Interaction of platinum agents, cisplatin, carboplatin and oxaliplatin against albumin in vivo rats and in vitro study using inductively coupled plasma-mass spectrometory.

The protein binding rates (PBR) of platinum-containing agents cisplatin (CDDP), carboplatin (CBDCA) and oxaliplatin (L-OHP) have been reported as 98%, 25-50% and 98%, respectively. To investigate the protein-binding properties of albumin with cisplatin, carboplatin and oxaliplatin, inductively coupled plasma mass spectrometry (ICP-MS) was used to measure their plasma concentration in rats over time. The study also examined the effects of cisplatin, carboplatin and oxaliplatin-binding on albumin in vitro, using CD spectrometry and native-polyacrylamide gel electrophoresis (native PAGE). The ratios of PBR to irreversible PBR, of cisplatin and oxaliplatin were 98%:98% and 90%:87%, respectively, indicating a higher affinity for irreversible binding with albumin. That of carboplatin was 25%:10%, indicating 60-70% reversible binding with albumin. The plasma protein binding rate concentrations of cisplatin, carboplatin and oxaliplatin after in vivo administration were 96%, 15% and 80%, respectively. The CD spectrometry of albumin was unaffected by cisplatin, carboplatin and oxaliplatin binding. Though similar protein binding rates were observed with oxaliplatin and cisplatin, oxaliplatin had a higher mobility rate during PAGE. It was confirmed that the binding of cisplatin and oxaliplatin with albumin affected its electric charge but not the structure. In conclusion, cisplatin and oxaliplatin bind irreversibly with albumin in plasma and may irreversibly interact with tissue protein and/or DNA. The difficulties involved with predicting the tissue concentrations of cisplatin and oxaliplatin from their plasma concentration inhibits their therapeutic drug monitoring. On the contrary, carboplatin, like some generic drugs, reversibly binds to plasma proteins. It is, therefore, possible to conduct therapeutic drug monitoring for carboplatin.

Development of a p38α-selective radioactive probe for qualitative diagnosis of cancer using SPECT.

OBJECTIVE: p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α activity for effective qualitative diagnosis of cancer. METHODS: Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [125I]m-YTM, was synthesized because m-YTM greatly inhibited the phosphorylation of p38α upon examining the inhibitory effects of the compounds. After investigating the binding affinity of [125I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α was performed to determine the binding site. Uptake of [125I]m-YTM into various cancer cell lines was investigated, and the pharmacokinetics was evaluated using tumor-bearing mice. RESULTS: The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective inhibitor. The binding site of [125I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because the [125I]m-YTM bound strongly to both activated p38α and inactive p38α. Various different cancer cells incorporated [125I]m-YTM; however, its accumulation was significantly reduced by treatment with SB203580. Pharmacokinetics study of [125I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tissues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24 h, respectively. The ratio of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also ≥ 1 at 1 h after the administration of the probe. CONCLUSIONS: This study suggests that [123I]m-YTM has potential as a p38α imaging probe effective for various cancer types.

A Novel Analytical Method of Cisplatin Using the HPLC with a Naphthylethyl Group Bonded with Silica Gel (πNAP) Column.

Cisplatin is the most widely used anticancer drug in the world. Mono-chloro and none-chloro complexes of cisplatin may be believed to be the activated compounds. The separation of these compounds using octa decyl silyl column or aminopropylsilyl silica gel column is difficult because of high-reactivity and structural similarity. In this study, cisplatin, hydroxo complexes, and OH-dimer were determined by HPLC using a naphthylethyl group bonded with silica gel (πNAP) column. The analytical conditions of HPLC were as follows: analytical column, πNAP column; wave length, 225 nm; column temperature, 40°C; mobile phase, 0.1 M sodium perchlorate, acetonitrile, and perchloric acid (290 : 10 : 3), flow rate, 1.0 mL/min. Sample (20 µL) was injected onto the HPLC system. Retention time of cisplatin, mono-chloride, OH-dimer, and none-chloride was 3.2, 3.4, 3.6, and, 4.3-6.6 min, respectively. Measurable ranges with this method were 1×10-5 to 4×10-3 M for cisplatin. Correlation coefficient of the calibration curves of cisplatin was 0.999 (p<0.01). The within- and between-day variations of coefficient of variation (CV) were 5% or lower. In this study, injectable formulations in physiological saline solution, water for injection, 5% glucose solution, and 7% sodium bicarbonate precisely were measured the stability and compositional changes upon mixing by πNAP column rather than C18 column. We successfully determined cisplatin, hydroxo complexes, and OH-dimer by HPLC using a πNAP column. Thus the measurement of cisplatin (cis-diamminedichloro-platinum(II), cis-[PtCl2(NH3)2]) (CDDP) should be done using a πNAP column rather than a C18 column or aminopropylsilyl silica gel column.

Highly efficient uptake into cisplatin-resistant cells and the isomerization upon coordinative DNA binding of anticancer tetrazolato-bridged dinuclear platinum(II) complexes.

We examined the cytotoxicity and cellular uptake in L1210 murine leukemia cells, as well as the coordinative reaction with the guanine derivative 9-ethylguanine (9EtG), of a series of µ-hydroxo-µ-tetrazolato dinuclear platinum(II) complexes (tetrazolato-bridged complexes), [{cis-Pt(NH3)2}2(µ-OH)(µ-tetrazolato-N1,N2)](2+) (5-H-X) and [{cis-Pt(NH3)2}2(µ-OH)(µ-5-R-tetrazolato-N2,N3)](n+), where R = H (5-H-Y), CH3 (1), C6H5 (2), CH2COOCH2CH3 (3), or CH2COO(-) (4), and n = 2 (5-H-Y, 1-3) or 1 (4). Most tetrazolato-bridged complexes overcame cross-resistance to cisplatin and were more efficiently taken up into cisplatin-resistant cells (L1210R) than into parental cisplatin-sensitive cells (L1210), whereas cisplatin uptake into L1210R was decreased compared with that into L1210. The cellular uptake was most likely controlled by the total charge of the complexes. There was no correlation between the cytotoxicity and the kinetics of the coordinative reactions of 1-4 with 9EtG, but the isomerization involved in the reactions could contribute to determining the higher order structures of the compacted DNA. The cytotoxicity of tetrazolato-bridged complexes appears to correlate with the efficiency of cellular uptake and DNA compaction.

Occurrence and fate of selected anticancer, antimicrobial, and psychotropic pharmaceuticals in an urban river in a subcatchment of the Yodo River basin, Japan.

Pollution status of six anticancer agents in the river water and effluents of sewage treatment plants (STPs) in Japan was surveyed with comparative analysis of the levels of four microbial and one psychotropic pharmaceuticals widely used for therapeutic medication. The area of survey is located in the Kanzaki-Ai River basin which is a major subcatchment of the Yodo River basin and is centered on a highly populated area that includes the middle and downstream reaches of the Yodo River. Selected cancer agents were bicalutamide, capecitabine, cyclophosphamide, doxifluridine, tamoxifen, and tegafur. A combination of strong anion solid-phase extraction cartridge under pH 11 for adsorption and optimization of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) system was necessary to ensure high recovery rates (63-124% for river water and 52-115% for STP effluent). The year-round survey of these compounds in four seasons showed that all anticancer compounds were detected at median concentrations ranged from not detected to 32 ng/L in the river water and from not detected to 245 ng/L in the effluents of sewage treatment plants not using ozonation. In the case of bicalutamide (an active antiandrogen used to treat prostate cancer), the maximum concentration detected was 254 ng/L in river water and 1032 ng/L in non-ozonated sewage treatment plant effluents. Based on the mass balance, sewage treatment plants were the primary sources of anticancer compounds as well as the other pharmaceuticals in the river, and the attenuation effect of the river water was small. Ozonation at sewage treatment plants was effective in removing these compounds. To the best of our knowledge, this study is the first to report the existence of bicalutamide, doxifluridine, and tegafur in the river environment.

Detection of peramivir and laninamivir, new anti-influenza drugs, in sewage effluent and river waters in Japan.

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN-in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.

The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.

Second- and higher-order structural changes of DNA induced by antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes with different types of 5-substituent.

Here, we used circular dichroism (CD) and fluorescence microscopy (FM) to examine the interactions of a series of antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes, [{cis-Pt(NH3)2}2(µ-OH)(µ-5-R-tetrazolato-N2,N3)](n+) (R=CH3 (1), C6H5 (2), CH2COOCH2CH3 (3), CH2COO(-) (4), n=2 (1-3) or 1 (4)), which are derivatives of [{cis-Pt(NH3)2}2(µ-OH)(µ-tetrazolato-N2,N3)](2+) (5-H-Y), with DNA to elucidate the influence of these interactions on the secondary or higher-order structure of DNA and reveal the mechanism of action. The CD study showed that three derivatives, 1-3, with a double-positive charge altered the secondary structures of calf thymus DNA but that 4, the only complex with a single positive charge, induced almost no change, implying that the B- to C-form conformational change is influenced by ionic attraction. Unexpectedly, single-molecule observations with FM revealed that 4 changed the higher-order structure of T4 DNA into the compact-globule state most efficiently, at the lowest concentration, which was nearly equal to that of 5-H-Y. These contradictory results suggest that secondary structural changes are not necessarily linked to higher-order ones, and that the non-coordinative interaction could be divided into two distinct interactions: (1) ionic attraction and (2) hydrogen bonding and/or van der Waals contact. The relationship between diffusion-controlled non-coordinative DNA interactions and cytotoxicities is also discussed.

Adenosine thiamine triphosphate (AThTP) inhibits poly(ADP-ribose) polymerase-1 (PARP-1) activity.

Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) has been demonstrated to result in various stress-related diseases, including diabetes mellitus. Deficiency of cellular nicotinamide adenine dinucleotide (NAD(+)) content, consumed by PARP-1 to add ADP-ribose moieties onto target proteins, contributes to pathophysiological conditions. Adenosine thiamine triphosphate (AThTP) exists in small amounts in mammals; however, the function(s) of this metabolite remains unresolved. The structure of AThTP resembles NAD(+). Recent experimental studies demonstrate beneficial impacts of high-dose thiamine treatment of diabetic complications. These findings have led us to hypothesize that AThTP may modulate the activity of PARP-1. We have chemically synthesized AThTP and evaluated the effect of AThTP on recombinant PARP-1 enzyme activity. AThTP inhibited the PARP-1 activity at 10 µM, and a structural model of the PARP-1-AThTP complex highlighted the AThTP binding site. The results provide new insights into the pharmacological importance of AThTP as an inhibitor of PARP-1.

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA.

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH(3))(2)}(2)(micro-OH)(micro-1,2,3-ta-N (1),N (2))](NO(3))(2) (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

Determination of albumin in bronchoalveolar lavage fluid by flow-injection fluorometry using chromazurol S.

A highly sensitive flow injection fluorometry for the determination of albumin was developed and applied to the determination of albumin in human bronchoalveolar lavage fluids (BALF). This method is based on binding of chromazurol S (CAS) to albumin. The calibration curve was linear in the range of 5-200 microg/ml of albumin. A highly linear correlation (r=0.986) was observed between the albumin level in BALF samples (n=25) determined by the proposed method and by a conventional fluorometric method using CAS (CAS manual method). The IgG interference was lower in the CAS flow injection method than in the CAS manual method. The albumin level in BALF collected from healthy volunteers (n=10) was 58.5+/-13.1 microg/ml. The albumin levels in BALF samples obtained from patients with sarcoidosis and idiopathic pulmonary fibrosis were increased. This finding shows that the determination of albumin levels in BALF samples is useful for investigating lung diseases and that CAS flow injection method is promising in the determination of trace albumin in BALF samples, because it is sensitive and precise.

[Current status of and future perspectives for platinum antitumor drugs].

Cisplatin, a simple inorganic compound, has been one of the leading antitumor drugs for near 30 years. However, cisplatin has several drawbacks such as toxicity and drug resistance. Therefore, much attention has been focused on the development of new platinum complexes with improved pharmacological properties and a broader spectrum of activity to tumors. The recent advance of research on the molecular mechanisms of drug action and the cellular mechanisms of the emergence of resistance to cisplatin assists the rational design of new classes of platinum antitumor drugs, though details of both mechanisms still remain elusive. Information on DNA binding mode of platinum complexes, recognition and repair of DNA damage is instructive. Since several not cis isomers but trans isomers and not neutral complexes but cation complexes have been found active in vitro and in vivo, the early empirical structure-activity relationships of cisplatin analogues should be reevaluated. The hypothesis that platinum complexes which bind to DNA in a different manner will have different pharmacological properties has been tested, and now cationic multi-nuclear complexes and even trans-platinum complexes comprise unique classes of antitumor platinum-based agents with chemical and biological properties different from cisplatin. These new type platinum complexes are often effective to acquired cisplatin resistant tumor cells. In conclusion, the following complexes appear to offer great potential as new antitumor agents: (1) Complexes with distinctively different DNA interaction modes from cisplatin, which may circumvent intrinsic and acquired resistance to cisplatin through eluding the vigilance of DNA repair systems and (2) complexes with different tissue distribution or mechanisms of membrane transport which may exhibit a different spectrum of activity.

Determination of desmosine in bronchoalveolar lavage fluids by time-resolved fluoroimmunoassay.

BACKGROUND: Urinary excretion of desmosine has been reported to be increased in patients with pulmonary fibrosis; however, several investigators have pointed out that measuring urinary desmosine is not a very useful indicator of lung wall destruction. We developed a sensitive time resolved fluoroimmunoassay (TR-FIA) to identify trace amounts of desmosine in bronchoalveolar lavage fluid (BALF), and applied this method to analyse BALF samples from healthy subjects and patients with interstitial lung diseases. METHODS: In the proposed TR-FIA, a polystyrene strip was coated with desmosine-conjugated gelatin. The strip was then incubated with rabbit anti-desmosine antibody and the test solution. The desmosine bound to the solid phase and free desmosine in the sample or standard solution were allowed to compete to bind to the anti-desmosine. The solid-phase antibody was detected by Eu-complex conjugated anti-rabbit IgG. RESULTS: The detectable limit of desmosine was 50 fmol/ml in the TR-FIA developed in this study. TR-FIA showed low cross-reactivity against amino acids. BALF desmosine levels were significantly higher in patients with idiopathic fibrosis and sarcoidosis compared with healthy subjects. CONCLUSIONS: Desmosine levels in BALF may be useful to investigate lung disease.

Fluorimetric determination of trace amounts of albumin in bronchoalveolar lavage fluid with eriochrome cyanine R.

A fluorimetric method using eriochrome cyanine R (ECR) was applied to determine trace amounts of albumin in bronchoalveolar lavage fluid (BALF). The ECR-albumin adduct showed intense fluorescence and the calibration curve was linear in the range of 2.5-100 microg/ml of human serum albumin (r=0.999). The sensitivity was high enough to determine trace amounts of albumin in BALF. In the present study, the results obtained by the ECR method were compared with those of an EIA, and a good linear correlation was observed between the two methods (r=0.946). The ECR method is simpler and more rapid than EIA. The concentrations of albumin in BALF samples were determined by the ECR method. The albumin level in BALF of healthy nonsmokers (n=9) was lower than that of healthy smokers (n=9) (42.9+/-20.7 and 48.3+/-15.7 microg/ml, respectively). On the other hand, the albumin level of BALF samples obtained from patients with hypersensitivity pneumonitis was increased versus in healthy subjects. The determination of albumin in BALF samples by the ECR method is useful for investigating lung diseases.

Molecular characterization of a high-affinity xylobiose transporter of Streptomyces thermoviolaceus OPC-520 and its transcriptional regulation.

Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an alpha-L-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular beta-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed high-level affinity for xylobiose (K(d) = 8.75 x 10(-9) M) and for xylotriose (K(d) = 8.42 x 10(-8) M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the -10 region of the bxl operon. The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose. This site was also present in the region located between stxI and stxIV and in the upstream region of stxII. BxlR specifically bound to the regions containing the inverted sequence. These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S. thermoviolaceus OPC-520.

New isomeric azine-bridged dinuclear platinum(II) complexes circumvent cross-resistance to cisplatin.

Four new isomeric azine-bridged complexes ([(cis-Pt(NH(3))(2)Cl)(2)(mu-pzn)]Cl(2) (1a) (pzn = pyrazine) and its corresponding nitrate salt (1b), [(cis-Pt(NH(3))(2)Cl)(2)(mu-pmn)]Cl(2) (2) (pmn = pyrimidine), and [(cis-Pt(NH(3))(2)Cl)(2)(mu-pdn)](NO(3))(2) (3) (pdn = pyridazine) have been newly synthesized as potential anticancer compounds. These complexes have been characterized by (1)H and (195)Pt NMR spectroscopy, and also the X-ray crystal structure of 1b has been determined. The reactions of 1a, 2, and 3 with guanosine-5'-monophosphate (GMP) have been monitored and kinetically investigated in D(2)O solutions at 310 K using (1)H NMR spectroscopy. Both 1a and 2 react with 2 equiv of GMP to form 1:2 complexes. The reactions involve a stepwise direct substitution of chloride ligands by GMP, with similar reaction rates for both complexes. On the other hand, the reaction of 3 with GMP results in the cleavage of one of the Pt-N(pyridazine) bonds to form an N7,O6-platinated polymer. The reaction products have been separated and have been characterized by (1)H and (195)Pt NMR spectroscopy. A cytotoxicity assay of the azine-bridged complexes (1a, 1b, 2, and 3) has been performed on human tumor cell lines and two L1210 murine leukemia cell lines (one sensitive to and one resistant to cisplatin). In general, the complexes show lower cytotoxicity than cisplatin for the human tumor cell lines except for the IGROV cell line. Their cytotoxicity for the mouse cell lines is comparable to or higher than that of cisplatin. Furthermore, these complexes appeared to largely or partly overcome the cross-resistance to cisplatin. Implications of these findings are discussed in the context of a structure-activity relationship for this class of compounds.

A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium, Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.

A novel isomerization on interaction of antitumor-active azole-bridged dinuclear platinum(II) complexes with 9-ethylguanine. Platinum(II) atom migration from N2 to N3 on 1,2,3-triazole.

The reactions of the dinuclear platinum(II) complexes, [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (1, pz = pyrazolate), [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (2, 1,2,3-ta = 1,2,3-triazolate), and a newly prepared [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-4-phe-1,2,3-ta-N1,N2)](NO(3))(2) (3, 4-phe-1,2,3-ta = 4-phenyl-1,2,3-triazolate), whose crystal structure was determined, with 9-ethylguanine (9EtG) have been monitored in aqueous solution at 310 K by means of (1)H NMR spectroscopy. The dinuclear platinum(II) complexes 1-3 each react with 9EtG in a bifunctional way to form 1:2 complexes, [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-pz)](3+) (4), [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-1,2,3-ta-N1,N3)](3+) (5), and [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-4-phe-1,2,3-ta-N1,N3)](3+) (6). The reactions of 2 and 3 involve a novel isomerization, in which the Pt atom, initially bound to N2 on the 1,2,3-ta, migrates to N3 after the first substitution by N7 of 9EtG. This isomerization reaction has been unambiguously characterized by 1D and 2D NMR spectroscopy and pH titration. The reactions of 2 and 3 with 9EtG show faster kinetics, and the second-order rate constants (k) for the reactions of 1-3are 1.57 x 10(-4), 2.53 x 10(-4), and 2.56 x 10(-4) M(-1) s(-1), respectively. The pK(a) values at the N1H site of 9EtG were determined for 4-6 from the pH titration curves. Cytotoxicity assays of 1-3 were performed in L1210 murine leukemia cell lines, respectively sensitive and resistant to cisplatin. In the parent cell line, 2 and 3 exhibit higher cytotoxicity compared to cisplatin, especially, 2 is 10 times as active as cisplatin. 1 was found to be less cytotoxic than cisplatin, but still in the active range and more active than cisplatin in a cisplatin-resistant cell line.

Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system.

Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.