Development of a nomogram incorporating serum C-reactive protein level to predict overall survival of patients with advanced urothelial carcinoma and its evaluation by decision curve analysis.

BACKGROUND: The purpose of this study is to investigate the prognostic impact of C-reactive protein (CRP) on patients with advanced urothelial carcinoma and to develop a novel nomogram predicting survival. METHODS: A total of 223 consecutive patients were treated at Tokyo Medical and Dental Hospital. A nomogram incorporating V was developed based on the result of a Cox proportional hazards model. Its efficacy and clinical usefulness was evaluated by concordance index (c-index) and decision curve analysis. RESULTS: Of the 223 patients, 184 (83%) died of cancer. Median follow-up periods of patients who died and those who remained alive were 5 and 11 months, respectively. We developed a novel nomogram incorporating Eastern Cooperative Oncology Group Performance Status, presence of visceral metastasis, haemoglobin and age. The c-index of the nomogram predicting survival probability 6 and 12 months after diagnosis was 0.788 and 0.765, respectively. Decision curve analyses revealed that the novel nomogram incorporating CRP had a superior net benefit than that without CRP for most of the examined probabilities. CONCLUSION: We demonstrated the prognostic impact of CRP that improved the predictive accuracy of a nomogram for survival probability in patients with advanced urothelial carcinoma.

Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus.

The gene coding for phenylalanine dehydrogenase [PDH; L-phenylalanine: NAD+ oxidoreductase (deaminating); EC 1.4.1.-] from Bacillus sphaericus SCRC-79a was cloned onto plasmid pUC9, and the nucleotide sequence of the 2-kb DNA region of the insert was determined. A 1143-bp open reading frame consisting of 381 codons was identified as a pdh gene coding for PDH.

Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli.

A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.

Showdomycin analogues: synthesis and antitumor evaluation.

The synthesis of N-beta-D-ribofuranosyl derivatives of maleimide, 3-methylmaleimide, and 3-chloromaleimide was accomplished in three steps from ribosylamine. The synthetic ribosides can be considered N-nucleoside analogues of showdomycin, which is an antitumor antibiotic of the C-nucleoside type. Although the three analogues were cytotoxic to cultured L1210 cells, no in vivo antitumor activity was found with the murine P388 leukemia test system. Drug transport studies were done in an attempt to trace the biological fate of the analogues.

Lymphotoxin cDNA clones from a HTLV-I-carrying T cell line HUT-102.

The conditioned medium of a HTLV-I-carrying T cell line HUT-102 showed cytotoxic activity against a mouse fibroblast cell line L-M. We prepared the cDNA library from HUT-102 poly(A)+ RNA and screened it using oligonucleotide probes that correspond to the amino acid sequences conserved in tumor necrosis factor (TNF) and lymphotoxin (LT). As a result we obtained two kinds of cDNA clones encoding LT in which amino acid residue 26t of mature LT is different; one is Asn and another is Thr. The sequence of the genomic clones obtained using a polymerase chain reaction method showed that the HUT-102 genome also contains two types of LT genes. Recombinant LTs expressed in Escherichia coli exhibited the same level of cytolytic activity against L-M cells. These results indicate that the cytotoxin constitutively produced by HUT-102 cells include two kinds of LT.

Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli.

The cloned xylE gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of approximately 15% of the total soluble protein. Of the total iron in the crude extract, 45% was on the enzyme. The crystallized enzyme from E. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme from P. putida mt-2. However, no differences were observed in physicochemical, protein-chemical, and kinetic properties between the two enzymes. The enzyme was a homotetramer, and no changes were observed in the values of M(r) (136,000 +/- 5,000) and Stokes radius (4.26 nm) in the concentration range from 0.36 nM to 2.8 microM, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range. The catalytic center activity and the Km values for catechol and dioxygen were 278 s-1, 1.87 and 7.45 microM, respectively, at pH 7.5 and 25 degrees C. The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity constants (approximately 200 microM-1.s-1) higher than that for catechol. Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme.

Production of eicosapentaenoic acid by marine bacteria.

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.

Characterization of thrombin- and plasmin-resistant mutants of recombinant human single chain urokinase-type plasminogen activator.

Recombinant human single-chain urokinase-type plasminogen activator (suc-PA) (SM0: wild type) and its variants resistant to plasmin and/or thrombin (SM1: Lys135 to Gln; SM3: Phe157 to Asp; and SM4: Lys135 to Gln and Phe157 to Asp) have been constructed by site-directed mutagenesis with the aim of producing more efficient thrombolytic agents [Miyake, T. et al. (1988) J. Biochem. 104, 643-647]. In the present study, we characterized the recombinant variant scu-PAs expressed in Escherichia coli. They appeared to have structural integrity because their heat-stabilities, immunological reactivities, and circular dichroism spectra were essentially identical to those of each other and of native scu-PA (nscu-PA). In the presence of thrombin, SM3 and SM4 showed efficient clot lysis by all of the assays used, compared with SM0, SM1, and nscu-PA. While in the absence of thrombin, when measured by a fibrin plate method in a purified system, SM3 and SM4 had lower specific activities than SM0, SM1, and nscu-PA, because of their catalytic constants for conversion to the two-chain form (tcu-PA) by plasmin are lower. However, SM4 lysed clots as efficiently as SM0 in plasma by retaining the single-chain form, whereas SM0 was partly converted to the two-chain form.

[A case of renal tuberculosis following bacillus Calmette-Guerin instillation therapy for bladder cancer].

A rare case of histopathologically revealed renal tuberculosis caused by intravesical bacillus Calmette-Guerin (BCG) therapy is reported. A 67-year-old man was admitted complaining of fever and micturition pain. He had been undergoing prophylactic BCG instillation therapy for recurrent superficial bladder tumor. Physical examination was unremarkable. The tuberculin skin test was negative. Mycobacterium tuberculosis (MT) was not demonstrated by acid-fast staining and culture of urine. However, MT was isolated by the polymerase chain reaction method. In the following 7 days, symptoms were dissolved with administration of isoniazid, rifampicin and piperacillin. Two months later, nephroureterectomy was performed because of left renal pelvic tumor. Tuberculomas were also found in the renal parenchyma which showed no MT by Ziehl-Neelsen's method. Anti-tuberculous medication was not given postoperatively. Two months after operation, he is free of disease with normal urine examination and positive tuberculin skin test measuring 12 x 10 mm.

Diffusion-weighted MRI as a potential imaging biomarker reflecting the metastatic potential of upper urinary tract cancer.

OBJECTIVE: To evaluate the role of diffusion-weighted MRI (DW-MRI) as an imaging biomarker for upper urinary tract cancer (UUTC) that has already metastasized or will metastasize soon. METHODS: 61 patients clinically diagnosed with UUTC were prospectively enrolled in this study. All the patients underwent MRI, including DW-MRI, prior to any interventions. Correlations between apparent diffusion coefficient (ADC) and other clinicopathological variables, including metastasis-free survival, were analysed. RESULTS: Median follow-up period was 938 days. Of the 61 patients, 12 had any metastases at the initial diagnosis. 11 patients developed metastases during the follow-up period. These 23 patients were categorized as "Metastatic". Of the remaining 38 patients, 35 with a follow-up period longer than 400 days were categorized as "Localized". ADC was significantly lower in the Metastatic category than in the Localized (p = 0.0002) category. Multivariate analysis of pre-operative variables identified ADC (cut-off value, 1.08 × 10(-3) mm(2) s(-1)) and clinical T stage based on T2 weighted MRI as an independent predictive factor of metastatic UUTC. 46 patients without any metastases during the initial diagnosis were stratified into a high-risk group (16 patients with low ADC and clinical T3-4) and a low-risk group (30 patients with high ADC or clinical Ta-2). The 3-year metastasis-free survivals were 45% and 93%, respectively. CONCLUSION: In the current study, UUTC with lower ADC value is more likely to have metastatic potential. Incorporating ADC with clinical T stage helps to differentiate metastatic UUTC at the initial diagnosis. ADVANCES IN KNOWLEDGE: DW-MRI is a potential imaging biomarker reflecting metastatic propensity of UUTC.

Prediction of the active sites of proteins from amino acid sequences.

The discriminant analysis of complementary units and repeated sequences of amino acids in an initial sample of 48 different enzymes produces practically useful empirical-functions which allow catalytic sites to be distinguished from non-catalytic sites. The independent variables in the discriminant functions were almost all composed of complementary units of amino acids, that is amino acid sequences whose nucleotide coding sequences were complementary to each other. In order to evaluate the validity of the functions, we applied them to the amino acid sequences of 17 different kinds of enzymes as well as 30 non-enzymes such as receptors, oncoproteins, cytokines, hormones and so on. The functions proved to be effective in predicting not only the catalytic sites of enzymes but also the binding sites of the other proteins. The result suggests that complementary units are evolutionarily conserved as a signal around the active sites of various proteins.

Phenylalanine dehydrogenase of Bacillus badius. Purification, characterization and gene cloning.

Phenylalanine dehydrogenase produced by Bacillus badius IAM 11059 was purified from the crude extract of B. badius to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.5 and a relative molecular mass, Mr, of 310,000-360,000. The enzyme is composed of identical subunits with an Mr 41,000-42,000. The substrate specificity of the enzyme in the oxidative deamination reaction was high for L-phenylalanine, but rather low in the reductive amination reaction, with phenylpyruvate, p-hydroxyphenylpyruvate, and 2-oxohexanoate. The gene for the enzyme was cloned into Escherichia coli with plasmid pBR322 as a vector. The enzyme was expressed in high level in E. coli. The enzyme produced by E. coli transformant was purified to homogeneity and shown to be identical to that of B. badius IAM 11,059 with respect to the specific activity, Mr, subunit structure and amino acid composition.

Antibacterial activity of (+/-) 6-benzyl-1-(3-carboxypropyl) indane; a possible way to identify leading novel anti-H. pylori agents.

Magainin 2, isolated from the skin of the Xenopus laevis, is an antimicrobial peptide which reacts directly with the biological membrane to lyse various bacteria from negative and positive microorganisms. In a previous report, we showed that (+/-)1-(4-aminobutyl)-6-benzylindane (PM2), which mimicked the conformation of the side-chains of a complementary unit on the amino acid sequence of magainin 2 analogs, expressed the in vitro antibacterial activity not only against Helicobacter, pylori (ATCC43526, ATCC43579), but also against Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC25923). In addition, PM2 caused human blood red cells (RBCs) to lyse at the minimum inhibitory concentration (MIC) value. Based on the antibacterial activities of 9-phenylnonanoic acid (pC9c), we further synthesized (+/-)-6-benzyl-1-(3-carboxypropyl) indane (PM2c), which replaced a positive charge of PM2 with a negative one, and tested the biological activities. PM2c had the ability to inhibit the growth of H. pylori strains, but its activity to inhibit the growth of E. coli and S. aureus was not detected and weak, respectively. Moreover, PM2c showed non-hemolytic activity against RBCs at the MIC value. These results indicate the possibility that PM2c may be more useful than PM2 either alone or in combination with well-known therapeutic agents for the treatment of H. pylori infection.

Synthesis of reversed magainin 2 analogs enhanced antibacterial activity.

Magainin 2 is an antimicrobial peptide found in the skin of Xenopus laevis. To find a reversed peptide comparable to the antibacterial activity of magainin 2 analogs, we have synthesized three reversed analogs, the peptide 53D, 87-ISM and A87-ISM, corresponding to the normal peptide D35, MSI-78 and MSI-78A, respectively. We examined their ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. Among the analogs, the A87-ISM, that is, the reverse of MSI-78A enhanced the amphiphilicity and the alpha-helical tendency of magainin 2, showed not only almost the same antibacterial activity against the bacteria as MSI-78A, but also stronger activity than other magainin 2 analogs. In addition, at the MIC (minimum inhibitory concentration) value, A87-ISM shows no hemolysis to human red blood cells, while both MSI-78 and MSI-78A cause strong hemolysis at the MIC value. This result indicates that a novel reversed peptide comparable or superior to normal magainin 2 analogs is available.

Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.

We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.

On the antibacterial activity of normal and reversed magainin 2 analogs against Helicobacter pylori.

Magainin 2 is an antimicrobial peptide isolated from the skin of Xenopus laevis. We have tested the antibacterial activities of normal and reversed magainin 2 analogs against two strains of Helicobacter pylori (ATCC 43526, ATCC 43579), compared with those against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Among these analogs, MSI-78A showed the strongest activity against H. pylori. The MIC (minimum inhibitory concentration) values were almost the same as those against E. coli and S. aureus. No or lesser activity was observed in all the reversed peptides compared to the corresponding normal magainin 2 analogs. Based on the CD (circular dichroism) measurement, the more active peptide tends to show a higher alpha-content. The positively-charged five amino acids (KILKK) positioned at the C terminus on the amphipathical alpha-helical structure play important roles in exerting the strong activity against H. pylori. This indicates that the net charge of the cell surface in H. pylori may be more negative than that of E. coli, though both strains belong to the same genus.

Growth inhibition and morphological changes of LLC-PK1 induced by ultimobranchial calcitonins.

Ultimobranchial calcitonins (CTs), known to stimulate cAMP production, inhibited the growth of a porcine kidney cell line LLC-PK1. This inhibition was accompanied by degenerative changes including vacuole formation and cell detachment. The electron microscopic study revealed marked swelling of rough endoplasmic reticulum (RER). Other cAMP-increasing agents such as human CT, arginine, vasopressin, and forskolin showed less growth inhibitory activities and no induction of the degenerative changes. These results indicate that the growth inhibition of LLC-PK1 by ultimobranchial CTs is mainly due to cellular death caused by the swelling of RER via a signalling pathway other than the cAMP-dependent event(s).

Antibacterial activity of two alkylamines integrated an indane scaffold: mimicry of a complementary unit on magainin 2.

Based on the antibacterial activity of 9-phenylnonylamine (pC9a) against Escherichia coli (ATCC29522) and Staphylococcus aureus (ATCC25923), we have further tested the inhibitory ability of the growth of the bacteria by (+/-)1-(4-aminobutyl)-6-benzylindane (PM2) and (+/-)1-benzyl-6-(4-aminobutyl) indane (PM3), that is, two kinds of 1,6-disubstituted indanes. In an in vitro assay, they showed almost the same antibacterial activities against the bacteria as pC9a, as well as that of magainin 2 analogs (i.e., the peptides MSI-78 and 87-ISM), except in the case of 87-ISM against S. aureus. At the MIC (minimum inhibitory concentration) values, however, their killing rate of E. coli is actually quicker than pC9a. This indicates that an indane scaffold, used as a template to mimic a part of the alpha-helical structure of magainin 2, can accelerate the killing rate. At present, however, it is unknown whether either the hydrophobicity or the alpha-helical structure, or both, of the indane scaffold is involved in accelerating the rate. Moreover, these two indanes also showed stronger antibacterial activity against two strains of Helicobacter pylori (ATCC43526, ATCC43579) than either pC9a or magainin 2 related peptides.

Synthesis and biological activity of four kinds of reversed peptides.

We have synthesized four kinds of reversed peptides of various physiologically active peptides, which inhibit TNF (tumor necrosis factor) cytotoxicity, produce NGF (nerve growth factor), exert antimicrobial activity and inhibit cell attachment, respectively. They were examined for their biological activity in comparison with that of normal peptides, that is, naturally occurring peptides. The reversed peptides induce similar activities, but to a lesser extent than those of the normal peptides, respectively. These results indicate that there may be conformationally ambiguous binding in some of the naturally occurring ligand-protein interactions. This method may be useful as a tool to rapidly generate a novel lead peptide with the desired biological function from a naturally occurring active peptide.

Biological activities of 1,1,6-trisubstituted indanes: beyond magainin 2.

MSI-78 is a peptide analog of naturally occurring magainin 2 isolated from the skin of Xenopus laevis. The peptide is known to have one of the strongest antibacterial activities in magainin 2 analogs against methicillin-resistant Staphylococcus aureus (MRSA). To find novel compounds superior to MSI-78, we have further designed, synthesizing 1,1-di(4-aminobutyl)-6-benzylindane (PM4) and 1,1-dibenzyl-6-(4-aminobutyl) indane (PM5), and tested their inhibitory ability of the growth of S. aureus. In an in vitro assay, PM4 showed the same antibacterial activity against the bacterium as MSI-78, and non-hemolytic activity against human red blood cells (RBCs) at the MIC (minimum inhibitory concentration) value, in contrast to the latter. On the other hand, PM5 showed stronger antibacterial activity than MSI-78, but being still accompanied with hemolysis at the MIC value. Otherwise, stronger decarboxylase activity for oxaloacetate was observed in PM5, rather than magainin 2 analogs or Oxaldie 1 as a control peptide, but not in PM4.