Nuclear magnetic resonance studies on polymer carbanions, 5. Polystyryl carbanion with lithium as counter cation and its dimer model compounds

Oligostyryllithium was synthesized by the reaction of sec-butyllithium with styrene in diethyl ether at ?40°C. Dimer model anions (counter cations: Li and K) of the growing chain ends were synthesized by metallation of 1-methoxy-1,3-diphenylpropane. The structures of the lithium compounds were corroborated by ¹³C NMR spectroscopy and compared with those of the potassium compounds. As for polystyryllithium and its model compounds, the charge density on the α-carbon increased and that on the phenyl ring of the growing end decreased with increasing molecular weight, while, in polystyrylpotassium and its model compounds, the charge density on the α-carbon increased and the one on the phenyl ring did not change with increasing molecular weight. The temperature change of the ¹H NMR spectrum of oligostyryllithium in diethyl ether indicated rotation around the C-1? C_α bond at lower temperatures than in tetrahydrofuran, but the activation energy was the same in both solvents. T₁ values of the phenyl carbons in model compound 2b were one to two orders of magnitude shorter than those of the protonated compound.     

The ventilatory threshold gives maximal lactate steady state

Summary The purpose of this study was to examine whether the ventilatory threshold (Th _v) would give the maximal lactate steady state ([1a]_{ss, max}), which was defined as the highest work rate (W) attained by a subject without a progressive increase in blood lactate concentration [1a]_b at constant intensity exercise. Firstly, 8 healthy men repeated ramp-work tests (20 W·min^–1) on an electrically braked cycle ergometer on different days. During the tests, alveolar gas exchange was measured breath-by-breath, and theW atTh _v (W _{Th v}) was determined. The results of two-way ANOVA showed that the coefficient of variation of a singleW _{Th v} determination was 2.6%. Secondly, 13 men performed 30-min exercise atW _{Th v} (Th _v trial) and at 4.9% aboveW _{Th v} (Th _v + trial), which corresponded to the 95% confidence interval of the single determination. The [1a]_b was measured at 15 and 30 min from the onset of exercise. The [1a]_b at 15 min (3.15 mmol·1^–1, SEM 0.14) and at 30 min (2.95 mmol·1^–1, SEM 0.18) were not significantly different inTh _v trial. However, the [1a]_b ofTh _v+ trial significantly increased (P<0.05) from 15 min (3.62 mmol·1^–1, SEM 0.36) to 30 min (3.91 mmol·1^–1, SEM 0.40). These results indicate thatTh _v gives the [1a]_ss,max, at which one can perform sustained exercise without continuous [1a]_b accumulation.     

Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.     

Mutation analysis of HNF-4 binding sites in the human glucose-6-phosphatase promoter

HNF-4, a member of the nuclear receptor superfamily, binds to HNF-4 response elements (HRE), consisting of a direct repeat of the hexameric half-sites spaced by 1 nt (direct repeat 1) and activates a number of genes, which play central roles in fatty acids and glucose metabolism. Glucose-6-phosphatase (G6Pase) catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. A previous study has shown that HNF-4 binds to two DR1s in the regions A (located between -266 and -234) and B (located between -306 and -274) on the human G6Pase promoter. We found that the region B contains the one more DR1 element, composed of the two half-sites, designated half-sites a and b, the latter of which overlaps with the previously identified DR1 consisting of two half-sites, designated half-sites b and c. In this study, electrophoretic mobility shift assay (EMSA) using point mutations in each half-site a, b, or c indicated that HNF-4 binds to the combination of half-sites a and b, but not to half-sites b and c. Furthermore, mutational analysis demonstrated that, in the context of the human G6Pase promoter, the half-sites a and b, but not the half-sites b and c, are required for the stimulatory effect of HNF-4. These results suggested that the DR1 element containing the half-sites a and b is a functional HRE that mediates the induction of hG6Pase promoter activity by HNF-4.     

In situ hybridisation with digoxigenin-labelled DNA probes for detection of viral genomes.

The applicability of a recently developed non-radioactive DNA labelling and detection method, which uses the digoxigenin (DIG) enzyme linked immunosorbent assay (ELISA) system, for the detection of viral infections in pathology specimens by in situ hybridisation, was examined. Its efficacy was compared with that of biotin and radioisotope labelling methods. Three cases of progressive multifocal leucoencephalopathy, two of verruca vulgaris, and seven cases of laryngeal papilloma were studied. The sensitivity of the DIG labelled probe was almost the same as that of a 35S-labelled probe in the dot-blot hybridisation test. Using in situ hybridisation with 35S-labelled and DIG labelled probes, the levels of the hybridised signals detected were similar. The biotin labelled probe was less sensitive, particularly in the cases of laryngeal papilloma. The DIG labelling and detection method was highly sensitive and applicable to the detection of viral infection by ISH, and is preferable to a radiolabelled probe, especially when in situ hybridisation is done in the pathology laboratory.     

Novel single nucleotide polymorphisms of the human nuclear factor kappa-B 2 gene identified by sequencing the entire gene

The nuclear factor kappa-B 2 (NFKB2) gene is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase of the inflammatory response and of immune responses. We identified three novel single nucleotide polymorphisms (SNPs) and determined their allelic frequencies, as determined by the sequencing of 48 alleles of the entire gene in a Japanese population sample. Two of the three polymorphisms were identified at nucleotide (nt) position 1837 (T/C) and nt position, 1867 (GG/G) in the upstream region of the gene. The other polymorphism was identified at nt position 2584 (G/T) within intron 1. These polymorphisms will be useful in genetic studies of the processes involved in inflammatory responses and in bone differentiation. Received: October 17, 2000 / Accepted: October 23, 2000     

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.     

Induction of angiogenesis by hyperplastic colonic mucosa adjacent to colon cancer

We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-alpha than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-beta inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-beta than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis.     

N-methyl-D-aspartate receptor antagonist reduces nitrotyrosine formation in caudate-putamen in rat focal cerebral ischemia-reperfusion

The aim of this study is to determine experimentally whether N-methyl-D-aspartate (NMDA) receptor is involved in nitrotyrosine formation in rat brain subjected to focal ischemia-reperfusion, by using the NMDA receptor antagonist MK-801. Halothane-anesthetized and artificially ventilated rats were given MK-801 (5 mg/kg, i.p.) or vehicle prior to 2 h of focal cerebral ischemia followed by 0.5 h of reperfusion. The brain was then removed and divided into four sections, cortical ischemic core, peri-ischemic cortex, lateral caudate-putamen and non-ischemic cortex. Tissue nitrotyrosine was measured by means of hydrolysis/HPLC. MK-801 significantly attenuated nitrotyrosine formation in the lateral caudate-putamen. We conclude that nitrotyrosine formation required activation of NMDA receptors, at least in part.     

Divergence of natural killer cell receptor and related molecule in the decidua from sporadic miscarriage with normal chromosome karyotype

The aim of this cohort study was to investigate immunophenotypic characteristics of natural killer (NK) cells by assessing specific molecules expressed in the decidua of sporadic miscarriages and induced abortions. The deciduae were obtained from 29 consecutively seen women whose pregnancies ended in first trimester miscarriages (MS), and the fetal chromosome karyotype of these MS was analysed. Additionally, 13 deciduae were obtained from induced abortion (IA) with informed consent. The expression of perforin, CD94, CD161, CD158a, CD158b, CD244 on CD3-CD56+NK cells, and perforin on CD3+CD8+ T cells was analysed by flow cytometry. The CD158a (mean+/-SD, 26.2+/-14.7%) and CD94 (50.2+/-25.7%) expressions in MS with normal chromosome karyotype (MSNK; n=11) were significantly decreased as compared with those (41.5+/-19.5%, 71.4+/-20.4%) in MS with abnormal karyotype (MSAK; n=18) and those (44.3+/-21.9%, 80.8+/-17.5%) in IA (n=13). Conversely, the perforin expression on CD3-CD8-CD56+NK cells (76.3+/-11.0%) and CD3+CD8+T cells (30.6+/-9.2%) in MSNK was significantly increased as compared with those (66.8+/-16.6%, 23.6+/-8.7%) in MSAK and those (62.9+/-11.6%, 19.7+/-8.1%) in IA. A positive correlation between CD94 and CD158a expressions on NK cells, negative correlations between CD94 on NK cells and perforin on NK cells/T cells, and between CD158a on NK cells and perforin on T cells were found in the decidua. A divergence of NK cell repertoire in the decidua might be related to aetiology of sporadic MSNK.