Isovaleric acid ameliorates ovariectomy-induced osteoporosis by inhibiting osteoclast differentiation

Osteoclasts (OCs) play important roles in bone remodelling and contribute to bone loss by increasing bone resorption activity. Excessively activated OCs cause diverse bone disorders including osteoporosis. Isovaleric acid (IVA), also known as 3-methylbutanoic acid is a 5-carbon branched-chain fatty acid (BCFA), which can be generated by bacterial fermentation of a leucine-rich diet. Here, we find that IVA suppresses differentiation of bone marrow-derived macrophages into OCs by RANKL. IVA inhibited the expression of OC-related genes. IVA-induced inhibitory effects on OC generation were attenuated by pertussis toxin but not by H89, suggesting a G_i-coupled receptor-dependent but protein kinase A-independent response. Moreover, IVA stimulates AMPK phosphorylation, and treatment with an AMPK inhibitor blocks IVA-induced inhibition of OC generation. In an ovariectomized mouse model, addition of IVA to the drinking water resulted in significant decrease of body weight gain and inhibited the expression of not only OC-related genes but also fusogenic genes in the bone tissue. IVA exposure also blocked bone destruction and OC generation in the bone tissue of ovariectomized mice. Collectively, the results demonstrate that IVA is a novel bioactive BCFA that inhibits OC differentiation, suggesting that IVA can be considered a useful material to control osteoclast-associated bone disorders, including osteoporosis.     

Involvement of histidine and tryptophan residues of glutamine binding protein in the interaction with membrane-bound components of the glutamine transport system of Escherichia coli

We treated the glutamine binding protein with diethyl pyrocarbonate (DEPC) and N-bromosuccinimide (NBS) to modify respectively the sole histidine and tryptophan residues and examined the effect of these modifications on the ability of the binding protein to bind glutamine as well as the ability to restore glutamine transport in membrane vesicles of Escherichia coli. Under the conditions used, both DEPC and NBS markedly inhibited the ability to restore glutamine transport in vesicles without any significant effect on glutamine binding. Moreover, saturating quantities of glutamine had no protective effect on the inactivation of the binding protein by DEPC or NBS. Fluorometric measurement and amino acid analysis indicate that the inactivation of the binding protein in restoring vesicle transport by NBS can be attributed to the oxidation of a single tryptophan residue. Similar analysis and the inability of hydroxylamine to reverse the effect of DEPC indicate that the effects of DEPC can probably be attributed to alterations of the sole histidine and/or one or more lysine residues of the binding protein. We conclude that the glutamine binding protein possesses at least two largely nonoverlapping functional domains, one responsible for glutamine binding and the other for the interaction with the other components of the glutamine transport system.     

NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.     

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.     

Telomere instability in a human tumor cell line expressing NBS1 with mutations at sites phosphorylated by ATM

Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1(S278A/S343A) gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1(S278A/S343A). The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1(S278A/S343A) promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.     

Clinical and genealogical characteristics in families with Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency and high predisposition for malignancies, particularly B-lymphoma. Clinical and genealogical analysis has been conducted in 7 families with NBS. Eight children with NBS (5 boys and 3 girls) were observed at the age from 7 months to 11 years. All the children were homozygous carriers for mutation 657del5. Oncohematological complications developed in 5 cases (4 cases of lymphoma and one case of lymphohystiocytosis) at the age of 6-12 years. NBS in probands is often accompanied with birth defects, especially with kidney pathologies. Considerable reproductive losts in the families with NBS were noted mainly among males who died at the age less than one year (4-6 events in the families). The cases of digestive system cancers (stomach, rectum, duodenum) were revieled in the family-trees. Consanguineous couple was observed in 1 case (marriage between third cousins) and 2 children had developed NBS in this family. Genealogical analysis seems to be very informative to predict somatic and reproductive disturbances in NBS families.     

A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes

Plant disease resistance (R) genes encode proteins in which several motifs of the nucleotide-binding region (NBS) are highly conserved. Using degenerate primers designed according to the kinase 1 (P-loop) and hydrophobic (HD) motifs of the R gene NBS domains, homologous sequences were cloned from moss (Physcomitrella patens; phylum Bryophyta) representing an ancient nonvascular plant. A novel gene family (PpC) with at least eight homologous members was found. Expression of five members was detected. The level of expression was dependent on the developmental stage of moss, being higher in the gametophyte tissue than in the protonema tissue. The PpCs contained the conserved motifs characteristic of the NBS regions of R genes, and a kinase domain was found upstream from the NBS region. Phylogenetic analysis using the deduced NBS amino acid sequences of the PpCs and the plant genes available in databanks indicated that the PpCs show the closest relationship with the TIR-NBS class of R genes. No significant similarity to plant genes other than R genes was observed. These findings shed novel light on the evolutionary history of the R gene families, suggesting that the NBS region characteristic of the TIR-NBS class of R-like genes evolved prior to the evolutionary differentiation of vascular and nonvascular plants.     

Natural variation, functional divergence, and local adaptation of nucleotide binding site sequences in Rhododendron (Ericaceae)

To further understand natural variation and local adaptation in the evolution of plant defense, we analyzed polymorphism data of nucleotide-binding site (NBS) sequences of Rhododendron at both the species and population levels. Multiple duplication events were found in NBS sequence evolution in Rhododendron genomes, which resulted in six clades: A–F. Our results of several NBS clade pair comparisons showed significant evolutionary rate changes based on differences in substitution rates between NBS-encoding protein clades (type I functional divergence). Pairwise comparisons of NBS clades further revealed that many amino acids displayed radical biochemical property changes causing a shift in amino acid preferences between NBS-encoding protein clades (type II functional divergence). Such divergent evolution of NBSs is likely a consequence of positive selection related to differentiation of recognition signals in response to different pathogens. Primers specific to clades B and C, which differed in the number of radical amino acid changes causing type II functional divergence and levels of nucleotide diversities, were further used to amplify population clades B and C NBS sequences of Rhododendron formosanum populations. Higher levels of net nucleotide divergences (measured by D _a) between R. formosanum populations were found based on NBS sequences of population clade B compared to population clade C, suggesting local adaptation of population clade B NBS sequences. Local adaptation can be further inferred for R. formosanum population clade B NBS sequences because of significant Φ _ST based on variation in nonsynonymous substitutions. Furthermore, local adaptation was also suggested by no significant correlation of population pairwise F _ST between population clades B and C in R. formosanum.     

Preservation of coronary reserve by ivabradine-induced reduction in heart rate in infarcted rats is associated with decrease in perivascular collagen

We tested the hypothesis that chronically reducing the heart rate in infarcted middle-aged rats using ivabradine (IVA) would induce arteriolar growth and attenuate perivascular collagen and, thereby, improve maximal perfusion and coronary reserve in the surviving myocardium. Myocardial infarction (MI) was induced in 12-mo-old male Sprague-Dawley rats, which were then treated with either IVA (10.5 mg.kg(-1).day(-1); MI + IVA) or placebo (MI) via intraperitoneal osmotic pumps for 4 wk. Four weeks of IVA treatment limited the increase in left ventricular end-diastolic pressure and the decrease in ejection fraction but did not affect the size of the infarct, the magnitude of myocyte hypertrophy, or the degree of arteriolar and capillary growth. However, treatment reduced interstitial and periarteriolar collagen in the surviving myocardium of MI + IVA rats. The reduced periarteriolar collagen content was associated with improvement in maximal myocardial perfusion and coronary reserve. Although the rates of proliferation of periarteriolar fibroblasts were similar in the MI and MI + IVA groups, the expression levels of the AT(1) receptor and transforming growth factor (TGF)-beta(1) in the myocardium, as well as the plasma level of the ANG II peptide, were lower in treated rats 14 days after MI. Therefore, our data reveal that improved maximal myocardial perfusion and coronary reserve in MI + IVA rats are most likely the result of reduced periarteriolar collagen rather than enhanced arteriolar growth.     

Structure-activity relationships of some selected beta-adrenergic blocking agents--oxidation with N-bromosuccinimide.

A relationship between in vitro rate of oxidation by N-bromosuccinimide (NBS) and the pharmacologic activity (pA2) of different beta-adrenergic blockers for different blocking agent-tissue combinations has been studied. The rates of oxidation of the alcoholic group in the drugs by NBS, as well as their molecular conformations as represented by molecular models, were studied in order to determine requirements for selectivity and potency of action of beta-adrenergic blocking agents. Using data from all 7 drugs studied--both nonselective and selective blocking agents--no significant correlation between pA2 and -log k2 (k2 is the second order rate constant for the oxidative reaction) was found. If data from only the 4 nonselective agents were used (16 drug-tissue combinations), a correlation significant at p less than 0.01 was found. Hypotheses are presented to account for the selective action of some beta-adrenergic blocking agents.     

Relative evolutionary rates of NBS-encoding genes revealed by soybean segmental duplication

It is well known that nucleotide binding site (NBS)-encoding genes are duplicate-rich and fast-evolving genes. However, there is little information on the relative importance of tandem and segmental NBS duplicates and their exact evolutionary rates. The two rounds of large-scale duplication that have occurred in soybean provide a unique opportunity to investigate these issues. Comparison of NBS and non-NBS genes on segments of syntenic homoeologs shows that NBS-encoding genes evolve at least 1.5-fold faster (~1.5-fold higher synonymous and approximately 2.3-fold higher nonsynonymous substitution rates) and lose their genes approximately twofold faster than the flanking non-NBS genes. Compared with segmental duplicates, tandem NBS duplicates are more abundant in soybean, suggesting that tandem duplication is the major driving force in the expansion of NBS genes. Notably, significant sequence exchanges along with significantly positive selection were detected in most tandem-duplicated NBS gene families. The results suggest that the rapid evolution of NBS genes may be due to the combined effects of diversifying selection and frequent sequence exchanges. Interestingly, TIR-NBS-LRR genes (TNLs) have a higher nucleotide substitution rate than non-TNLs, indicating that these types of NBS genes may have a rather different evolutionary pattern. It is important to determine the exact relative evolutionary rates of TNL, non-TNL, and non-NBS genes in order to understand how fast the host plant can adjust its response to rapidly evolving pathogens in a coevolutionary context.     

NBS1 cooperates with homologous recombination to counteract chromosome breakage during replication

Nijmegen breakage syndrome (NBS) is characterized by genome instability and cancer predisposition. NBS patients contain a mutation in the NBS1 gene, which encodes the NBS1 component of the DNA double-strand break (DSB) response complex MRE11/RAD50/NBS1. To investigate the NBS phenotype in more detail, we combined the mouse mimic of the most common patient mutation (Nbs1^ΔB/ΔB) with a Rad54 null mutation, which diminishes homologous recombination. Double mutant cells were particularly sensitive to treatments that cause single strand breaks (SSBs), presumably because these SSBs can be converted into detrimental DSBs upon passage of a replication fork. The persistent presence of nuclear RAD51 foci and increased levels of chromatid type breaks in metaphase spreads indicated that replication-associated DSBs are repaired inefficiently in the double mutant cells. We conclude that Nbs1 and Rad54 function cooperatively, but in separate pathways to counteract this type of DNA damage and discuss mechanistic implications of these findings.     

NBS1 prevents chromatid-type aberrations through ATM-dependent interactions with SMC1

Antoccia, A., Sakamoto, S., Matsuura, S., Tauchi, H. and Komatsu, K., NBS1 Prevents Chromatid-Type Aberrations through ATM-Dependent Interactions with SMC1. Radiat. Res. 170, 345-352 (2008).Nijmegen breakage syndrome shares several common cellular features with ataxia telangiectasia, including chromosomal instability and aberrant S- and G(2)-phase checkpoint regulation. We show here that after irradiation, NBS1 interacts physically with both BRCA1 and SMC1, a component of the cohesin complex, and that their interactions are completely abolished in AT cells. It is noted that BRCA1 is required for the interaction of NBS1 with SMC1, whereas the reverse is not the case, since BRCA1 is able to bind to NBS1 in the absence of an NBS1/SMC1 interaction as observed in MRE11- or RAD50-deficient cells. This indicates that ATM and BRCA1 are upstream of the NBS1/SMC1 interaction. Furthermore, the interaction of NBS1 with SMC1 requires both conserved domains of NBS in the N-terminus and the C-terminus, since they are indispensable for binding of NBS1 to BRCA1 and to MRE11/ATM, respectively. The interaction of NBS1 with SMC1 and the resulting phosphorylation are compromised in the clones lacking either the N- or C-terminus of NBS1, and as a consequence, chromatid-type aberrations are enhanced after irradiation. Our results reveal that ATM plays a fundamental role in promoting the radiation-induced interaction of NBS1 with SMC1 in the presence of BRCA1, leading to the maintenance of chromosomal integrity.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.     

Recruitment of NBS1 into PML oncogenic domains via interaction with SP100 protein

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, chromosomal instability, radiation sensitivity, and an increased incidence of malignancies. NBS1, the protein responsible for NBS, forms a complex with MRE11 and RAD50, and plays a vital role in DNA repair, cell cycle checkpoint, and telomere maintenance. Here, we show that a BRCA carboxyl terminus (BRCT) domain-containing region of NBS1 interacts with a nuclear dots-associated protein, SP100. The SP100 and NBS1 proteins co-localized in PODs and APBs in normal human fibroblast MRC5 and ALT line VA13 at G2 phase, respectively. Introduction of PML and SP100 into NT2 cells, which express no detectable amount of PML or SP100 proteins, resulted in localization of NBS1 in ectopically expressed PODs. These results indicate that NBS1 is recruited into PODs via interaction with SP100 protein. Thus, interaction between the NBS1 and SP100 proteins may be involved in genomic stability and telomere maintenance.     

Genetic variation within and between winter wheat genotypes from Turkey, Kazakhstan, and Europe as determined by nucleotide-binding-site profiling

The genetic diversity within wheat breeding programs across Turkey and Kazakhstan was compared with a selection of European cultivars that represented the genetic diversity across eight European countries and six decades of wheat breeding. To focus the measure of genetic diversity on that relevant to disease-resistant phenotypes, nucleotide-binding-site (NBS) profiling was used to detect polymorphisms associated with the NBS motifs found within the NBS--leucine-rich repeat (LRR) class of resistance (R) genes. Cereal-specific NBS primers, designed specifically to the conserved NBS motifs found within cereal R-genes, provided distinct NBS profiles. Although the genetic diversity associated with NBS motifs was only slightly higher within the Eastern wheat genotypes, the NBS profiles produced by Eastern and European wheat lines differed considerably. Structure analysis divided the wheat genotypes into four groups, which compared well with the origin of the wheat genotypes. The highest levels of genetic diversity were seen for the wheat genotypes from the Genetic Resource Collection held in Ankara, Turkey, as wheat genotypes within breeding programs were genetically more similar. The wheat genotypes from Kazakhstan were the most similar to the European cultivars, reflecting the significant number of eastern European cultivars used in the breeding program in Kazakhstan. In general, the NBS profiles suggested that NBS-LRR R-gene usage in winter wheat breeding in Turkey and Kazakhstan differed from that deployed in European cultivars.     

Macrophage catabolism of lipid A is regulated by endotoxin stimulation

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.     

Endotoxin biosynthesis in Pseudomonas aeruginosa: enzymatic incorporation of laurate before 3-deoxy-D-manno-octulosonate.

Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria.     

Group IVA phospholipase A2 is associated with the storage of lipids in adipose tissue and liver

Prostaglandin (PG) E(2) is considered to participate in the storage of fat in adipocytes and hepatocytes, but roles of group IVA phospholipase A(2) (PLA(2)), a key PLA(2) isozyme in the arachidonic acid cascade, remain unclear. The present study examined the possible involvement of the enzyme using group IVA PLA(2)-deficient mice (C57BL/6 background, 22 weeks of age) fed a normal diet (5.3% fat). The ratio of epididymal fat pad weight to body weight was significantly reduced in group IVA PLA(2)-deficient mice compared to wild-type mice. Histological analysis revealed that in group IVA PLA(2)-deficient mice, the adipocytes were smaller, and hepatocytes bearing cytoplasmic vacuolation were scarce. Hepatic triglyceride content and the serum levels of PGE(2) in the deficient mice were also lower. However, there was no difference in the serum levels of insulin, glucose, non-esterified free fatty acid, or total cholesterol between the deficient and wild-type mice. Our findings suggest that group IVA PLA(2) is involved in the storage of lipids in the adipose tissue and liver and in determining circulating PGE(2) levels.     

RAD50/MRE11/NBS1 proteins in relation to tumour development and prognosis in patients with microsatellite stable colorectal cancer

RAD50/MRE11/NBS1 complex is essential for DNA double-strand break repair and for maintaining genomic integrity. In this study, we immunohistochemically examined MRE11, NBS1 and RAD50 expression in primary CRCs (n=208), the corresponding distant (n=41) and adjacent normal mucosa (n=130), and lymph node metastases (n=26), and investigated their clinicopathological significance in colorectal cancers (CRCs). We found that the intensity and percentage of MRE11 and NBS1 in primary CRCs were positively correlated with each other and with RAD50 (P<0.0001). Strong expression of MRE11, NBS1 or combined RAD50/MRE11/NBS1 was related to MSS, positive hMLH1 expression, earlier tumour stage (TNM stage I and II) and favourable survival (P<0.05). A high percentage of MRE11 expression was associated with less local recurrence and high apoptotic activity (P<0.05). In MSS CRCs, the expression of MRE11 and NBS1 was stronger than that in normal mucosa (P<0.05), and strong expression of NBS1 in primary tumour was related to favourable survival of patients in TNM stage I and II (univariate analysis: P=0.03; multivariate analysis: P=0.07). In MSI CRCs, neither MRE11 nor NBS1 expression showed differences among normal mucosa, primary tumour and metastasis, or among clinicopathological variables. In conclusion, RAD50/MRE11/NBS1 proteins interacted with each other, which had different clinicopathological significance in MSS and MSI CRCs, and further, each component of the complex might have additional roles. NBS1 might be a prognostic factor for patients with MSS tumour in TNM stage I and II.