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.     

Enhancement of plaque formation of herpes simplex virus (HSV) and varicella-zoster virus (VZV) by subinhibitory dose of acyclovir (ACV)

Acyclovir (9-/2-hydroxyethoxymethyl/guanine) enhanced the plaque formation of HSV and VZV at subinhibitory dose and inhibited at higher concentration but not in thymidine kinase deficient viruses. The enhancement was neutralized by thymidine. Induction of viral thymidine kinase activity was not affected with ACV. These results suggest that the enhancement may be mediated by viral thymidine kinase.     

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.     

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

Summary Azathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.     

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.