Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1

Cdc34, a ubiquitin-conjugating enzyme in Saccharomyces cerevisiae, is required for cell cycle progression. Sic1, an S-phase cyclin-dependent kinase (CDK) inhibitor, is a critical target of Cdc34-mediated ubiquitination. Other essential target protein(s) could be defined since cdc34 sic1 double mutants still arrest in G2 phase. To identify proteins which function in the Cdc34-dependent ubiquitin pathway, a series of extragenic suppressors of the cdc34-1 sic1 double mutations was isolated. One of them was found to be defective in GRR1, which is involved not only in glucose repression but also in G1 cyclin destabilization. However, neither lack of glucose repression nor stabilization of G1 cyclin caused the suppression of cdc34-1 sic1. Conversely, Grr1 overproduction in cdc34-1 sic1 cells impaired colony formation, even at the permissive temperature. A multicopy suppressor, MGO1, which rescued the growth defect associated with Grr1 overproduction was isolated, and found to be identical to SKP1. Furthermore, Grr1 bound Skp1 directly in vitro. These results strongly suggest that Grr1 functions in the ubiquitin pathway through association with Skp1.     

Female-limited responses in remating rate and mating duration in the experimental evolution of a beetle Callosobruchus chinensis

Mating rate optima often differ between the sexes: males may increase their fitness by multiple mating, but for females multiple mating confers little benefit and can often be costly (especially in taxa without nuptial gifts or mala parental care). Sexually antagonistic evolution is thus expected in traits related to mating rates under sexual selection. This prediction has been tested by multiple studies that applied experimental evolution technique, which is a powerful tool to directly examine the evolutionary consequences of selection. Yet, the results so far only partly support the prediction. Here, we provide another example of experimental evolution of sexual selection, by applying it for the first time to the mating behaviour of a seed beetle Callsorobruchus chinensis. We found a lower remating rate in polygamy-line females than in monogamy-line (i.e. no sexual selection) females after 21 generations of selection. Polygamy-line females also showed a longer duration of first mating than monogamy-line females. We found no effect of male evolutionary lines on the remating rate or first mating duration. Though not consistent with the original prediction, the current and previous studies collectively suggest that the observed female-limited responses may be a norm, which is also consistent with the conceptual advances in the last two decades of the advantages and limitations of experimental evolution technique.     

Homologous Desensitization of Serotonin 5-HT2 Receptor-Stimulated Intracellular Calcium Mobilization in C6BU-1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Abstract: Serotonin 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time-and concentration-dependent manner. The desensitization of 5-HT₂ receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT₂ receptor-mediated Ca²⁺ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.     

On the reaction of 2-aminopropionitrile in aqueous media

The reactions of 2- and 3-aminopropionitrile (APN), and 2,2-iminodipropionitrile (IDPN) were carried out in aqueous ammoniacal media. 2-APN was found to give IDPN, N-(1-cyanoethyl)alanine amide, N-(1-cyanoethyl)alanine, N-(1-carbamoylethyl)alanine, 2,2-iminodipropionic acid, alanine amide, and alanine. Compounds of biological significance such as peptides and amino acids other than alanine were not formed. The results were well consistent with those obtained for aminoacetonitrile. IDPN which can be formed easily from 2-APN in aqueous media, also yielded the same products as with 2-APN. On the other hand, 3-APN gave 3-alanine via 3-alanine amide under similar conditions. 3-APN was found to be more stable than 2-APN in aqueous media.     

Nucleotide sequence of Dictyostelium small nuclear RNA Dd8 not homologous to any other sequenced small nuclear RNA

The cellular slime mold Dictyostelium discoideum, a lower eukaryote, was shown to contain several species of small nuclear RNA (Takeishi, K., and Kaneda, S. (1981) J. Biochem. (Tokyo) 90, 299-308; Wise, J. A., and Weiner, A. M. (1981) J. Biol. Chem. 256, 956-963). One of these RNAs, Dd9 or D2, was sequenced and found to be homologous to mammalian nucleolar U3 RNA. In the present study, the nucleotide sequence of another Dictyostelium small nuclear RNA Dd8 was determined by direct analysis. The sequence is: (formula; see text) Dd8 RNA contains high proportions of A (34%) and U (30%). No modified nucleotide could be detected in the internal region. Computer-assisted analysis of sequence homology indicated that Dd8 RNA is not homologous to any other small nuclear RNA species sequenced so far.     

Carboxyl-terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin

The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin-affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 micrograms/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 x 10(-7) Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-molecular-weight DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave ultraviolet light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave ultraviolet light irradiation. The arrest of chain elongation at the replicon-size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.     

Gas chromatography with surface ionization detection: a highly sensitive method for determining underivatized codeine and dihydrocodeine in body fluids

Underivatized codeine and dihydrocodeine in human plasma and urine have been determined with a high degree of accuracy by capillary gas chromatography (GC) with surface ionization detection (SID). The drugs were extracted with the aid of Sep-Pak C₁₈ cartridges. Recovery of both drugs was 90%. The calibration curves obtained with dimemorfan as an internal standard showed linearity in the range 4.5–72.3 and 3.0–75.5 ng/ml of plasma for codeine and dihydrocodeine, respectively. The detection limit was about 100 pg on column (2.5 ng/ml sample). Codeine was determined quantitatively in plasma and urine obtained from a volunteer who had received 10 mg codeine phosphate orally 3 h before the sampling: the levels were found to be 14.1 and 142 ng/ml, respectively. The present GC-SID method has been compared carefully with GC-NPD (nitrogen-phosphorus detection) using the same extracts; the sensitivity of GC-SID was more than ten times greater than that of GC-NPD, with background noise correspondingly lower.