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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.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.

The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2(-/-) mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR-induced apoptosis. The IR-induced G(1)/S cell cycle checkpoint, but not the G(2)/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2(-/-) mice. IR-induced stabilization of p53 in Chk2(-/- )cells was 50-70% of that in wild-type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2(-/-) cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.

We used transport and Raman scattering measurements to identify the insulating state of self-doped spin (1/2) two-leg ladders of Sr14Cu24O41 as a weakly pinned, sliding density wave with nonlinear conductivity and a giant dielectric response that persists to remarkably high temperatures.

Members of the FOXO family of mammalian forkhead transcription factors, including AFX, FKHRL1, and FKHR, are homologs of DAF-16, which regulates genes that contribute both to longevity and to resistance to various stresses (including oxidative stress) in Caenorhabditis elegans. We have generated mouse myoblastic C2C12 cell lines in which expression of a constitutively active form of AFX (AFX-TM) is inducible by Cre-mediated recombination at loxP sites. Here we show that forced expression of AFX-TM blocked cell cycle progression at the G(1) and G(2) phases and that FOXO family members regulated the expression of stress-inducible genes such as GADD45. AFX and FKHRL1 each directly activated the GADD45 promoter through interaction with FOXO binding motifs. Oxidative stress activated the GADD45 promoter in a FOXO-dependent manner, resulting in an increased abundance of GADD45 mRNA and protein as well as G(2) arrest. These responses were evident in cells in which the tumor suppressor protein p53 was inactivated. Our results suggest that the FOXO family of transcription factors plays an important role in the regulation of GADD45 in response to oxidative stress and thereby contributes to G(2)-M checkpoint.

We report momentum-resolved charge excitations in a one-dimensional (1D) Mott insulator studied using high resolution inelastic x-ray scattering over the entire Brillouin zone for the first time. Excitations at the insulating gap edge are found to be highly dispersive (momentum dependent) compared to excitations observed in two-dimensional Mott insulators. The observed dispersion in 1D cuprates ( SrCuO2 and Sr2CuO3) is consistent with charge excitations involving holons which is unique to spin-1/2 quantum chain systems. These results point to the potential utility of momentum-resolved inelastic x-ray scattering in providing valuable information about electronic structure of strongly correlated insulators.

A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.

The low-energy electronic structure of the nearly optimally doped trilayer cuprate superconductor Bi(2)Sr(2)Ca(2)Cu(3)O(10+delta) is investigated by angle-resolved photoemission spectroscopy. The normal state quasiparticle dispersion and Fermi surface and the superconducting d-wave gap and coherence peak are observed and compared with those of single- and bilayer systems. We find that both the superconducting gap magnitude and the relative coherence-peak intensity scale linearly with T(c) for various optimally doped materials.

The two-magnon (2M) excitation at 3000 cm(-1) in Sr14Cu24O41 two-leg ladder is studied by Raman scattering. A slight anisotropy of the superexchange coupling J(perpendicular)/J(parallel) approximately 0.8 with J(parallel) = 110+/-20 meV is proposed from the analysis of the magnetic scattering. The resonant coupling across the charge transfer gap increases the 2M intensity by orders of magnitude. The anisotropy of Raman scattering is dependent upon the excitation energy. The 2M relaxation is found to be correlated with the temperature dependent electronic Raman continuum at low frequencies.

Chimeric mice were prepared from embryonic stem cells transfected with IgH genes as transgenes and RAG-2-deficient blastocysts for the purpose of identifying the cis-acting elements responsible for the induction of somatic hypermutation. Among the three transgene constructs used, the V(H) promoter, the rearranged V(H)-D-J(H), an intron enhancer/matrix attachment region, and human Cmu were common to all, but the 3'-untranslated region in each construct was different. After immunization of mice with a T cell-dependent Ag, the distribution and frequency of hypermutation in transgenes were analyzed. The transgene lacking the 3' untranslated region showed a marginal degree of hypermutation. Addition of the 3' enhancer resulted in a slight increase in the number of mutations. However, the transgene containing DNase I-sensitive regions 3b and 4 in addition to the 3' enhancer showed more than a 10-fold increase in hypermutation, reaching levels comparable to those observed in endogenous V(H)186.2 genes of C57BL/6 mice.

Examination of the effects of water temperature on the inactivation of Cryptosporidium parvum oocysts with ozone, ozonation experiments were conducted in a semi-batch mode with a wide temperature range of 3-30 degrees C. Inactivation was assessed in terms of mice infectivity and in vitro excystation. The temperature dependency of the CT products by a reduction in infectivity of 2 log10 could be described successfully by the Arrhenius equation, 1/CT = 1.086 x 10(18)e-12520/K where CT is the integrated ozone concentration over the contact time (mg/min/L) and K is the Kelvin temperature of water. As for the reduction in viability assessed by the excystation assay, protocol B, the obtained regression equation, 1/CT = 1.802 x 10(18)e-12640/K, was almost identical to that observed for the infectivity. Thus, the CT products required for a 2 log10 reduction in both infectivity and viability increased by an average factor of 4.2 for every 10 degrees C decrease in water temperature. Additionally, our findings suggested that the viability, as determined by protocol B, could substitute for animal infectivity in evaluating the effects of environmental factors on the efficacy of ozonation.

We report strong instantaneous photoinduced absorption in the quasi-one-dimensional Mott insulator Sr2CuO3 in the IR spectral region. The observed photoinduced absorption is to an even-parity two-photon state that occurs immediately above the absorption edge. Theoretical calculation based on a two-band extended Hubbard model explains the experimental features and indicates that the strong two-photon absorption is due to a very large dipole coupling between nearly degenerate one- and two-photon states. Room temperature picosecond recovery of the optical transparency suggests the strong potential of Sr2CuO3 for all-optical switching.

Caspases, which play crucial roles during apoptosis, are activated from their inactive proforms in a sequential cascade of cleavage by other members of the caspase family. Caspase-9 is autoprocessed by the Apaf-1/cytochrome c pathway and acts at an early point in this cascade, whereas Bcl-xL, an antiapoptotic member of the Bcl-2 family, prevents activation of caspases in vitro. Little is known, however, about the relation between caspase-9 and Bcl-xL during development of the mammalian nervous system. We used antisera against two cleavage sites in mouse caspase-9 that recognize only the activated form of mouse caspase-9, and we examined immunohistochemically the activation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse embryos. Mouse caspase-9 is processed at both D(353) and D(368), but it is processed preferentially at D(368) during apoptosis of cultured cells induced by various stimuli and in the nervous system of Bcl-x-deficient mouse embryos. We show that Bcl-xL protects against caspase-9- and/or caspase-3-dependent apoptosis in the caudal portion of the ventral hindbrain, anterior horn cells, and dorsal root ganglia neurons of the normal mouse embryos and against caspase-9/caspase-3-independent apoptosis in the dorsal region of the nervous system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation of caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5.

Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

Caspases and Bcl-xL, the mammalian homologues of the Caenorhabditis elegans (C. elegans) ced-3 and ced-9 genes, respectively, regulate apoptosis of various cells. Caspase-3 is processed into an active form (p20 or p17 and p12) during apoptosis. We investigated the relation between caspase-3 and Bcl-xL during development by examining activation of caspase-3 and apoptotic cells in Bcl-x-deficient (bcl-x(-/-)) mice at embryonic (E) day 11.5. We used a double-staining technique with a cleavage site-directed antibody against caspase-3 (anti-p20/17) and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Bcl-xL-deficiency increased both numbers of p20/17-positive and -negative apoptotic cells in dorsal root ganglia (DRG); the numbers of p20/17-positive apoptotic cells in the caudal parts of the ventral hindbrain and ventral spinal cord; and the numbers of p20/17-negative apoptotic cells in the dorsal midbrain, dorsal hindbrain, and dorsal spinal cord. Thus, Bcl-xL blocks the caspase-3-dependent apoptotic pathway in the restricted regions of the nervous system during development. Furthermore, these observations suggest that Bcl-xL protects against activation of the caspase-3-independent apoptotic pathway. Other caspases or apoptotic mechanisms may also be activated in the nervous systems of bcl-x(-/-) mice., (Copyright 1999 Elsevier Science B.V.)

bcl-x is a member of the bcl-2 gene family, which regulates apoptotic cell death in various cell lineages. There is circumstantial evidence suggesting that bcl-x might play a role in the apoptosis of erythroid lineage cells, although there is no direct evidence. In this study, we used Bcl-X null mouse embryonic stem (ES) cells, and showed that Bcl-X is indispensable for the production of both embryonic primitive erythrocytes (EryP) and adult definitive erythrocytes (EryD) at the end of their maturation. In vivo, bcl-x-/- ES cells did not contribute to circulating EryD in adult chimeric mice that were produced by blastocyst microinjection of the bcl-x-/- ES cells. bcl-x-/- EryP and EryD were produced by in vitro differentiation induction of ES cells on macrophage colony-stimulating factor-deficient stromal cell line OP9, and further analysis was carried out. The emergence of immature EryP and EryD from bcl-x-/- ES cells was similar to that from bcl-x+/+ ES cells. However, prominent cell death of bcl-x-/- EryP and EryD occurred when the cells matured. The data show that the antiapoptotic function of bcl-x acts at the very end of erythroid maturation.

The adenovirus E1B19K protein inhibits apoptosis induced by E1A and other divergent signals. The cellular proteins that interact with E1B19K have been analyzed by isolating cDNA clones by the yeast two hybrid system. One of these clones encodes B5 which consists of 219 amino acid residues and contains the putative BH3 and transmembrane regions. B5 binds strongly to Nip3 and itself, weakly to E1B19K, but not to Bcl-2 and localizes in nuclear envelope, endoplasmic reticulum and mitochondria. B5 has sequence homology with Nip3 in the middle and C-terminal regions, but not in the N-terminal region. Unlike other E1B19K binding BH3 proteins so far characterized, B5 does not induce apoptosis, but inhibits apoptosis induced by Nip3. However the deletion mutant B5Delta1-31 lacking the N-terminus does induce apoptosis, although weaker than does Nip3, suggesting that the N-terminal region is masking the apoptosis-inducing capacity of B5.

Lyn kinase-deficient (lyn-/-) mice show several abnormalities such as reduced numbers of circulating B cells, hyper-IgM, and low proliferative responses induced by CD40 ligand. Lyn-/- mice also develop splenomegaly, produce autoreactive Abs with age, and finally develop glomerulonephritis. Another abnormality observed in lyn-/- mice is that their disability to form germinal centers (GCs). It has been considered that GCs play an important role in affinity maturation and differentiation to B cell memory upon immunization with thymus-dependent Ag. Since Lyn kinase has been thought to be downstream of the signals from the B cell Ag receptor as well as CD40, we studied whether or not lyn-/- mice could exhibit normal Ag-specific class switching and affinity maturation following somatic hypermutation. The mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-CG). Production of NP-specific IgG1 Abs was slightly reduced but clearly detectable. The affinity of Abs produced was comparable to that in wild-type mice. Furthermore, somatic hypermutation occurred in the heavy-chain variable region at the same level as that in wild-type mice. Therefore, we conclude that isotype switching and affinity maturation occur normally in lyn-/- mice without the formation of GCs. The results lead to a speculation that Lyn may not play a role in induction of isotype switching or affinity maturation, despite being downstream of the signals from the B cell Ag receptor complex and CD40, and that GC architecture may not be absolutely essential for affinity maturation.

bcl-x is a member of the bcl-2 gene family, which is expressed at high levels in the embryonic brain. The targeted disruption of bcl-x results in massive cell death of immature neurons in the developing mouse brain (Motoyama et al., 1995). bcl-x-deficient mice die around embryonic day 13 (E13), probably secondary to their inability to produce mature red blood cells. To determine whether the death of immature neurons in the bcl-x-deficient brain is cell autonomous, we examined primary telencephalic cell cultures from E12.5 homozygous mutant (bcl-x-/-), heterozygous mutant (bcl-x+/-), and wild-type (bcl-x+/+) mice. bcl-x-/- telencephalic cells cultured in 0.5 or 2.0% fetal calf serum (FCS)-containing medium for 48 hr showed increased apoptosis, defined by abnormal bisbenzamide staining and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and decreased numbers of microtubule-associated protein-2-immunoreactive neurons compared with bcl-x+/- and bcl-x+/+ cultures. Cycloheximide treatment of bcl-x-/- telencephalic cell cultures failed to prevent the increased cell death observed in low FCS-containing medium, suggesting a protein synthesis-independent apoptosis. There were no significant differences among bcl-x-/-, bcl-x+/-, and bcl-x+/+ telencephalic cells grown for 48 hr in 5% FCS-containing medium or in a chemically defined serum-free medium (ITS). bcl-x-/- neurons generated in ITS showed increased susceptibility to subsequent serum deprivation. These results indicate that bcl-x is important for both neuron maturation and survival.

During thymic development, T cells that can recognize foreign antigen in association with self major histocompatibility complex (MHC) are selected for survival (positive selection) and autoreactive T cells are eliminated (negative selection). Both of these selective events are mediated by interaction between the T-cell receptor (TCR) and the peptide-MHC complex. But the signalling pathways that lead to cell survival or to cell death are still unclear. ZAP-70 is a protein tyrosine kinase (PTK) that is associated with the TCR signalling subunits (CD3 and zeta) and is expressed in T cells and natural killer cells. It has been shown that ZAP-70 plays a crucial role in T-cell activation and development. Here we show that mice lacking ZAP-70 had neither CD4 nor CD8 single-positive T cells, but human ZAP-70 reconstituted both CD4 and CD8 single-positive populations. Moreover, ZAP-70-/- thymocytes were not deleted by peptide antigens. Natural killer cell function was intact in the absence of ZAP-70. These data suggest that ZAP-70 is a central signalling molecule during thymic selection for CD4 and CD8 lineage.

Rat liver and house fly, Musca domestica L., glutathione S-transferases were purified by affinity chromatography and chromatofocusing. The specific activity with alpha-naphthyl acetate (a substrate, for general esterase activity) was determined. Some of the esterase activity that is normally assayed for resistance is apparently caused by the activity of the glutathione S-transferase.

bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

Purpose: This study was undertaken to compare the pathoclinical findings in gastric adenocarcinoma with serum IgG antibody to Helicobacter pylori., Materials and Methods: We examined 185 patients with histologically established gastric cancer. The presence of immunoglobulin (Ig)G antibody in the high molecular cell-associated antigen of H. pylori was determined by enzyme-linked immunosorbent assay. Pepsinogens I and II were measured by radioimmunoassay. The distribution of H. pylori on the gastric mucosa was assessed by the Campylobacter-like organism test and phenol red dye spraying., Results: H. pylori IgG antibody was detected in 93.1% of patients with gastric cancer (mean age 61.7 years), 94.3% of patients with early gastric cancer and 91.2% with advanced gastric cancer. No statistical difference in serology was observed between type of gastric cancer, depth of cancer invasion, tumor size or histology. Only in patients with diffuse-type cancer of the cardia was there a lower percentage of positive results (80.0%). The ratio of pepsinogen I to pepsinogen II was higher in the patients who exhibited no H. pylori antibodies., Conclusions: H. pylori antibodies were common in patients with gastric cancer, and were not correlated with histological type nor stage of cancer. In the Niigata district, a higher percentage of patients with gastric carcinoma displayed H. pylori antibodies compared with other districts in Japan.

Transgenic mice containing the chloramphenicol acetyl transferase (CAT) gene driven by the immunoglobulin (Ig) VH promoter and the heavy chain intron enhancer were prepared to increase our understanding of the mechanism responsible for somatic mutation. The transgene showed similar tissue specificity in terms of expression as endogenous Ig genes. Hybridomas were prepared after multiple immunization of a transgenic mouse with (4-hydroxy-3-nitrophenyl)acetyl-chicken gammaglobulin, a thymus-dependent antigen. We analyzed mutations in the CAT coding region as well as in the region 5' upstream of the promoter after amplification of DNA using the PCR followed by sequencing of cloned DNA. Mutation detection enhancement gel electrophoresis was also used to detect mutations. Only a single band was observed in PCR products from the region 5' upstream of the promoter and from the enhancer, whereas in those from the CAT coding region, three out of 11 hybridomas showed multiple bands. In DNA sequences of the CAT coding region from these three hybridomas, a total of six nucleotide substitutions were observed, but none in the region 5' to the promoter. The results of DNA sequencing and the electrophoresis were in good agreement, suggesting that mutation occurred only in the CAT gene but not in the region 5' to the promoter or the enhancer. The rate of mutation in the CAT gene was estimated to be 1.6 x 10(-5) per base pair per cell division, which was a lower limit of the rate for somatic mutation reported for Ig genes. Location and distribution of mutations were similar to that of the Ig gene, suggesting that (i) mutation in the CAT gene was induced by a mechanism similar to that occurring in the Ig gene and (ii) the mutator mechanism can operate in a non-Ig gene provided it is flanked by the VH promoter and heavy chain intron enhancer.

HRF20 (CD59) is a membrane glycoprotein which protects cells from the membrane attack reaction of homologous complement. A patient who is completely deficient in HRF20 expression and is suffering from paroxysmal nocturnal hemoglobinuria (PNH) was studied. His parents are cousins and both have decreased HRF20 expression, suggesting that the deficiency is genetic. We established a cultured cell line (NCU1) which is HRF20 deficient from the patient's lymphocytes by Epstein-Barr-virus (EBV) infection. Northern blot analysis revealed HRF20 mRNA signals, indicating that HRF20 mRNA were transcribed. HRF20 cDNA was amplified by the polymerase chain reaction (PCR) method. Sequencing of the cDNA from the NCU1 showed two single-base deletions at amino acid 16 and 96 from the N terminus of the mature protein. Deletion in the genomic DNA of peripheral blood lymphocytes was confirmed by the DNA sequence of an HRF20 open reading frame containing amino acid 16. Furthermore, the patient's parents and sister possessed both intact and deleted genomic HRF20 DNA while his brother's DNA was intact. These findings demonstrate that the HRF20 deficiency was genomic in origin, and that complete deletion was brought about by a homozygous abnormality in the HRF20 gene. The base deletion caused a codon frame shift resulting in failure to produce intact HRF20 protein in the patient.

Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag-binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag-binding to Fc.

A unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogenous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000. The molecular weight of the native enzyme was 45,000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8 X 10(-4) M, 1.4 X 10(-4) M, and 1.7 X 10(-4) M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8 X 10(-4) M and 2.0 X 10(-4) M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L(+)tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.

The role of prostaglandin I2 (PGI2) in the control of DNA synthesis during the cell cycle was investigated in cultured rabbit aortic smooth muscle cells (SMC). SMC at confluency in the G0 state reached the S phase about 16 h after stimulation with serum, as judged by measurement of [3H]thymidine incorporation into DNA (DNA synthesis). Cyclooxygenase inhibitors such as indomethacin and aspirin enhanced DNA synthesis, suggesting that endogenously synthesized prostaglandins inhibit DNA synthesis. Added PGE1 or PGE2 had little effect on DNA synthesis. PGI2 inhibited DNA synthesis only when added from 10 to 16 h after stimulation of SMC in the G0 state with serum. Addition of CS-570, a stable PGI2 analogue, inhibited DNA synthesis at any time after serum stimulation. The endogenous syntheses of PGI2 and DNA were negatively correlated. These results suggest that PGI2 inhibits DNA synthesis by acting on the progression stage of the G1 state.

The principal biochemical mechanisms of resistance to insecticides involve either modified, less sensitive cholinesterase, esterase action, glutathione S-transferase action or cytochrome P-450-dependent monooxygenation. Both quantitative and qualitative differences in cytochrome P-450 isozymes are under genetic control and both are related to resistance. Recent characterization studies involving ligand binding and multiplicity of isozymes in Musca domestica (the housefly) are discussed in relation to resistance. The recent demonstration that multiple isozymes of glutathione S-transferase exist in susceptible and resistant insects is of interest, and some re-examination of their role in the mechanism of resistance is required. Esterases are a heterogeneous group of enzymes whose role in resistance has often been suggested but seldom rigorously defined. Purification studies in the green rice leafhopper, Nephotettix cincticeps, have involved an enzyme with carboxylesterase, phosphotriesterase and pyrethroid esterase activities. A similar enzyme, but without pyrethroid esterase activity, is also found in the housefly. In resistance such enzymes may serve either to catalyse hydrolysis or as binding proteins. It has been suggested, from time to time, that regulator genes, enzyme induction and gene magnification all play a part in controlling biochemical mechanisms of resistance, although clearly defined evidence has not always been brought forward. These hypotheses are re-examined.

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.