggest that the eosinophil number may be regulated by different mechanisms in W/W(v) and +/+ mice, and that the effect of SUN-C8257 on nematode-induced eosinophilia is probably due to chymase inhibition. CONCLUSIONS: Chymase released by activated mast cells may play a role in helminth-induced eosinophilia.     

Determination of medullasin levels for the diagnosis of multiple sclerosis

Medullasin levels in granulocytes of patients with neurological diseases and healthy volunteers were determined by the enzyme immunoassay using mouse monoclonal antibodies against human medullasin and o-phenylenediamine-H2O2 as the detection system of the enzyme activity. One hundred twenty-one out of 159 patients with multiple sclerosis (76.1%) showed positive results (above means of normals + 2SD) in this test, while only 16.9% (24/142) of patients with non-inflammatory neurological diseases had positive results. This enzyme immunoassay method for medullasin is considered to be an useful paraclinical test for the diagnosis of multiple sclerosis.     

CDX2 expression is progressively decreased in human gastric intestinal metaplasia, dysplasia and cancer.

Intestinal metaplasia is a key event in multistep gastric carcinogenesis. CDX2, a master regulator of intestinal phenotype, was shown to play a tumor-suppressive role in colon cancer. However, it was reported to be expressed in nearly all gastric intestinal metaplasia and many gastric cancers. As CDX2 is differentially expressed in normal stomach and intestine, we sought to relate the CDX2 expression to gastrointestinal differentiation along gastric carcinogenesis. The expression of CDX2 protein in gastric intestinal metaplasia, dysplasia and cancer was examined and related to their gastrointestinal differentiation. CDX2 expression was significantly decreased in incomplete intestinal metaplasia, which expresses both gastric mucins (MUC5AC and MUC6) and intestinal mucin (MUC2), compared with complete intestinal metaplasia, which expresses intestinal mucin (MUC2) only. Although incomplete intestinal metaplasia morphologically resembles colon, its CDX2 expression was apparently lower than that in the normal colon. Moreover, CDX2 expression was progressively reduced in gastric dysplasia and cancer. The CDX2 expression in gastric cancer was also inversely correlated with the expression of gastric mucins. As incomplete intestinal metaplasia is associated with higher risk of gastric cancer, its lower CDX2 expression compared with that in complete intestinal metaplasia and normal colon epithelium resolved the current contradiction between the tumor-suppressive role of CDX2 in the colon and the high prevalence of CDX2 in intestinal metaplasia. Further decrease of CDX2 expression in gastric dysplasia and cancer suggests that CDX2 plays a similar anticarcinogenic role in intestinal metaplasia as it does in colon. Intestinal metaplasia or dysplasia with low expression of CDX2 may serve as predictive markers for gastric cancer.     

Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress

The identification of rare monogenic forms of Parkinson's disease (PD) has provided tremendous insight into the molecular pathogenesis of this disorder. Heritable mutations in alpha-synuclein, parkin, DJ-1 and PINK1 cause familial forms of PD. In the more common sporadic form of PD, oxidative stress and derangements in mitochondrial complex-I function are considered to play a prominent role in disease pathogenesis. However, the relationship of DJ-1 with other PD-linked genes and oxidative stress has not been explored. Here, we show that pathogenic mutant forms of DJ-1 specifically but differentially associate with parkin, an E3 ubiquitin ligase. Chemical cross-linking shows that pathogenic DJ-1 mutants exhibit impairments in homo-dimer formation, suggesting that parkin may bind to monomeric DJ-1. Parkin fails to specifically ubiquitinate and enhance the degradation of L166P and M26I mutant DJ-1, but instead promotes their stability in cultured cells. The interaction of parkin with L166P DJ-1 may involve a larger protein complex that contains CHIP and Hsp70, perhaps accounting for the lack of parkin-mediated ubiquitination. Oxidative stress also promotes an interaction between DJ-1 and parkin, but this does not result in the ubiquitination or degradation of DJ-1. Parkin-mediated alterations in DJ-1 protein stability may be pathogenically relevant as DJ-1 levels are dramatically increased in the detergent-insoluble fraction from sporadic PD/DLB brains, but are reduced in the insoluble fraction from parkin-linked autosomal recessive juvenile-onset PD brains. These data potentially link DJ-1 and parkin in a common molecular pathway at multiple levels that may have important implications for understanding the pathogenesis of inherited and sporadic PD.     

Simple method for the identification of oxidative fibers in skeletal muscle

Skeletal muscle is composed of several different types of myofiber: slow oxidative (SO), fast glycolytic oxidative and fast glycolytic. However, the classification is usually determined by myosin heavy chain typing rather than by metabolic index. In this study, the oxidative metabolic index was investigated as a possible method of myofiber typing. Myoglobin, which is involved in oxygen transport and storage in myofibers, and mitochondria, which are the central organelles for oxidative metabolism, were studied. High levels of myoglobin and mitochondria are believed to exist in SO fibers, but the current study showed that they are considerably richer in some fast type fibers. As myofiber typing using the oxidative metabolic index is important physiologically, an attempt was made to find a simple method for this purpose. Some mitochondrial proteins have been observed to auto-fluoresce but until now this effect was too faint to detect easily. Owing to the recent advances in cooling charge-coupled device technology, such auto-fluorescence can now be used for myofiber typing, and the simple and rapid method for doing so is reported here.