Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

Chromatography of guanidino compounds.

Guanidino compounds involved in the urea and guanidine cycles have been found in serum of nephritic patients, and some guanidino compounds have been suspected to be uremic toxins. The simultaneous analysis of naturally occurring metabolites is important for diagnosis of diseases. In this review, liquid chromatographic analysis of natural metabolites of guanidino compounds are described. the information about arginine as a precursor of nitric oxide are included. The reports of pharmaceutical compounds having a guanidino group, peptides containing arginine and aminoglycosides are summarized in Table 1.

Isoproterenol-induced myocardial injury resulting in altered S100A4 and S100A11 protein expression in the rat.

S-100 proteins (S100) are characterized by calcium-binding ability with two structural EF hands. Several S100 are expressed in cardiomyocytes and thought to play a crucial role in calcium signaling. To examine whether the expression of S100 is a response to detectable myocardial damage or regeneration, we investigated, immunohistochemically, the expression of S100A4 and S100A11 in the isoproterenol (ISP)-treated rat heart. Definite expression of S100A4 and S100A11 was demonstrated in normal cardiomyocytes, and their staining patterns were enhanced in the ISP-treated rat heart, suggesting the possible involvement of S1-A4 and S100A11 in ISP-induced myocardial damage.

Rapid analysis of guanidino compounds in serum from nephritic patients using column-switching with isocratic elution.

The rapid method for baseline separation of ten guanidino compounds in serum from nephritic patients was designed using a single eluent with a column switching system. A porous graphitic carbon column and an octadecyl-bonded silica gel columns were used, (50 mm x 4.6 mm i.d.). Separation was completed within 15 min. The stable baseline permitted highly sensitive detection with excellent reproducibility. The system was applied to analyze guanidino compounds in sera from 175 nephritic patients. The hemodialysis process could not completely eliminate guanidino compounds, and the degree of removal varied between patients. The correlation among metabolites indicated the differences in disease.

Liquid chromatography of guanidino compounds using a porous graphite carbon column and application to their analysis in serum.

The retention mechanism of guanidino compounds on a porous graphitic carbon seemed to be mainly hydrophobic interaction, according to the retention factors in buffer solutions and the results of an analysis by computational chemical calculation using molecular mechanics (MM2). The baseline separation of ten guanidino compounds was achieved by the addition of a hydrophobic counterion. The retention mechanism may be dynamic ion-exchange. The stable system was applied to the analysis of guanidino compounds in serum from nephritic patients. The effluent was monitored by a post-column labeling detection method using ninhydrin. The detection limit of guanidino compounds was a few picomoles; however, that of creatinine was one hundredth of those of the other compounds. The reproducibilities of the peak height and area of the ten guanidino compounds using gradient elution were quite high, and the standard deviations were within a few percent (n=5), except for creatinine. The recovery of the compounds from serum was more than 90% (n=5). The reproducibility of retention times was within 1% (n=5).

The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors.

Octamer binding transcription factors (Oct factors) play important roles in activation of transcription of various genes but, in some cases, require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses showed that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH was also shown to interact with POU domains of Oct-1 and Oct-2. MAT1 is shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific, suggesting that, in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (i) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (ii) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (iii) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions.

Site- and strand-specific nicking at oriT of plasmid R100 in a purified system: enhancement of the nicking activity of TraI (helicase I) with TraY and IHF.

We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, TraI and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the TraI protein and Mg2+. The products were complex DNA molecules with a protein covalently linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the TraI protein alone, indicating that the protein attached at the 5' end of the nick is the TraI protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the TraI and TraY proteins at oriT.

Specific DNA binding of the TraM protein to the oriT region of plasmid R100.

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.

Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100.

We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100. The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein. This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100. The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick. The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA.

Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100.

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.

Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid.

The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning. In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene. The purity of the final preparation was more than 95%, as determined by densitometry. The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene. Therefore, it was concluded that the protein was the sopB gene product. Using anti-SopB serum, the SopB protein was detected in the cell lysates of F+, F', and Hfr strains. The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322.

Identification and characterization of the products from the traJ and traY genes of plasmid R100.

The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with beta-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.

[Intraoperative radiotherapy of stomach cancer].

UNLABELLED: Clinical results of intraoperative radiotherapy for gastric cancer were reported. One hundred and five cases of gastric cancer were treated by intraoperative radiotherapy. Indications for the intraoperative radiotherapy were determined as follows: 1) The primary lesion (tumor) located at the level of gastric corpus or antrum (M, A); 2) At least, the primary tumor should be resected; 3) Neither liver metastasis nor peritoneal dissemination was noticed; 4) Lymph node metastasis were limited to the second group (less than N3); 5) Cases of which tumor-infiltration-depth reached to the serosal surface (S3) of the posterior gastric wall and infiltrated to the pancreatic capsule were included. However, infiltrations to the serosal surface of the anterior gastric wall were not subjected to the treatment, because of having peritoneal disseminations. Irradiation of 2800-4000 rad of electron beam from betatron was performed after resection of the stomach and the regional lymph node as possible. Five-year survival rate was studied on 58 cases (consisting 5 cases of stage I, 27 cases of stage II, 12 cases of stage III and 14 cases of stage IV). Comparisons were done with the cases receiving surgical operation only. RESULTS: Whatever the stage of the patient was, 3-year survival rate was found to be better in the radiotherapy group than that of the control group (treated surgical resection only). Five year survival rate of the stages III and IV in the radiotherapy group was better than the control group. Unfavorable side effects were observed in 4 cases out of 105 cases. In one case, penetration of postoperative peptic ulcer into the irradiated aortic wall was found by autopsy. Two cases of bile duct stenosis and one case of ileus due to acutely developed peritonitis carcinomatosa were experienced. In conclusion, intraoperative radiotherapy immediately after surgical resection for the treatment of gastric cancer was found to be an effective method. The most effective application of the method appears to be to cases of stage II and III without liver metastasis and peritoneal disseminations (H0P0, M, A).