Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Lithium chloride-tolerant character of the heterobasidiomycetous yeastRhodotorula glutinis

The heterobasidiomycetous yeastRhodotorula glutinis was able to grow in medium containing a high concentration of LiCl. This character ofR. glutinis was presumed to be attributable to its ability to incorporate [¹⁴C]-adenine and [¹⁴C]-leucine into nucleic acids and proteins, respectively, in the presence of LiCl. Intracellular levels of Li⁺ and Cl^– ions, production and accumulation of glycerol as an osmoregulator, and respiration in the LiCl-stressed condition were almost the same in the tolerant yeastR. glutinis and the sensitive yeastRhodosporidium sphaerocarpum.     

Proline specific endo- and exopeptidases

Summary Peptidases which are specific for proline residues have been described and include endopeptidases (post-proline cleaving enzyme and proline specific endopeptidase), N-terminal exopeptidases (post-proline dipeptidyl aminopeptidase, proline iminopeptidase, aminopeptidase P), C-terminal exopeptidases (prolylcarboxypeptidase, and carboxypeptidase P) and dipeptidases (prolyl dipeptidase and proline dipeptidase). The properties, distinguishing characteristics, and possible significance of these proline specific endo- and exopeptidases are discussed. In addition, reference is made to a series of enzymes which can hydrolyze proline containing peptide bonds, but which are not specific for proline.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Alterations of lymphocyte count and platelet volume precede cerebrovascular lesions in stroke-prone spontaneously hypertensive rats

Abstract

Background: Cerebral small vessel disease (CSVD) is associated with future stroke. Although pathological alteration in small vessels of patients with CSVD can be detected by neuroimaging, diagnosis of CSVD is delayed because it is an asymptomatic disease. The stroke-prone spontaneously hypertensive rat (SHRSP) show similar pathological features to human CSVD and develop stroke-related symptoms with advancing age.

Objective: We investigated the time course of haematological parameters in Wistar rats and SHRSP.

Material and Methods: Blood cells were analysed using an automated haematological analyser.

Results: SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14?weeks of age. Lymphocyte counts were gradually decreased at 3?weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The both mean platelet volume and large platelet ratio gradually increased at 3?weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP.

Conclusion: Our pilot study suggests that alterations of lymphocyte count and platelet volume predictive indicators for asymptomatic CSVD and symptomatic stroke in SHRSP.     

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum)

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.     

The Neurospora Peptide:N-Glycanase Ortholog PNG1 Is Essential for Cell Polarity despite Its Lack of Enzymatic Activity

Secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. The cytoplasmic peptide:N-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 S proteasome. In contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:N-glycanase activity remains unclear. Here we show that the PNG1 ortholog from the filamentous ascomycete Neurospora crassa is an essential protein, and its deletion results in strong polarity defects. PNG1 and its predicted binding partner RAD23 have distinct functions in N. crassa and are involved in cell wall integrity and DNA repair, respectively. Moreover, wild type PNG1 has substitutions in essential catalytic amino acids, and its deglycosylation activity is lost. These substitutions are conserved in many PNG1 orthologs of the fungal kingdom, implying a so far unrecognized enzyme-independent function of PNG1 that may only become apparent in highly polar cells such as fungal hyphae.     

Localization of Thy-1�Cpositive Cells in the Perichondrium During Endochondral Ossification

We elucidated the localization of Thy-1–positive cells in the perichondrium of fetal rat limb bones to clarify the distribution of osteogenic cells in the process of endochondral ossification. We also examined the formation of calcified bone-like matrices by isolated perichondrial cells in vitro. At embryonic day (E) 15.5, when the cartilage primodia were formed, immunoreactivity for Thy-1 was detected in cells of the perichondrium adjacent to the zone of hypertrophic chondrocytes. At E17.5, when the bone collar formation and the vascular invasion were initiated, fibroblast-like cells at the sites of vascular invasion, as well as in the perichondrium, showed Thy-1 labeling. Double immunostaining for Thy-1 and osterix revealed that Thy-1 was not expressed in the osterix-positive osteoblasts. Electron microscopic analysis revealed that Thy-1–positive cells in the zone of hypertrophic chondrocytes came in contact with blood vessels. Perichondrial cells isolated from limb bones showed alkaline phosphatase activity and formed calcified bone-like matrices after 4 weeks in osteogenic medium. RT-PCR demonstrated that Thy-1 expression decreased as calcified nodules formed. Conversely, the expression of osteogenic marker genes Runx2, osterix, and osteocalcin increased. These results indicate that Thy-1 is a good marker for characterizing osteoprogenitor cells. (J Histochem Cytochem 58:455–462, 2010)     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.