Insights into the divergent evolution of the oceanic squid Sthenoteuthis oualaniensis (Cephalopoda: Ommastrephidae) from the Indian Ocean.

Sthenoteuthis oualaniensis is known for its complex population structure with three major transoceanic forms (viz. middle-sized, dwarf, and giant forms) whose taxonomic status has been disputed for decades. This integrated taxonomic study examines these prevenient morphotypes gathered on cruises in the Indian Ocean to ascertain their status in the evolutionary history of the species. Molecular analyses employing mitochondrial (COI, ND2) and nuclear (H3) markers revealed four genetically distinct and novel lineages of the species in the Indian Ocean, representing three morphotypes from the Arabian Sea and one from the Southern Indian Ocean. The mitochondrial-based phylograms revealed two distinct clades in the species: "dwarf forms + giant form" and "middle-sized forms," which further branch into geographically structured evolutionary units. Species delimitation analyses recovered five distinct clades, namely, the Arabian Sea giant and dwarf forms, Equatorial, Eastern Typical, and Other Middle-sized forms, representing the consensus molecular operational taxonomic units. H3 being heterozygous could not resolve the phylogeny. Haplotype network and AMOVA analysis of mtDNA genes indicated explicit phylogeographic structuring of haplotypes, whereas these outputs and PCA results were incongruent with the morphological grouping. Phenetic features distinguishing the morphotypes were sometimes plastic and mismatched with the genotypes. The giant form was genetically close to the dwarf forms, contradicting the earlier notion that it descended from the middle-sized form. It may be assumed that the dwarf form evolved following sympatric speciation and adaptation to warm equatorial waters, while the focal features of the Western Arabian Sea guide toward allopatric speciation of the giant form.

Spatio-temporal variability of physico-chemical variables, chlorophyll a, and primary productivity in the northern Arabian Sea along India coast.

The present study attempts to understand the seasonal and spatial variations in the physico-chemical (temperature, pH, salinity, dissolved oxygen, and nutrients) and productivity characteristics of the northern Arabian Sea off the Indian coast. Samples were collected from four different sites off the Veraval coast. The values of the physical and chemical variables were higher during the summer season, whereas nutrient concentrations were high during the winter season due to the maturity of intake nutrients during post-monsoon and winter convective mixing during the northeast monsoon. The dissolved oxygen (DO) concentration was strongly and positively correlated with the net primary productivity (NPP) and chlorophyll a (Chl-a) content to support productivity along the region. Dissimilarity in study variables was observed between the inshore and offshore locations. Principal component analysis revealed a strong relationship between nutrients and productivity variables (Chl-a and NPP). Nutrient levels were high at inshore sites, which can be attributed to the heavy nutrient load from land-based anthropogenic activities and impact due to natural processes like water mixing, sedimentation, and wave activities. Nutrients were strongly and positively correlated with the productivity variables, i.e., Chl-a and NPP. Chl-a positively correlated with NPP (r = 0.90), which indicates that it is a principle productivity pigment in the marine ecosystem.

Pharmacokinetic analysis and antitumor efficacy of MKT-077, a novel antitumor agent.

MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [14C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of 14C after i.v. administration of [14C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of 14C decreased with a T1/2 of approximately 5 min. In the second and terminal phase, the T1/2 of 14C was 2.8-4.6 h and 16.2 h, respectively. Cmax (1 min after injection) increased from 0.3 to 1.5 microg/ml linearly, but less than proportionately between the doses. The AUC(0-infinity) at 0.3, 1 and 3 mg/kg were 0.030 +/- 0.002, 0.60 +/- 0.12 and 1.73 +/- 0.25 microg x h/ml, respectively. Plasma clearance was approximately 1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), 14C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), 14C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [14C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors.

Determination of MKT-077, a novel antineoplastic agent, in plasma samples by high-performance liquid chromatography and its application to pharmacokinetics in rats.

A simple high-performance liquid chromatographic method was developed for determination of a novel antineoplastic agent MKT-077 in plasma. MKT-077 was extracted from 50 microl of plasma with acetonitrile containing 1 ml trifluoroacetic acid per liter. Chromatographic separation was achieved within 13.5 min using a reverse-phase Puresil C18 analytical column. A visible detector operated at 490 nm was used. The linearity of the calibration curve was obtained (r2 = 0.99986) over the analytical range of 10-500 ng/ml(-1). The intra- and inter-assay precision was in the range of 0.9-11.1 and 0.3-4.4%, respectively. The intra- and inter-assay bias ranged from -7.3 to 11.1% and from 0.4 to 11.6%, respectively. The utility of this assay was demonstrated after the administration of a single dose of MKT-077 to rats. The plasma elimination half-life of MKT-077 was 1.8-4 h.

Selective antitumor activity of MKT-077, a delocalized lipophilic cation, on normal cells and cancer cells in vitro.

BACKGROUND AND OBJECTIVES: 1-Ethyl-2-¿[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-+ ++oxothiazolidin-2-ylidenemethyl¿pyridium chloride (MKT-077, formerly known as FJ776), a delocalized lipophilic cation, is known to accumulate in the mitochondria, according to the negative potential inside the mitochondria, and exert its cytotoxicity. METHODS: The single-cell suspensions of human cancer cell lines, human spleen cells, and fresh cancer specimens obtained from patients with gastric carcinoma were used for the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. RESULTS: The antitumor activity of MKT-077 was dose and concentration related, and 50% inhibitory concentrations (IC50) ranged from 1.7 to 14.3 microg/ml, with a mean +/- standard deviation (SD) of 8.4 +/- 4.6 microg/ml. The IC50 of fresh surgical spleen-cell specimens ranged from 0.34 microg/ml to >100 microg/ml in a 48 h incubation, with a mean +/- SD of 66.5 +/- 37.7 microg/ml. When the antitumor activity of MKT-077 was compared between gastric cancer cells and spleen cells obtained from the same patient, the concentration-dependent antitumor activity of this agent was obvious in the cancer cells, while no significant cytotoxicity was observed in the spleen cells. The fresh surgical specimens of gastric cancer showed higher sensitivity to MKT-077 than did spleen cells at a concentration of 30 microg/ml, with a statistically significant difference at P < 0.05. CONCLUSIONS: The selective antitumor activity of MKT-077 was confirmed using fresh surgical specimens and warrants further investigation.

MKT-077, localized lipophilic cation: antitumor activity against human tumor xenografts serially transplanted into nude mice.

BACKGROUND: In in vitro experiments, the localized lipophilic cation, MKT-077, demonstrated time- and concentration-dependent antitumor activity. In the present experiment, the in vivo antitumor activity of MKT-077 was evaluated using human tumor xenografts serially transplanted into nude mice. MATERIALS AND METHODS: The antitumor efficacy of MKT-077 against five xenografts inoculated subcutaneously into nude mice was studied. Treatment with MKT-077 was initiated when the tumors started exponential growth. The antitumor activity of MKT-077 was assessed by a) the lowest value of the relative mean tumor weight of the treated:control tumors (T/CRW), where relative weight (RW) represented the change from baseline weight; and by b) the unadjusted weight of treated tumors at the end of the experiment. RESULTS: When MKT-077 was administered continuously using the Osmotic Micropump, antitumor activity was positively correlated with exposure time and drug concentration from 7.5 to 40 mg/kg/day. The maximum tolerated dose was 20 mg/kg/day for 7 days. Co-4 (human colon cancer), St-4 (human gastric cancer), and CRL1420 (human pancreatic cancer) were evaluated as sensitive to MKT-077 in tested 5 strains. CONCLUSION: The antitumor activity of MKT-077 was confirmed using human tumor xenografts in nude mice.

Structure-activity of novel rhodacyanine dyes as antitumor agents.

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Photoactivation enhances the mitochondrial toxicity of the cationic rhodacyanine MKT-077.

In this study, the mitochondrial phototoxicity of the cationic rhodacyanine MKT-077 was investigated by comparing its effects on the inhibition of mitochondrial respiration and the structural integrity of mitochondrial DNA (mtDNA) in the presence and absence of added high-intensity visible light (7.5 J/cm2). Results indicate that photoirradiation significantly enhances the mitochondrial toxicity of MKT-077 at both the biochemical and DNA levels. For example, the concentration of MKT-077 required to achieve one-half maximal inhibition of ADP-stimulated respiration was observed to be 6-fold lower in the presence versus absence of high-intensity light (one-half maximal inhibition at 2.5 versus 15 microg MKT-077/ mg, respectively). In addition, photoirradiation produced a 25-fold increase in inhibition of succinate-cytochrome c reductase activity by MKT-077 (one-half maximal inhibition at 2 versus 50 microg MKT-077/ml, +/-light, respectively) and a 6-fold increase in inhibition of cytochrome oxidase activity (one-half maximal inhibition at 5 versus 30 microg MKT-077/ml, +/-light, respectively). Furthermore, the combination of 25 microg/ml MKT-077 and 7.5 J/cm2 visible light caused significant degradation of mtDNA in isolated rat liver mitochondria, whereas the same concentration of dye in the absence of light had only a modest effect on mtDNA. Evaluation of light-induced MKT-077 lipid peroxidation in mitochondrial membrane fragments by the thiobarbituric acid test and by measurement of nonrespiratory-linked oxygen uptake suggests that mitochondrial phototoxicity by MKT-077 may be the result of lipid peroxidation via reactive oxygen species. These results have important implications with regard to the potential use of MKT-077 in photochemotherapy.

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation.

MKT-077 (formerly known as FJ-776) is a newly synthesized, highly water-soluble ( > 200 mg/ml) rhodacyanine dye that exhibits significant antitumor activity in a variety of model systems. In culture, MKT-077 inhibits the growth of five human cancer cell lines (colon carcinoma CX-1, breast carcinoma MCF-7, pancreatic carcinoma (CRL 1420, bladder transitional cell carcinoma EJ, and melanoma LOX) but not monkey kidney CV-1, an indicator cell line for normal epithelial cells. In nude mice, MKT-077 inhibits the growth of s.c. implanted human renal carcinoma A498 and human prostate carcinoma DU145 and prolongs the survival of mice bearing i.p. implanted human melanoma LOX (tumor:control = 344%). Subcellular localization indicates that MKT-077 is taken up and retained by mitochondria, and flow cytometric analysis suggests that CX-1 cells take up MKT-077 to a much greater extent than CV-1 cells. Quantitation of MKT-077 uptake by ethanol extraction shows that CX-1 cells accumulate 65-fold more MKT-077 than do CV-1 cells. MKT-077 is the first delocalized lipophilic cation with a favorable pharmacological and toxicological profile in preclinical studies. MKT-077 is now being investigated in Phase I clinical trials.

Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077.

We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

In vivo administration of MKT-077 causes partial yet reversible impairment of mitochondrial function.

The effects of in vivo administration of a pharmacologically toxic dose of the lipophilic cationic compound, MKT-077, were investigated in selected vital organs of the rat. MKT-077 (15 mg/kg body weight), administered by bolus i.v. injection every day for 5 days, did not detectably influence rat heart and kidney mitochondrial respiration. Although the same dosage of MKT-077 significantly decreased respiratory rates in rat liver mitochondria relative to untreated controls, complete recovery was evident within 3 days following drug withdrawal. Whereas the mitochondrial DNA of rat kidney and liver appeared to be unaffected by MKT-077 treatment, levels of heart mtDNA were noticeably less than control levels in the immediate interval following drug administration. However, this latter effect was partially reversed as early as 10 days following treatment and completely reversed within a 30-day posttreatment period. These results strongly suggest that a pharmacologically toxic dose of MKT-077 minimally affects the overall functional integrity of mitochondria in such critical, although highly vulnerable, tissues as the heart, liver, and kidney.

[Study on clinical standard consistency of the luting cement].

Various properties of dental luting cement are influenced by powder/liquid ratio. The standard consistency of luting cement is determined in Japanese industrial standard and American dental association's specifications. However, it is not considered that a constant consistency is best for luting cement of all kinds. This study were performed on suitable consistency of individual cement with regard to bond strength and disintegration. Most suitable consistency was almost the same as standard consistency by the specification in the case of Zinc phosphate cement (GC's Elite cement 100). But, in the case of Polycarboxylate cement (Shofu's HY-Bond carbo cement) and Glass alkynoate cement (GC's Fuji ionomer Type I Liv), maximum bond strength can be obtained with a more powder/liquid ratio than standard consistency by the specification. Especially, Glass alkynoate cement shows this tendency strongly. Therefore, it must be manipulated quickly after mixing.