Effect of cyclic AMP,theophylline and caffeine on the glucose repression of sporulation inSaccharomyces cerevisiae

Summary Repression of the sporulation ability ofSaccharomyces cerevisiae by glucose present in the presporulation medium was studied. Glucose lowered sporulation ability when added to the presporulation medium containing yeast extract but did not do so when added to the presporulation medium without glucose. The glucose-repressed sporulation ability was recovered by the addition of cyclic AMP, and theophylline or caffeine to the presporulation culture. Theophylline promoted the action of cyclic AMP, but caffeine did not. The effect of caffeine to reverse glucose repression was greater than that of cyclic AMP and theophylline.     

DNA polymorphism in active gene and pseudogene of the cytosolic phosphoglucose isomerase (PgiC) loci in Arabidopsis halleri ssp. gemmifera

DNA variations in two PgiC loci were investigated in 15 strains of Arabidopsis halleri ssp. gemmifera. In a 5.5-kb region of the PgiC1 locus, 127 nucleotide substitutions and 33 length variations were observed. In a 6.0-kb region of the PgiC2 locus, 138 nucleotide substitutions and 33 length variations were observed. Frame shift, novel stop codons, and large length variations were observed in the PgiC2 coding region. These findings suggested that PgiC2 may be a pseudogene. The nucleotide diversities (pi) for the entire regions of both PgiC loci were approximately 0.0033. Tajima's test of both PgiC loci yielded significantly negative results. In the coding regions, the high proportions of replacement substitutions caused significant deviations from neutrality in McDonald and Kreitman's test. An excess of singletons and a high proportion of replacement polymorphic sites have been observed in the Adh and ChiA regions of A. halleri ssp. gemmifera. Thus, the A. halleri ssp. gemmifera population may not have reached equilibrium, and thus nonneutral patterns of DNA polymorphism were observed.     

Voided urine cytology of papillary renal cell carcinoma and renal calculus: report of a case with emphasis on the importance of cytologic screening in high-risk individuals

BACKGROUND: Preoperative diagnosis of cases of renal calculus complicated with papillary renal cell carcinoma (RCC) by image analysis is usually difficult. CASE: A 50-year-old man who had a past history of renal calculus suffered from macrohematuria and abdominal pain for one month was admitted to our hospital. Ultrasonographic examination revealed a 4-cm tumor shadow in the right kidney; it was hypovascular in arteriography. Papillary cell clusters with abundant cytoplasm were found by the cytologic examination of voided urine. Their nuclei were oval and situated eccentrically in the cytoplasm. The nuclear/cytoplasmic ratio was increased. Fine, granular chromatin was distributed evenly, and the nuclear membrane was thin and nearly smooth. Several small nucleoli were evident. All these findings were indicative of a diagnosis of papillary RCC. Histology of nephrectomy specimens confirmed the diagnosis. CONCLUSION: Voided urine cytology can be useful for screening and follow-up of patients with papillary RCC.     

A Novel Protein Associated with Membrane-type 1 Matrix Metalloproteinase Binds p27kip1 and Regulates RhoA Activation, Actin Remodeling, and Matrigel Invasion

Pericellular proteolysis by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor cell invasion. Localization of MT1-MMP at the invasion front of cells, e.g. on lamellipodia and invadopodia, has to be regulated in coordination with reorganization of the actin cytoskeleton. However, little is known about how such invasion-related actin structures are regulated at the sites where MT1-MMP localizes. During analysis of MT1-MMP-associated proteins, we identified a heretofore uncharacterized protein. This protein, which we call p27RF-Rho, enhances activation of RhoA by releasing it from inhibition by p27^kip1 and thereby regulates actin structures. p27^kip1 is a well known cell cycle regulator in the nucleus. In contrast, cytoplasmic p27^kip1 has been demonstrated to bind GDP-RhoA and inhibit GDP-GTP exchange mediated by guanine nucleotide exchange factors. p27RF-Rho binds p27^kip1 and prevents p27^kip1 from binding to RhoA, thereby freeing the latter for activation. Knockdown of p27RF-Rho expression renders cells resistant to RhoA activation stimuli, whereas overexpression of p27RF-Rho sensitizes cells to such stimulation. p27RF-Rho exhibits a punctate distribution in invasive human tumor cell lines. Stimulation of the cells with lysophosphatidic acid induces activation of RhoA and induces the formation of punctate actin structures within foci of p27RF-Rho localization. Some of the punctate actin structures co-localize with MT1-MMP and cortactin. Down-regulation of p27RF-Rho prevents both redistribution of actin into the punctate structures and tumor cell invasion. Thus, p27RF-Rho is a new potential target for cancer therapy development.Malignant tumor cells grow invasively and form distant metastases after moving through multiple tissue barriers. Invasion requires cell locomotion together with degradation of the extracellular matrix (ECM)² by matrix metalloproteinases (MMPs) (1). MT1-MMP (MMP-14) is an integral membrane protease that degrades a variety of protein components within the extracellular milieu (2). The substrates of MT1-MMP include a variety of components of the ECM, membrane proteins including cell adhesion molecules, and growth factors and cytokines (3). To degrade the ECM barrier in advance of an invading cell, MT1-MMP localizes to the leading edge of invasion (4) and cellular protrusions called invadopodia (5–7). Therefore, it is of particular interest how reorganization of actin structures is regulated at sites where MT1-MMP localizes.During mass spectrometric analysis of proteins co-purified with MT1-MMP, we identified a protein of unknown function (8). Although this protein did not affect MT1-MMP activity, we observed that enhanced expression or down-regulation of this protein affected activation of RhoA. Thus, we became interested in the possibility that this protein mediates focal reorganization of actin structures close to sites where MT1-MMP localizes.RhoA plays a pivotal role in signal transduction pathways that regulate reorganization of actin structures and does so by assuming active GTP-bound and inactive GDP-bound states, with the transition between the two forms finely regulated by many cellular proteins (9, 10). In addition to the classical modulators, recent studies have revealed that p27^kip1 also regulates activation of RhoA and Rac1 (11, 12). p27^kip1 has been characterized as a cyclin-dependent kinase inhibitor localized to the nucleus, but phosphorylation of p27^kip1 by protein kinase B/Akt or kinase-interacting stathmin (KIS) mediates its translocation from the nucleus to the cytoplasm. Cytoplasmic p27^kip1 binds RhoA and prevents activation of RhoA by GEFs (12, 13). However, it is not known how inhibition of RhoA by p27^kip1 is released to allow activation. The protein we identified binds p27^kip1, thereby preventing its binding to RhoA (schematically illustrated in supplemental Fig. S1). We named this protein p27RF-Rho (p27^kip1 releasing factor from RhoA) based on this activity.     

Genetical Analysis of Chromosomal Interaction Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

By combining ten second and ten third chromosomes, we investigated chromosomal interaction with respect to the action of the modifier factors on G6PD and 6PGD activities in Drosophila melanogaster. Analysis of variance revealed that highly significant chromosomal interaction exists for both enzyme activities. From the estimated variance components, it was concluded that the variation in enzyme activity attributed to the interaction is as great as the variation attributed to the second chromosome but less than attributed to the third chromosome. The interaction is not explained by the variation of body size (live weight). The interaction is generated from both the lack of correlation of second chromosomes for third chromosome backgrounds and the heterogeneous variance of second chromosomes for different third chromosome backgrounds. Large and constant correlation between G6PD and 6PGD activities were found for third chromosomes with any second chromosome background, whereas the correlations for second chromosomes were much smaller and varied considerably with the third chromosome background. This result suggests that the activity modifiers on the second chromosome are under the influence of third chromosome factors.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.     

Comparative Genome Mapping of the Ataxia–Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Chromosomal locations of theAtm(ataxia–telangiectasia (AT)-mutated) andAcat1(mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4–qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice.Atm, Acat1,andNpat,which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among theAtm, Npat, Acat1,andD9Mit6loci, and these loci were mapped 2.0 cM distal toD9Mit99and 1.3 cM proximal toD9Mit102.Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion betweenEts1andAtm–Npat–Acat1and that the inversion of MMU9 originated from the chromosomal breakage at the boundary betweenGria4andAtm–Npat–Acat1on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with theRckgene.     

Intragastric administration of capsiate, a transient receptor potential channel agonist, triggers thermogenic sympathetic responses

The sympathetic thermoregulatory system controls the magnitude of adaptive thermogenesis in correspondence with the environmental temperature or the state of energy intake and plays a key role in determining the resultant energy storage. However, the nature of the trigger initiating this reflex arc remains to be determined. Here, using capsiate, a digestion-vulnerable capsaicin analog, we examined the involvement of specific activation of transient receptor potential (TRP) channels within the gastrointestinal tract in the thermogenic sympathetic system by measuring the efferent activity of the postganglionic sympathetic nerve innervating brown adipose tissue (BAT) in anesthetized rats. Intragastric administration of capsiate resulted in a time- and dose-dependent increase in integrated BAT sympathetic nerve activity (SNA) over 180 min, which was characterized by an emergence of sporadic high-activity phases composed of low-frequency bursts. This increase in BAT SNA was abolished by blockade of TRP channels as well as of sympathetic ganglionic transmission and was inhibited by ablation of the gastrointestinal vagus nerve. The activation of SNA was delimited to BAT and did not occur in the heart or pancreas. These results point to a neural pathway enabling the selective activation of the central network regulating the BAT SNA in response to a specific stimulation of gastrointestinal TRP channels and offer important implications for understanding the dietary-dependent regulation of energy metabolism and control of obesity.     

DNA binding of tilorone: 1H NMR and calorimetric studies of the intercalation

The fluorene derivative tilorone has received great attention as a DNA intercalator and has been widely recognized as an inducer of interferon. The biological activity of tilorone is known to be related to its binding mode with DNA; however, few structural and thermodynamic studies have elaborated on this issue. This paper presents two-dimensional (2-D) NMR and isothermal titration calorimetry (ITC) for the tilorone/DNA complex, coupled with circular dichroism (CD) spectroscopy and viscosity measurements. NMR investigation suggests that tilorone binds to DNA through intercalation, showing greater affinity for insertion between AT base pairs than between CG pairs. CD spectral changes were observed for T/B (tilorone/DNA base pair molar ratio) ratios greater than the stoichiometric ratio generally expected for intercalators (i.e., T/B = 0.5, according to the neighbor-exclusion principle). However, there was a clear plateau in the CD intensity between T/B < 0.35 and T/B > 0.45. From comparison with NMR and other measurements, we postulate that CD changes below the plateau should be related to the intercalation and the latter to electrostatic interactions and nonspecific bindings. ITC data showed that DeltaH < -TDeltaS < 0, which indicated that tilorone/DNA binding is enthalpy controlled. The magnitude of Kb (the binding constant) was of the same order as that of ethidium bromide. The stoichiometric number, obtained from ITC, CD, and UV data, implied a relatively smaller value (0.28-0.35) than that of the neighbor-exclusion principle. This is because side chains located in the groove disrupt further intercalation to the adjacent sites.     

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone

A Wilms tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a prophylactic model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a therapeutic setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a therapeutic model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.H. Nakajima and K. Kawasaki contributed equally to this study.