Proprotein convertases regulate activity of prostate epithelial cell differentiation markers and are modulated in human prostate cancer cells

Prostate derived factor (PDF) is a member of transforming growth factor-beta (TGF-beta) superfamily proteins involved in differentiation of the prostate epithelium. Proprotein convertases (PCs) such as furin are thought to mediate the processing of TGF-beta superfamily. In the present study, we demonstrated for the first time that human prostate cancer cell lines differentially synthesize and secret prostate derived factor (PDF), and that PDF secreted by LNCaP is processed by PCs. Exposure of LNCaP cells to the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK), a synthetic furin-like protease inhibitor, inhibited PDF processing and resulted in the loss of luminal cell phenotype and induction of basal cell phenotype in LNCaP cells as demonstrated by alternations in the expression of cytokeratins 8, 14, 18, and 19, markers of prostate epithelial cell differentiation. These results suggest that proprotein convertases may be involved in the regulation of prostate epithelial cell differentiation, and may be an important target of prostate cancer therapy.     

Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast

Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.     

Relationship between adrenomedullin and vasopressin-aquaporin system under general anesthesia

AIM: The roles of adrenomedullin (AM) in body fluid balance under general anesthesia were investigated. METHODS: Time course changes in plasma osmolality, AM, arginine vasopressin (AVP), and urinary aquaporin 2 (AQP2) in 17 patients undergoing abdominal surgery under general anesthesia were examined. RESULTS: Increases in plasma AM levels were observed in parallel with increases in the levels of urinary AQP2/creatinine (Cr) before induction and 90 and 180 min after initiation of anesthesia. Significant correlations between plasma AM and urinary AQP2/Cr (r = 0.62, p < 0.0001) as well as urinary AVP/Cr and AQP2/Cr (r = 0.60, p < 0.0001) were uncovered. Multivariate stepwise analysis identified plasma AM as the critical independent factor affecting urinary AQP2/Cr level. CONCLUSION: A novel correlation of AM and AQP2 which overlays an AVP-AQP2 system may play a key role in fluid homeostasis during general anesthesia.     

Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability

The association of the p27(Kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(Kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for approximately 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was approximately 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively. The phosphorylation of p27(Kip1) was markedly reduced when the positions of Ser(10) and Pro(11) were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser(10). The extent of Ser(10) phosphorylation was markedly increased in cells in the G(0)-G(1) phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser(10) was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.     

Participation of testicular spermatids in development upon intracytoplasmic injection into eggs of the sawfly, Athalia rosae (Insecta, hymenoptera)

Fertilization by intracytoplasmic injection of mature sperm into mature eggs has previously been achieved in the sawfly, Athalia rosae (Insecta, Hymenoptera). In the present study, we examined the potential of spermatids, premature male gametes, for participating in development. When round spermatids and elongating spermatids from pupal testes were injected into the anterior end of mature eggs, about 5% of the total injected eggs developed into chimeric embryos (independent participation in development of the egg and spermatid nuclei). Some of them developed further, hatched, and pupated, with 1-2% of the total injected eggs becoming haploid chimeric male adults in which both the egg-derived and injected spermatid-derived nuclei contributed to the germline. No fertilized embryos were obtained by these injections. Elongated spermatids (immature sperm) from newly eclosed adult male testes upon injection did produce fertilized embryos that developed into normal diploid females (about 7% of the total injected). These results indicate that insect spermatids (round and elongating) have the potential to participate in development, but only independently of the egg nucleus. J. Exp. Zool. 286:181-192, 2000.     

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.     

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3)

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.