Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay(MLA) for detecting in vitro clastogens and spindle poisonsand to compare it with the in vitro chromosomal aberration test(CA), we conducted an international collaborative study of theMLA that included 45 Japanese laboratories and seven overseaslaboratories under the cooperation of the Ministry of Healthand Welfare of Japan and the Japanese Pharmaceutical Manufacturer'sAssociation. We examined 40 chemicals; 33 were reportedly positivein the CA but negative in the bacterial reverse mutation assay,six were negative in both assays and one was positive in both.We assayed mutations of the thymidine kinase (TK) locus (tk)of L5178Y tk^+/– mouse lymphoma cells using the microwellmethod. According to our standard protocol, cells were exposedto the chemical for 3 h, cultured for 2 days and TK-deficientmutants were expressed in 96-well plates under trifluorothymidine.Each chemical was coded and tested by two or three laboratories.Among the 34 CA-positive chemicals, positive MLA results wereobtained for 20 and negative results were obtained for nine.The remaining five chemicals were inconclusive or equivocalbecause of discrepant inter-laboratory results or reproduceddiscrepant results, respectively. Among the six CA-negativechemicals, one was negative in the MLA, two were positive andthree were inconclusive. Thus, the MLA could detect only 59%(20/34) of CA-positive chemicals. We concluded that the MLAwas not as sensitive as the CA. Some MLA-negative chemicalsevoked positive responses in the CA only after long continuoustreatment. These might also be genotoxic in the MLA with longcontinuous treatment. Improvement of the MLA protocol, includingalteration of the duration of the treatment, might render theMLA as sensitive as the CA. ⁸ To whom correspondence should be addressed. Tel: +81 3 37009847; Fax: +81 3 3700 2348; Email: sofuni{at}nihs.go.jp     

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.     

Evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased; however, the mechanisms involved in the pathogenesis of NAFLD have not been thoroughly investigated in humans. In this study, we evaluated the expression of fatty acid metabolism-related genes in NAFLD. Real-time RT-PCR was performed using liver biopsy samples from 12 NAFLD patients. The target genes studied were: acetyl-CoA carboxylase (ACC) 1, ACC2, and fatty acid synthase (FAS) for the evaluation of de novo fatty acid synthesis; carnitine palmitoyltransferase 1a (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), and long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase alpha (HADHalpha) for beta-oxidation in the mitochondria; peroxisome proliferator-activated receptor- (PPAR-) alpha and cytochrome P450 2E1 (CYP2E1) for oxidation in peroxisomes and microsomes (endoplasmic reticulum) respectively; and diacylglycerol O-acyltransferase 1 (DGAT1), PPAR-gamma, and hormone sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, expression of ACC1 and ACC2, but not FAS was increased, indicating that de novo fatty acid synthesis is enhanced in NAFLD. In contrast, expression of CTP1a, a rate-limiting enzyme, was remarkably decreased, indicating that beta-oxidation in the mitochondria was decreased, although the expression of LCAD and HADHalpha was increased. Expression of PPAR-alpha was increased, whereas that of CYP2E1 was reduced. The expression of DGAT1, PPAR-gamma, and HSL was enhanced. These data suggest that in NAFLD, increased de novo synthesis and decreased beta-oxidation in the mitochondria lead to accumulation of fatty acids in hepatocytes, although the extent of oxidation in peroxisomes and microsomes remains unclear.     

Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk κ‐casein digests

The inhibitory effects of bovine milk κ‐casein and its enzymatic digests on the proliferative responses of mouse spleen lymphocytes induced or not induced by mitogens were studied with a colorimetric assay using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT). κ‐Casein and its glyco‐macropeptide (residues 106–169) inhibited the lipopolysaccharide (LPS)‐induced proliferative response, but the inhibitory effect was lost significantly after neuraminidase and chymotrypsin digestions. In contrast, trypsin and pronase digestions of K‐casein increased inhibitory effects. The pronase digest inhibited the proliferative responses not only induced by LPS but also in the absence of mitogen or when induced by concanavalin A and phytohemagglutinin. The pronase digest seemed to possess weak cytotoxity for lymphocytes. The inhibitory peptide was a glycopeptide(s) having specific size, and the inhibitory effects were reduced significantly by neuraminidase and chymotrypsin digestions. Moreover, similar inhibitory effects on proliferation of lymphocytes were observed in pronase‐digested bovine milk αs₁‐casein and β‐casein. These findings suggest that some peptides produced from milk caseins by the action of gastrointestinal proteinases might contribute to down‐regulate the immune response of neonates.     

The properties of the Kir6.1–6.2 tandem channel co-expressed with SUR2A

Functional ATP-sensitive K (KATP) channels have an octameric subunit structure with four pore-forming subunits (Kir6.x) and four sulfonylurea receptors (SURx). In the present study, the properties of the heteromeric KATP channel whose pore subunits are composed of Kir6.1 and Kir6.2 were examined using a heterologous expression system. In COS7 cells co-transfected with Kir6.1, Kir6.2 and SUR2A at a ratio of 1:1:2, KATP channels showed various unitary conductances between those of Kir6.1/SUR2A (33.6+/-4.2 pS) and Kir6.2/ SUR2A (67.1+/-1.6 pS). Kir6.1-6.2 tandem protein, constructed by fusing the C-terminus of Kir6.1 to the N-terminus of Kir6.2 with a ten glutamine linker sequence, also formed a channel with an intermediate conductance (58.9+/-1.5 pS). Kir6.2 and Kir6.1-6.2 showed similar sensitivity to ATP4-: half-maximal inhibition (IC50) was obtained at 14.1+/-12.8 microM and 17.6+/-9.6 microM, respectively. In the presence of Mg2+, Kir6. 1-6.2 was significantly less sensitive than Kir6.2 to MgATP (IC50=95.5+/-49.6 microM versus 18.9+/-5.0 microM). These results suggest that Kir6.1 and Kir6.2 are endowed with the potential to form a heteromeric KATP channel, which has a low sensitivity to MgATP.     

HLA-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method

We developed a system for discriminating HLA-B40, B18, B27, and B37 alleles using a two-step PCR method followed by SSCP analysis. Fragments (0.8 kb) including exon 2, intron 2, and exon 3 were amplified in the first PCR. We used two sets of primers, one specific for HLA-B60-related alleles and the other specific for HLA-B6l-related, B18, B27, and B37 alleles. No amplifications of other class I genes or pseudogenes were observed. In the second PCR, exon 2 and exon 3 were amplified separately, using diluents of the first PCR products as templates. HLA-B6l-related, B18, B27, B37, and B60-related alleles were clearly discriminated in the SSCP analysis of the second PCR products. In a population study in which B6l alleles were analyzed, B^*4003 was detected in two Japanese individuals in addition to two B6l alleles previously reported to occur in Japanese, B^*4002 and B^*4006. The relative frequencies of B^*4002, B^*4006, and B^*4003 in Japanese were 58, 35, and 6%, respectively. The individuals having B^*4003 are the first non-South Americans in whom this allele has been detected. The SSCP banding patterns of 18 HLA-B60-positive Japanese population samples were identical to those of a B^*40012 sample for both exon 2 and exon 3. We also demonstrated that the B37 allele occurring in some Japanese is B^*3701.     

Analysis by fluorescence in situ hybridization with a mouse gamma satellite DNA probe of isolated micronuclei induced in mice by two clastogens and two spindle poisons

The presence of centromeric DNA was studied in micronuclei isolatedfrom the blood of male ddY mic after five weekly intraperitonealinjections of mitomycu C (MMC), 1-ß-D-arabinofuranosylcytosine(Ara-C), colchi cine (COL) or vinblastine sulfate (VBL). Inagreement with our earlier findings, about half of the micronucleiisolate* from vehicle control mice showed centromere signalsa analyzed by fluorescence in situ hybridization (FISH) witha mouse major (gamma) satellite DNA probe. In an earlie experimentwith mice acutely exposed to the same chem icals, the clastogensMMC and Ara-C did not reduce thi proportion of micronuclei withcentromere signals. In the present study, however, MMC and Ara-Cdecreased the proportion of micronuclei with centromeres. Incontrast the spindle poisons COL and VBL increased the proportionof micronuclei that contained centromeres. ³To whom correspondence should be addressed     

Plasmacytoma of Lymph Node Recurrent Lymphadenopathy Terminating in Plasma Cell Dyscrasia with Polyneuropathy and Endocrine Disturbances

A case with lymphadenopathy of the left side of the neck in a 38-year-old male is described. He had a history of several relapses of about 10 years duration. Swollen lymph nodes were histologically similar to those of the hyaline-vascular type of Castleman's disease, but contained clear-cut lymph sinus and a sheet-like proliferation of plasma cells. Lymph follicles showed proliferation and atrophic germinal centers, in which cellular hypertrophy in the wall of ramifying small blood vessels, called angiosclerosis, was frequently encountered. During its progress, the patient developed plasmacytoma of the lymph nodes with varied clinical manifestations such as polyneuropathy, disturbance of gait, unusual perspiration, hirsuitism, gynecomastia, bilateral papilledema, and albumino-cytologic dissociation in cerebrospinal fluid.     

Adjuvant Activity of 6-Amino-6-Deoxy-Muramyldipeptides and Their Acylamino Derivatives on the Induction of Delayed Hypersensitivity to Azobenzenearsonate-N-Acetyl-l-Tyrosine in Guinea Pigs

The 6-amino-6-deoxy-N-acetylmuramyldipeptides and their 6-acylamino derivatives were shown to be active as adjuvants on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine in guinea pigs. However, 6-acylamino-6-deoxy-N-(acyl)muramyldipeptides were inactive as adjuvants.