Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.     

Transformation of thiols to disulfides by epolactaene and its derivatives

In this paper we report a disulfide formation of thiols induced by epolactaene and its derivatives. We previously reported the disulfide formation of N-acetylcysteine methyl ester by epolactaene in a 1:1 MeOH/0.5M NaHCO(3) aq solution. The present studies reveal that the disulfide formation proceeds under mild conditions such as in PBS at pH 7.3, suggesting that epolactaene may induce disulfide formation of cellular thiols. This compound induces the disulfide formation of several thiols in a 1:1 MeOH/0.5M NaHCO(3) aq solution at room temperature. Moreover, our results show that the acyl side-chain of epolactaene greatly influences the products of the reaction. We analyzed the reaction mechanism by using thiolysis products of epolactaene derivatives and propose a new reaction mechanism.     

Triticone A: a novel bioactive lactam with potential as a molecular probe

Triticone A is one member of a family of novel compounds which are spirocyclic lactams produced by several plant pathogenic fungi including Drechslera tritici repentis on wheat. It undergoes racemization to form triticone B and when tested, the enantiomeric mixture causes chlorosis and necrosis on a wide range of plants. Fluorescein diacetate treated protoplasts in conjunction with various triticone treatments allowed for accurate quantitation of the biological activity of the toxin. Various physiological functions of the wheat cell are impaired including the Hill and CO2 fixation reactions in photosynthesis. In addition, triticone A inhibits enzymes that have SH functional groups as part of their active site, eg., the protease-ficin. Neither triticone C or D had any activity in the enzyme or protoplast assays. It is apparent that triticone A has some potential as a molecular probe in a variety of biological systems.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

Maturation and spawning processes of anadromous and resident pond smelt in lake ogawara

Pond smelt,Hypomesus nipponensis McAllister, in Lake Ogawara demonstrate alternative life history strategies, as evidenced by the coexistence of anadromous and resident fish. However, it is unknown if anadromous and resident groups interbreed. In this study, maturation and spawning processes were examined and compared between anadromous and resident groups. Histological observations indicated negligible variation in the maturational stage composition of oocytes, the frequency of oocyte diameter being unimodal for all specimens at different maturational stages. Oocytes were absent in the ovaries of spent fish. Accordingly, the species can be considered a semelparous spawner with unimodal oocyte diameter distribution. Temporal changes in the proportion of spent fish were compared between anadromous and resident groups. Spawning of both groups began in late March and peaked over April 8–12. Although both groups did not differ significantly in the period of peak spawning, anadromous fish finished spawning earlier than resident ones. Anadromous fish were not able to spawn upon migration into Lake Ogawara, and quickly matured after immigration, contrasting with resident fish.     

Mode analysis of a fatty acid molecule binding to the N-terminal 8-kDa domain of DNA polymerase beta. A 1:1 complex and binding surface.

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C(18)-linoleic acid (LA) or C(24)-nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an Omega-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K(D)) of 1.02 or 2.64 mM, respectively.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

DNA Data Bank of Japan dealing with large-scale data submission.

The DNA Data Bank of Japan (DDBJ) (http//:www.ddbj.nig.ac.jp) has developed a software system for mass submissions to cope with a recent expansion of EST and genome data submissions. The system is composed of four parts, the WWW data submission, large-scale submission, submission management and storing. Using this system one can submit data on a large number of sequences or a very long sequence while checking the consistency between the annotation and sequence without much effort. DDBJ has received large scale data of Homo sapiens, Arabidopsis and Pyrococcus from Japanese researchers who made full use of the new submission system.     

Functional analysis of the p57 KIP2 gene mutation in Beckwith-Wiedemann syndrome

p57 ^KIP2 is a potent tight-binding inhibitor of several G₁ cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57 ^KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57 ^KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57 ^KIP2 in patients with BWS that were transmitted from the patients’ carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57 ^KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57 ^KIP2 would have existed, which might have caused the disorders in BWS patients. Received: 7 November 1998 / Accepted: 19 December 1998     

Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60 degrees C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade p-chlorobiphenyl, suggesting the existince of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra- and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA.