Positional cloning of the gene for Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT. Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis. Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region. The gene comprises 50 kb and encodes a protein of 754 amino acids. The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins. The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination. We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.

The combination of hyperthermia or chemotherapy with gimeracil for effective radiosensitization.

PURPOSE: 5-chloro-2,4-dihydroxypyridine (gimeracil) is a component of the oral fluoropyrimidine derivative S-1. Gimeracil was originally added to S-1 to yield prolonged 5-fluorouracil (5-FU) concentrations in serum and tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We previously demonstrated that gimeracil enhances the efficacy of radiotherapy through the suppression of homologous recombination (HR) in DNA double strand repair. The goal of this paper was to examine the effects of gimeracil on the sensitivity of anticancer drugs and hyperthermia in order to obtain effective radiosensitization. MATERIALS AND METHODS: Various cell lines, including DLD 1 (human colon carcinoma cells) and cells deficient in HR or nonhomologous end-joining (NHEJ), were used in clonogenic assays. The survival of these cells after various treatments (e.g., drug treatment, heat treatment, and radiation) was determined based on their colony-forming ability. RESULTS: Gimeracil enhanced cell-killing effects of camptothecin (CPT), 5-FU, and hydroxyurea. Gimeracil sensitized effects of CPT or 5-FU to cells deficient in HR or NHEJ to a similar extent as in other cells (DLD1 and a parent cell), indicating that its sensitizing mechanisms may be different from inhibition of HR or NHEJ. Combination of gimeracil and CPT or 5-FU sensitized radiation more effectively than each modality alone. Gimeracil also enhanced heat sensitivity at 42°C or more. The degree of heat sensitization with gimeracil increased as the temperature increased, and the combination of gimeracil and heat-sensitized radiation was more effective than each modality alone. CONCLUSION: Gimeracil enhanced sensitivity of CPT, 5-FU, and hyperthermia. Combination of these modalities sensitized radiation more efficiently than each modality alone.

Gut microbiota development of preterm infants hospitalised in intensive care units.

Gut microbiome development affects infant health and postnatal physiology. The gut microbe assemblages of preterm infants have been reported to be different from that of healthy term infants. However, the patterns of ecosystem development and inter-individual differences remain poorly understood. We investigated hospitalised preterm infant gut microbiota development using 16S rRNA gene amplicons and the metabolic profiles of 268 stool samples obtained from 17 intensive care and 42 term infants to elucidate the dynamics and equilibria of the developing microbiota. Infant gut microbiota were predominated by Gram-positive cocci, Enterobacteriaceae or Bifidobacteriaceae, which showed sequential transitions to Bifidobacteriaceae-dominated microbiota. In neonatal intensive care unit preterm infants (NICU preterm infants), Staphylococcaceae abundance was higher immediately after birth than in healthy term infants, and Bifidobacteriaceae colonisation tended to be delayed. No specific NICU-cared infant enterotype-like cluster was observed, suggesting that the constrained environment only affected the pace of transition, but not infant gut microbiota equilibrium. Moreover, infants with Bifidobacteriaceae-dominated microbiota showed higher acetate concentrations and lower pH, which have been associated with host health. Our data provides an in-depth understanding of gut microbiota development in NICU preterm infants and complements earlier studies. Understanding the patterns and inter-individual differences of the preterm infant gut ecosystem is the first step towards controlling the risk of diseases in premature infants by targeting intestinal microbiota.

Homologous recombination repair is regulated by domains at the N- and C-terminus of NBS1 and is dissociated with ATM functions.

The proteins responsible for radiation sensitive disorders, NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme C-terminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM-independent fashion.

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

Nijmegen breakage syndrome shares several common cellular features with ataxia telangiectasia, including chromosomal instability and aberrant S- and G2-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.

Chk2 activation dependence on Nbs1 after DNA damage.

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation.

Vascular endothelial growth factor (VEGF) has been identified as a peptide growth factor specific for vascular endothelial cells. In this study, we demonstrated the expression of the KDR gene transcript, which encodes a cell surface receptor for VEGF, in normal human hematopoietic stem cells, megakaryocytes, and platelets as well as in human leukemia cell lines, HEL and CMK86. Moreover, we showed the expression of VEGF gene transcript in these normal fresh cells and cell lines. To elucidate biological functions of VEGF on hematopoiesis, we determined whether this growth factor has mitogenic activity to hematopoietic cells or the ability to suppress apoptotic cell death. The liquid culture and colony-formation assay revealed that VEGF suppressed apoptotic cell death of both CMK86 cells and normal hematopoietic stem cells caused by gamma-ray irradiation, although mitogenic activity of VEGF was not detected. The ability of VEGF to suppress apoptotic cell death was independent of the change of cell cycle distribution. These data suggest that VEGF may play an important role in survival or maintenance of hematopoietic stem cells due to the prevention of apoptotic cell death caused by some stresses such as ionizing radiation and that VEGF may give leukemia cells some abilities of resistance against radiotherapy in an autocrine or paracrine manner.

Mutations of a novel human RAD54 homologue, RAD54B, in primary cancer.

Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination. The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis. However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer. Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases. The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene. The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination. These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities. Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer. These findings suggest that some cancers arise through alterations of the RAD54B function.

Age-related alteration of renal brush border leucine aminopeptidase in rat kidney cortex.

Renal brush border enzyme activities were significantly decreased with age. The decrease was observed in the homogenate and brush border fractions. Purified leucine aminopeptidase was significantly decreased in Vmax and Km value for leucyl-beta-naphthylamide in the old rats. Heat stability of leucine aminopeptidase from both old and young rats showed biphasic, and the enzyme from old rats was more stable at 60 degrees C than that of the young. However, other properties such as molecular weight, antigenicity, charge, and optimum pH were not significantly different between young and old rats. From these results, it is suggested that age-related alteration of leucine aminopeptidase is due to a conformational change of enzyme molecule; the conformational change might occur at the active sites of the enzyme.

Difference in activation by Tris(hydroxymethyl)aminomethane of Ca,Mg-ATPase activity between young and old rat skeletal muscles.

Fresh weight of rat skeletal muscles (M. tibialis anterior) was decreased with age. Specific activity of myosin-ATPase in the homogenate was decreased significantly at later stages of age, but not Ca,Mg-ATPase activity. The activity of Ca,Mg-ATPase was activated by high concentration (more than 75 mM) of Tris(hydroxymethyl)aminomethane. The degree of the activation was observed to be an age-related change; the activation of Ca,Mg-ATPase activity in old rats was lower than that of young rats.

Changes in hepatic cell mitochondria during parabiosis between old and young rats.

Electron-micrographic measurement studies have been made on the hepatic cell mitochondria of the long-lived parabiotic unions between old and young rats. The mitochondria of the younger partners were significantly larger in size and those of the older partners were significantly larger in number and smaller in size compared with those of young and old control rats, respectively. The mitochondrial cristae seemed to become more compactly arranged in the younger partners and more sparsely in the older ones. The influence of parabiosis between different aged rats upon age changes in mitochondria and their function is discussed.

Change of the hepatic cells in parabiosis between old and young rats.

The hepatic cells of the parabiotic unions 3 to 9 months after uniting between old and young rats were micrometrically examined. In the older parabiotic partners, it cannot be denied that the character of the findings tended in part towards that of the young controls. In the younger parabiotic partners, the estimated number of hepatic cells was apparently decreased, size of the hepatic cells and their nuclei was significantly increased, and number of binucleate hepatic cells was markedly increased. The findings of the hepatic cells in the younger partners were similar to those in the old controls. From the results, it is inferred that the proliferation of the hepatic cells of the younger parabiotic partners is inhibited by the inhibitory factors for cell division secreted from the hepatic cells of the older partners.

Effect of environmental conditions upon age changes in the human liver.

Age changes in the liver of the Hawaii-Japanese have been compared with those of native Japanese and Caucasians in the United States. Among the Japanese in Hawaii, all cases over 70 years of age were migrant Japanese to Hawaii, i.e. Issei, those under 60 years of age were their Hawaii born descendents, i.e. Nisei, and cases in the 7th decade were composed of Nisei and Issei. Aging changes of the liver seem to begin earlier in the Nisei and Caucasians than in the native Japanese. The earlier onset of aging can be explained on the basis of earlier maturation due to better nutritional conditions. The marked decrease in the number of hepatic cells in the Issei may well be a reflection of their nutritional state during their early years. They, in this sense, resemble the native Japanese.

Age changes of mitochondria of rat kidney.

Age-related changes of the mitochondria in the renal proximal convoluted tubules, using 2-, 12-, 18- and 25-29-month-old rats, were morphometrically examined with reference to the mitochondrial function. Mitochondrial volume per cell was significantly larger in 12- and 18-month-old rats compared with that in 2- and 25-29-month-old animals. The total volume of mitochondria in the proximal convoluted tubules was estimated to be maximal in the 18-month-old rats, and decreased significantly in the 25-29-month-old ones; the mitochondrial cristae were arranged most sparsely in the former case and became significantly compact in the latter. The number of epithelial cells in the 25-29-month-old rats was also significantly smaller than in the 18-month-old rats. From the above, in the 25-29-month-old rats, each mitochondrion seems to function more actively to compensate for the numerical loss of cells and their mitochondria.

Age changes in human vocal muscle.

Micromeasuring studies have been made on the senile changes of the vocal muscle in 109 Japanese males of various ages (18--97 years). For the microscopic differentiation between red and white fibers, paraffin section were stained with phosphotungstic-hematoxylin. The decrease in number after 50 years of age was similar in both red and white fibers, while the volume of both types of fiber somewhat increased with age. However, a slight difference was recognized in the changes between the red and white fibers. The white fibers significantly decreased in number first in the earlier stage of life, and noticeably decreased in number after 80 years of age, with no appreciable increase in volume. In the red fibers, on the other hand, the decrease in number after 80 years was highly significant, and was accompanied by a significant increase in their volume.

Accumulation of lipofuscin pigment in human hepatic cells from different races and in different environmental conditions.

The changing pattern with age of lipofuscin pigment deposition in the hepatic cells of native and Hawaii Japanese and caucasians in the U.S.A. was micrometrically examined. The amount of pigment was generally largest in the Hawaii Japanese, and smallest in the native Japanese; the age-related increase of the pigment was most marked in the latter. The accumulation of pigment in human hepatic cells is considered to be an age-related change in the hepatic cells, but not necessarily of the individual, and seems to be influenced by a relationship between nutritional conditions and constitution of the individual. Though a possible relationship between age and lipofuscin deposition may be noticed, the accumulation of pigment in the hepatic cells is not necessarily an inherent part of the aging process of the individual.

Fine structural analysis of lipofuscin in various tissues of rats of different ages.

Comparative fine structural analysis of intracellular dense inclusions in various tissues of rats of different ages (2, 11 and 29.5 months old) was made. The majority of dense inclusions observed in 2-month-old rats were of the granular type. With advancing age, granular type inclusions gradually decreased in frequency, while compound type inclusions, such as granular-homogeneous and granular-lamellated type increased except for spleen. In the bronchial epithelia, granular-lamellated inclusions appeared at 2 months of age. On the other hand, in the heart, liver, spleen, and skeletal muscle, lamellated or granular-lamellated inclusions were not observed at any of the three age levels. Dense inclusions seemed to have various patterns of distribution, frequency of appearance, and source of organelles in each tissue and cell. They also did not show the maturity of pigment in the process of pigment formation. These findings suggested that these inclusions were not merely age-related granules, but seemed to be influenced by their relationship to the cell metabolism and other functions.

Morphometric studies on the age changes of autofluorescent granules and lysosomes in the epithelial cells of rat kidneys.

Morphometric analysis of the lysosomal system in the proximal convoluted tubules of the kidney of rats of different ages was studied, with special regard to the accumulation of autofluorescent pigment in the epithelial cells. After maturity, the lysosomal amount in the epithelial cells was significantly increased in the 25-29-month-old group; however, the amount of autofluorescent pigment granules was not significantly increased.

Age-related change in activation by Tris (hydroxymethyl) aminomethane on myosin-ATPase activity of human minor pectoral muscles.

Age related change of the human minor pectoral muscles was biochemically demonstrated. Myosin-ATPase activity was significantly decreased with age, and was activated with Tris. The degree of the activation by Tris was observed to be lower in old aged patients than in the young. Furthermore, at low concentration of CaCl2 (less than 100 microM), myosin-ATPase activity was higher in the young age than in the old, while at high concentration of CaCl2 (more than 1 mM) no significant difference was observed between young and old age. Decrease with age in activation by primary amine such as Tris would play an important role in the muscle working capacity in old age.