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Japan SLC was commissioned by the Council for SAM Research to produce and market in Japan and South Korea five strains (SAMP1, SAMP6, SAMP8, SAMP10 and SAMR1) of Senescence-Accelerated Mouse (SAM) in the summer of 2002. Japan SLC undertook microbiological control and genetic monitoring of these five strains and at the same time maintained and produced them under specific pathogen-free (SPF) conditions in a uniformly controlled breeding environment for market. The company used the basic “traffic-light-system” for mass production of SAM mice in their actual maintenance and production. In the selection of maintenance lines, by checking the specific biological characteristics and pathological phenotype of each strain in addition to the system, the principle in selection was to be able to supply mice of high reproducibility with the phenotypical characteristics of each strain.

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO 2 − ) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO 2 − -hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO 2 − tolerance. A mutant in which the pdh gene cluster was disrupted (Δ pdh mutant) could not grow in the presence of 100 mM NO 2 − . Nitrite decreased the cellular NADH/NAD + ratio and the cellular ATP level. These defects were more severe in the Δ pdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO 2 − -tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO 2 − . These findings demonstrate a link between NO 2 − tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO 2 − -tolerating mechanism in this strain.

Bone density is inherited as a complex polygenic trait. Previously, we identified two quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected by bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains: SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. In this study, we report the effects of the Chr 13 QTL on peak bone density (Pbd2). First, we constructed a congenic strain P6.P2-Pbd2b, which carried a single genomic interval from the Chr 13 of SAMP2 on an SAMP6-derived osteoporotic background, to dissect this polygenic trait into single gene factors. This congenic strain had a higher bone density than the background strain using three measurement methods with different principles for bone density. Next, we measured the peak relative bone mass of the AKR/J strain and the 13 senescence-accelerated mouse (SAM) strains, which are considered to be a series of recombinant-like inbred (RI) strains derived from the AKR/J strain and other unspecified strains. We then determined the microsatellite marker haplotypes of these strains around the Pbd2 locus, in which three strains with a high relative bone mass shared the same haplotype over the 26-centimorgan (cM) region. In the Pbd2 locus, a high relative bone mass was associated with alleles of the unknown strain, whereas a low relative bone mass was associated with the alleles from the AKR/J strain. These results confirmed the existence of a Pbd2 locus regulating bone density in the SAM strains.

The effects of aminoguanidine supplementation on senescence acceleration in vitro were examined in fibroblasts from the dorsal dermis of newborn SAMP II (accelerated senescence-prone mice), and were compared to the effects in cell lines from SAMRI (accelerated senescence-resistant) mice. Four millimow aminoguanidine supplementation signijicantly de­ layed the senescencelcrisis in eel/lines from SAMP 11 mice, but did not affect the senescencelcrisis in cell lines from SAMRI mice. Flow cytometric analysis of the DNA content of confluent cells revealed that aminoguanidine supplementa­ tion signijicandy decelerated the increase in the number of tetraploid cells until senescencelcrisis. Although mean concen­ trations of lipid peroxides in the primary cultures did not differ signijicantly, considerably higher lipid peroxidatWn was ob­ served in some SAMPll cultures, and aminoguanidine supplementation reduced them to the levels in SAMRI cultures. These results strongly suggest that oxidative stress derivedfrom polyamine catabolism may contribute to the senescence ac­ celeration in vitro in eel/lines from SAMPII mice.

Nucleoside diphosphates linked to moiety X (Nudix) hydrolase functions were investigated in hypoxic Aspergillus nidulans cells. Among three nudix hydrolase isozymes, NdxA transcription was up-regulated under oxygen (O2)-limited conditions. A gene disruptant of the NdxA-encoding gene (NdxAΔ) accumulated more NADH and ADP-ribose than the wild type (WT) under the same conditions. These results indicate that NdxA hydrolyzes these nucleotides in hypoxic fungal cells, which accords with the thesis that NdxA hydrolyzes NADH and ADP-ribose. Under O2-limited conditions, NdxAΔ decreased glucose consumption, the production of ethanol and lactate, cellular ATP levels, and growth as compared with WT. WT cultured under hypoxia converted exogenously added fructose 1,6-bisphophate, a glycolytic intermediate, to glyceraldehyde 3-phosphate (GAP). The hypoxic NdxAΔ cells accumulated 3.0- to 4.2-fold more GAP than WT under the same conditions, indicating that NdxA increased GAP oxidation by a glycolytic mechanism. Steady-state kinetics indicated that NADH and ADP-ribose competitively inhibited fungal GAP dehydrogenase (GAPDH) with Ki values of 34- and 55-µM, respectively. These results indicate that NdxA hydrolyzes cellular NADH- and ADP-ribose, derepresses GAPDH activity, and hence up-regulates glycolysis in hypoxic A. nidulans cells. That NdxAΔ consumed less pyruvate and tricarboxylate cycle intermediates than WT suggests that NdxA-dependent hydrolysis of nucleotides controls the catabolism of these carbon sources under O2-limited conditions.

Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv /Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6 f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb.

This study investigated temporal variations in the potential maximum quantum yield of photosystem II (F(v)/F(m) ratio) and growth-phase dependent cellular protein expressions of Chattonella antiqua under laboratory conditions. Despite the culture conditions, significant positive correlations between the F(v)/F(m) ratio and daily growth rate were observed. Threshold F(v)/F(m) ratios associated with positive cell growth were calculated to be0.44,0.44, and0.37, and those associated with active cell growth (growth rate0.5 div. d(-1)) were0.58,0.60, and0.49 under control culture, low nutrient and intense light conditions, respectively. Proteome profiles obtained by two-dimensional gel electrophoresis (2-DE) indicated that 42 protein spots were differentially expressed at various growth phases of C. antiqua, which indicates changes in cellular physiological status throughout the growth cycle, and suggests that oxygen evolving enhancer 1 and 2-cysteine peroxiredoxin play roles in maintaining the positive growth of C. antiqua.

Telomeres, at the end of chromosomes, shorten with each cell division, resulting in cellular senescence. Tumor cells, unlike normal somatic cells, express a telomerase that maintains the telomere length. Deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma (RCC) and reintroduction of a normal chromosome 3 into an RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental RCC23. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells.

Cellular levels of NAD(+) and NADH are thought to be controlled by de novo and salvage mechanisms, although evidence has not yet indicated that they are regulated by NAD(+) degradation. Here we show that the conserved nudix hydrolase isozyme NdxA hydrolyzes and decreases cellular NAD(+) and NADH in Aspergillus nidulans. The NdxA-deficient fungus accumulated more NAD(+) during the stationary growth phase, indicating that NdxA maintains cellular NAD(+)/NADH homeostasis. The deficient strain also generated less of the secondary metabolites sterigmatocystin and penicillin G and of their gene transcripts than did the wild type. These defects were associated with a reduction in acetylated histone H4 on the gene promoters of aflR and ipnA that are involved in synthesizing secondary metabolites. Thus, NdxA increases acetylation levels of histone H4. We discovered that the novel fungal sirtuin isozyme SirA uses NAD(+) as a cosubstrate to deacetylate the lysine 16 residue of histone H4 on the gene promoter and represses gene expression. The impaired acetylation of histone and secondary metabolite synthesis in the NdxA-deficient strain were restored by eliminating functional SirA, indicating that SirA mediates NdxA-dependent regulation. These results indicated that NdxA controls total levels of NAD(+)/NADH and negatively regulates sirtuin function and chromatin structure.

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.

We performed the whole-genome scan for quantitative trait loci (QTLs) in SAMP6 mice that exhibits a significantly low peak bone mass. We found two significant loci on Chrs 11 and 13 (designated Pbd1 and Pbd2, respectively) and one suggestive locus on Chr X (designated Pbd3 ) that were linked to the bone mass. Next, we clarified that Pbd1 is related to the bone geometry, Pbd2 to the formation of bone before maturation, and Pbd3 to the bone loss after maturation. In particular, we showed that secreted frizzled-related protein 4 (Sfrp4) encoded by a gene within the Pbd2 locus on Chr 13 was responsible, at least in part, for the lower bone mineral density (BMD) in the SAMP6 mice, by inhibiting the proliferation of osteoblasts.

We discovered the phenyllactate (PLA)-producing fungal strain Wickerhamia fluorescens TK1 and purified phenylpyruvate reductase (PPR) from fungal cell-free extracts. The PPR used both NADPH and NADH as cofactors with more preference for the former. The enzyme reaction as well as the fungal culture produced optically active d-PLA. The gene for the PPR (pprA) was cloned and expressed in Escherichia coli cells. Purified preparations of both native and recombinant PPR used hydroxyphenylpyruvate, glyoxylate and hydroxypyruvate as substrates but not pyruvate, oxaloacetate or benzoylformate. The predicted PPR protein had sequence similarity to proteins in the d-isomer-specific 2-hydroxyacid dehydrogenase family. Phylogenetic analyses indicated that the predicted PPR protein together with fungal predicted proteins constitutes a novel group of glyoxylate/hydroxypyruvate reductases. The fungus efficiently converted phenylalanine and phenylpyruvate to d-PLA. These compounds up-regulated the transcription of pprA, suggesting that it plays a role in fungal phenylalanine metabolism.

Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD + and NADPH/NADP + under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD + and NADP + regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

To construct a panel of radiation hybrids (RHs) for human chromosome 3p mapping, mouse microcell hybrid cells, A9(neo3/t)-5, containing a single copy of human chromosome 3p with pSV2neo plasmid DNA integrated at 3p21-p22 were irradiated and fused to mouse A9 cells. A panel of 96RHs that retain several sizes and portions of human chromosome 3p segments was used to map 25 DNA markers for chromosome 3p. Eight of them, H28, H29, H32, H33, H35, H38, H48, and H64, were cloned from Alu -primed PCR products using A9(neo3/t)-5 cell DNA as a template. The most likely order of the 24 markers, except for H28, based on the statistical ordering method proposed by Falk, was cen-D3S4-D3S3-D3S30-H29-D3S13-D3S2-H48-D3F15S2-D3S32-D3S23-CCK-H35-H33-D3S11-D3S12-RARB-THRB(ERBA2-pBH302)-H64-H38-RAF1-D3S18-H32-D3S22-pter. The order and location of these markers were in good agreement with those previously determined by other mapping methods, suggesting that a panel of these 96 RHs is a valuable source for a rapid mapping of human chromosome 3p markers.

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

The tripeptide glutathione is involved in cellular defense mechanisms for xenobiotics and reactive oxygen species. This study investigated glutathione-dependent mechanisms in the model organism Aspergillus nidulans. A recombinant dimeric protein of A. nidulans glutathione reductase (GR) contained FAD and reduced oxidized glutathione (GSSG) using NADPH as an electron donor. A deletion strain of the GR gene (glrA) accumulated less intracellular reduced glutathione (GSH), indicating that the fungal GR contributes to GSSG reduction in vivo. Growth of the deletion strain of glrA was temperature-sensitive, and this phenotype was suppressed by adding GSH to the medium. The strain subsequently accumulated more intracellular superoxide, and cell-free respiration activity was partly defective. Growth of the strain decreased in the presence of oxidants, which induced glrA expression 1.5-6-fold. These results indicated that the fungal glutathione system functions as an antioxidant mechanism in A. nidulans. Our findings further revealed an initial proteomic differential display on GR-depleted and wild type strains. Up-regulation of thioredoxin reductase, peroxiredoxins, catalases, and cytochrome c peroxidase in the glrA-deletion strain revealed interplay between the glutathione system and both the thioredoxin system and hydrogen peroxide defense mechanisms. We also identified a hypothetical, up-regulated protein in the GR-depleted strains as glutathione S-transferase, which is unique among Ascomycetes fungi.

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.

To determine whether sensitivity to polyethylene particles varies among patients, we studied 25 patients who had undergone total hip arthroplasty. We used pelvic radiographs to measure annual polyethylene wear and the area of osteolysis. The ratio of the area of osteolysis to the volumetric polyethylene wear was defined as sensitivity index. Adherent cells from peripheral blood were cocultured with polyethylene particles, and the amount of bone-resorptive cytokines was measured. The amount of interleukin-6, but not of interleukin -1 β or tumor necrosis factor- α , released from adherent cells in the in vitro experiment correlated with the in vivo sensitivity indices. This technique appears capable of predicting the development of polyethylene-induced osteolysis, allowing surgeons to avoid using polyethylene as the bearing surface in patients at risk for osteolysis.

© 2015 Muraguchi et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

We segregated a QTL for peak BMD on Chr 13 by generating congenic sublines of the senescence-accelerated mouse SAMP6. Sfrp 4 within this locus was responsible for lower BMD of SAMP6. Introduction: Our genome-wide linkage study using SAMP6 and SAMP2 showed a significant quantitative trait locus (QTL) for peak BMD on chromosome (Chr) 13. To verify the gene that regulates peak BMD, we generated a congenic strain, P6.P2-Pbd2b, which carried a 15-cM SAMP2 interval on an osteoporotic SAMP6 background, and showed that this Pbd2 locus increased peak BMD in SAMP6. Materials and Methods: To narrow down this interval, we generated a new congenic subline P6.P2–13. We studied the effect of this locus on morphological and histomorphological features in vivo and on osteoblasts in vitro. The levels of expression of all genes in the segregated interval were examined, and we clarified the effect of the candidate gene, secreted frizzled-related protein (Sfrp4), on osteoblasts in vitro. Results: The new congenic strain, P6.P2–13, retained the 2.4-Mb SAMP2 interval on the SAMP6 background, and 11 genes existed in this interval. In morphometrical analysis, P6.P2–13 increased the bone area fraction (BA/TA) by 6.6% at the diaphysial cortex (p < 0.001) and increased the trabecular bone volume (BV/TV) by 54.2% at the distal metaphysis (p < 0.05) in the femora compared with those of SAMP6. The bone formation rate of P6.P2–13 was markedly increased at the periosteal surface of femoral cortex and that was caused by a higher proliferation rate of osteoblasts in P6.P2–13 compared with those in SAMP6. Quantitative RT-PCR analysis of calvaria tissue showed ∼40-fold higher levels of expression of Sfrp4 in SAMP6 than in P6.P2–13. Taken together with the result that recombinant Sfrp4 suppressed the proliferation of osteoblasts, we hypothesized that Sfrp4 inhibited the proliferation of osteoblasts through its antagonistic effect on Wnt signaling. TCF/β-catenin–dependent reporter activity in osteoblasts derived from SAMP6 showed lower responsiveness for the Wnt ligand, Wnt3A, than that in osteoblasts from P6.P2–13. Conclusions: In SAMP6 mice, Sfrp4 negatively regulates bone formation and decreases BMD through the inhibition of Wnt signaling.

Background The wear rate of polyethylene against alumina was demonstrated to be lower than against metal, and the results of early clinical studies of alumina-on-polyethylene combination were as good as expected, but the long-term results of alumina ceramic heads were not as good, as reported by several authors. The purpose of this study was to assess the clinical results over 10 years of cemented total hip arthro-plasty (THA) with two types of alumina head: old and current alumina. Methods We compared the clinical results of these two types of alumina used for cemented THA. The first type was old alumina (containing 7% yttria), and 77 hips with a 28-mm head (group I) were included. The second type was current alumina (highly purified alumina), and 50 hips with a 26-mm head (group II) and 88 hips with a 22-mm head (group III) were included. The mean follow-up was 17 years 4 months in group I, 12 years 3 months in group II, and 10 years in group III. Results The Kaplan-Meier survival analysis, with revision for any reason as the endpoint, predicted 10-year survival rates of 90.8%, 100%, and 97.5% for groups I, II, and III, respectively. The survival curves differed significantly only between groups I and II. The probabilities of 10-year survival of the pros-theses with radiological loosening as the endpoint were 77.2%, 91.6%, and 96.5%, respectively. The survival curves showed significant differences only between groups II and III. Conclusions The old alumina showed a higher wear rate and rougher surface on the femoral head than did the current alumina in our previous study. The clinical results also indicated superiority of current alumina over old alumina. The difference in the size of the femoral head (26 vs. 22 mm) did not affect the clinical results.

Acute displacement of the cemented acetabular component of a hip arthroplasty after attempted reduction of a dislocation is described. The causes of displacement of the socket, which was not radiologically loose, are discussed. Potential causes include impingement of the femoral head and the ceramic screw head overhanging the acetabular bone, which was used to fix the bone graft.

In immortal cells (LCS-AF.1-3) which originated from cultured skin fibroblasts from a patient with Li-Fraumeni syndrome, a specific deletion has been identified on chromosome 6. To examine the relationship between this deletion and the loss of function in aging genes, we performed microcell mediated chromosome transfer (MMCT) of normal human chromosome 6 into LCS-AF.1-3 cells and analyzed their characteristics. Transferred human chromosome 6 induced morphological changes in the cells and cellular senescence with growth suppression. In a revertant clone that had escaped from induced senescence, a specific deletion was found at D6S309 in the transferred chromosome 6. High molecular weight telomeric sequences were lost in a clone that had been induced to senescence by introduction of human chromosome 6. On the other hand, human chromosome 7 induces senescence in immortal cells by suppressing the alternative lengthening of telomere (ALT) mechanism. We also performed MMCT of chromosome 7 and discuss the effect of chromosome transfer by comparing the two chromosomes. LCS-AF.1-3 cells containing a transferred chromosome 7 showed no changes in immortality. These results suggest that genes which are new candidates for aging and which suppress the ALT mechanism are located in the region D6S309 in human chromosome 6.

Previously, we identified two significant quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected for bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains, SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. We recently designated the Chr 13 locus as Pbd2 (Peak bone density 2) and constructed a congenic strain, P6.P2-Pbd2(b), which carried a single genomic interval from the Chr 13 of SAMP2 on a SAMP6-derived osteoporotic background. In this study, we have constructed a congenic strain, P2.P6-Pbd2(a), carrying a SAMP6-derived susceptible interval on a SAMP2-derived resistance background. This congenic strain had a lower bone density than the background strain, SAMP2, based on three measurement methods, each utilizing a different principle for evaluating bone density: MD, DXA, and pQCT. Next, a candidate gene approach was used to find polymorphisms of Bmp6 (bone morphogenetic protein 6). The CAG trinucleotide repeat numbers in exon 1 of this gene differ among SAM strains. We found an association of CAG repeat length with relative peak bone mass in mice.

Maintenance of telomeres, commonly through expression of telomerase activity, is necessary but may not be sufficient for human cells to escape from the cellular senescence program and become immortal. We report here that human tumor cells could undergo cellular senescence in the presence of telomerase activity when a specific normal human chromosome was introduced via microcell-mediated chromosome transfer. The cell models studied include SiHa (uterine cervical carcinoma cells expressing E6 and E7 oncoproteins of human papillomavirus type 16) with a transferred chromosome 2, CC1 (choriocarcinoma cells expressing an amino-terminally truncated p53 protein) with a transferred chromosome 7, and JTC-32 (bladder carcinoma cells) with a transferred chromosome 11. The microcell hybrids with the indicated chromosomes ceased to divide after five to 10 population doublings and showed senescence-associated β-galactosidase activity but still expressed the genes encoding three components of human telomerase, consistent with the retention of telomerase activity. These results are evidence for barriers to human cell immortalization, which involve activation of unidentified senescence-inducing genes that function independently of inactivation of telomerase. Mol. Carcinog. 25:249–255, 1999. © 1999 Wiley-Liss, Inc.

SAMP6 mice are a murine model for senile osteoporosis, characterized by low peak bone mass seen at 4 or 5 months of age. Parathyroid hormone (PTH)-induced c- fos expression was examined in the bones, bone-marrow cells and kidney tissues of 2-month-old male SAMP6 mice. SAMP2 mice, which have a higher peak bone mass, were used as controls. The expression of c- fos in the bone peaked at 30 min after 60 μg/kg of human PTH(1–34) administration. After peaking, the expression fell quickly in SAMP2 mice. This decrease in expression was delayed in SAMP6 mice and the expression was higher at 1 h than in SAMP2 mice. The phenomenon observed in the bone appears to be tissue specific as it was not seen in the bone-marrow cells or kidney tissue. Immunohistochemical studies showed that c-Fos protein was localized to the nuclei of some of the osteocytes and a few of the osteoblasts in the cortical bone, and that osteocytes expressing c-Fos protein increased after PTH treatment. These results suggest that osteocytes might contribute to the maintenance of higher levels of c- fos expression in the bones of SAMP6 mice and may be related to cortical osteopenia in these mice by modulating bone remodeling and/or modeling.

The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series.

The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.

Telomeres, which are the repeated sequences located on both ends of chromosomes in eukaryotes, are known to shorten with each cell division, and their eventual loss is thought to result in cellular senescence. Unlike normal somatic cells, most tumor cells show activation of telomerase, a ribonucleoprotein enzyme that stably maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. The KC12 cell line derived from a renal cell carcinoma in a patient with von Hippel-Lindau disease showed telomerase activity and loss of heterozygosity on the short arm of chromosome 3. Introduction of a normal human chromosome 3 into KC12 cells by microcell fusion induced cellular senescence, accompanied by suppression of telomerase activity and shortening of telomere length. Microcell hybrids that escaped from cellular senescence maintained telomere length and telomerase activity similar to those of the parental KC12 cells. We previously showed a similar suppression of telomerase activity by introduction of chromosome 3 into another renal cell carcinoma cell line, RCC23. The putative telomerase repressor gene was mapped to chromosome region 3p14.2-p21.1 by deletion mapping of KC12 + chromosome 3 revertants that escaped from cellular senescence and by transfer of subchromosomal fragments of chromosome 3 into RCC23 cells.

Human telomerase, a ribonucleoprotein that adds TTAGGG repeats onto telomeres and compensates for their shortening, is repressed in most normal human somatic cells. Human somatic cells are considered to have a limited proliferation capacity because of the telomere shortening. Although immortalization of somatic cells is often associated with telomerase reactivation, there are some immortal cells in which telomerase activity is undetectable. In these cells, telomeres may be maintained by an unknown mechanism other than telomerase reactivation. To examine the genetic regulation of telomerase activity, we constructed hybrids between immortal cells with (HepG2) and without (KMST6) telomerase activity. These two cell lines had relatively short and long telomeres, respectively. The hybrid cells continued to proliferate without detectable telomerase activity even after 100 population doublings. Telomerase-positive subpopulations occasionally appeared after serial passages. Southern blot analysis revealed that the hybrids had long terminal restriction fragments similar to that of KMST6, regardless of telomerase activity, and fluorescence in situ hybridization with a telomeric probe showed high-intensity hybridization signals on telomeres, indicating relatively long telomeric repeats. These results suggest that the telomerase-negative immortal cells contain a gene or genes functioning as a telomerase repressor and maintain telomere length by a dominant mechanism other than telomerase reactivation.

The YL-1 gene, encoding a novel nuclear protein with transcription factor-like features, has been isolated from the human chromosome 1q21, one of the regions supposedly carrying a transformation suppressor gene(s) for Kirsten sarcoma virus-transformed NIH3T3 (DT) cells. To test the suppressive activity of the YL-1 gene product, we forced the expression of human YL-1 cDNA in DT cells. The anchorage-independent growth (colony-forming ability in soft agar medium) was markedly suppressed in cells highly expressing the exogenous human YL-1 protein. Moreover, the soft agar clones, which were rarely originated from these cells, expressed reduced levels of exogenous YL-1 or none, with or without the loss/rearrangement of the introduced cDNA. In control experiments, cells carrying an introduced vector alone or an antisense-strand expression plasmid grew in soft agar as efficiently as parental DT cells. In contrast to the suppression of anchorage-independent growth, the forced expression of YL-1 did not effect the transformed phenotypes in adherent culture and tumorigenicity in nude mice. These findings not only indicated that the YL-1 protein functions as a transformation suppressor, but also suggest that it may be important for elucidating anchorage independence under separate genetic control from other transformed phenotypes.

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms. © 1994 Wiley-Liss, Inc.

Cytogenetic and molecular studies of human renal cell carcinoma (RCC) have suggested that the genetic and functional losses of one or more putative tumor suppressor genes on the short arm of chromosome 3 play a crucial role in the development of this disease. To examine whether the introduction of chromosome 3 has any effects on the biology of RCC cells, we introduced either chromosome 3, 7, or 11 from normal human fibroblasts into a newly established human RCC cell line with loss of heterozygosity for 3p, via microcell-mediated chromosome transfer. Microcell hybrids containing an introduced, intact chromosome 3 showed a significant reduction in in vitro growth rate and saturation density together with morphological alteration; these properties were not altered in microcell hybrids containing an introduced chromosome 7 or 11. During long-term cultivation, one of the clones that had lost the introduced chromosome 3 showed growth properties and morphology similar to those of the parental cell lines. Thus, our findings provide additional evidence for the presence of a putative tumor suppressor gene or genes on normal chromosome 3p and indicate that the gene is a dominant, negative growth regulator whose loss promotes progressive features of the neoplastic phenotype. © 1994 Wiley-Liss, Inc.

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with 60Co gamma-rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22--qter or del(8)q23--qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybrids, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1--q11.1.

The human gene encoding pituitary adenylate cyclase activating polypeptide (PACAP) was isolated and its nucleotide sequence was determined. By comparison with a human PACAP cDNA, the exon/intron organization of PACAP gene was determined. The last exon encoded the longer form of PACAP, PACAP38 and 3′-untranslated sequences, suggesting that the shorter form of PACAP, PACAP27 is not generated by alternative splicing mechanisms. The 5′-flanking region of the PACAP gene contains several sequence motifs homologous to CRE, TRE, and GHF-1. On the basis of DNA isolated from mouse A9 microcell hybrid clone containing a single human chromosome, the PACAP gene was assigned to human chromosome 18. Furthermore, we determined the locus of the gene to be 18p11 by the chromosomal in situ hybridization technique.

The selective polymerization behavior of O-ethyl S-4-vinylbenzyl xanthate (1) was investigated on the basis of polymerization kinetics in the dark and in the presence of UV-light. S-Benzyl O-ethyl xanthate (2) was used as a non-polymerizable model compound of 1 to evaluate chain transfer constants. In the homopolymerization and the copolymerization of 1 initiated with AIBN in the dark, styryl groups selectively take part in the polymerization, and xanthate groups are not responsible for it. The monomer reactivity ratios (r1 = 0,51 and rMMA = 0,41) of 1 with methyl methacrylate (MMA) are very close to those of styrene with MMA. The photolysis of 2 was investigated by 1H NMR. It was found that photodecomposition of 2 takes place at CH2S (a) and SC( = S) bonds (b) with the ratio: a/b = 1/5. The polymerization of MMA was carried out with 1 upon photoirradiation. Number-average degrees of polymerization of the polymers obtained increase linearly with conversion. Block and graft copolymers were prepared by using these macrophotoinitiators.

A leukocytosis promotion-inhibiting factor (LPIF) was isolated and purified from crude bovine bile. The purified LPIF was homogeneous on 15% polyacrylamide gel electrophoresis. It inhibits the effects of leukocytosis-promoting substance in rabbits. The molecular weight of LPIF was estimated to be 1.2×104, and the isoelectric point was at pH 8.2. The active component dissociated into two subcomponents with molecular weights of 9000 and 3000 on sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis.

In order to obtain further information on the effects of cadmium (Cd) on the mechanism of activation of vitamin D3 in the kidney, the serum concentrations of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] were determined by radioimmunoassay techniques. The serum concentration of 1,25(OH)2D3 in Cd-exposed rats was always higher than that in control rats. The concentrations of serum 1,25(OH)2D3 in parathyroidectomized rats (PTX), both in the control and in the Cd-exposed groups, were markedly lower than those in non-PTX rats. On the other hand, the concentration of serum 24,25(OH)2D3 in Cd-exposed rats was less than that in control rats. In other words, the pathway from secretion of parathyroid hormone (PTH) to synthesis of 1,25(OH)2D3 or 24,25(OH)2D3 was normal, but the pathway from stimulation of PTH to secretion of PTH was abnormal. These results suggest that rats exposed to Cd for 90 days were in a state of either deficiency of 1,25(OH)2D3 or excess secretion of parathyroid hormone. Although hypophosphatemia occurred in the Cd-exposed rats, the 1,25(OH)2D3 serum level in rats was not increased by PTX. On the basis of these results, it is suggested that hypophosphatemia occurring after the exposure of rats to Cd is a secondary hypophosphatemia.

Both photopolymerization and thermal polymerization of a variety of vinyl monomers with bis(isopropylxanthogen) disulfide (BX) as initiator and as chain-transfer agent were carried out in benzene. The results of photopolymerizations to high conversion with BX support the polymerization mechanism given in a previous paper of this series. Telechelic isopropyl xanthate-terminated polymers (TXP) having a different backbone have been used as macroinitiator for the polymerization of vinyl monomers. The rate of polymerization of the functional polymers was found to be markedly dependent on the monomer species: block polymerization of methyl acrylate or methyl methacrylate proceeded rapidly compared to the chain-extension polymerization of styrene. However, in all cases the molecular weight of the polymers obtained was found to increase linearly with conversion, which is an interesting property for the design of block copolymers and which is similar in polymerization with BX. It is also found that the mo...