Toxicological assessment of Xanthigen® nutraceutical extract combination: Mutagenicity, genotoxicity and oral toxicity.

Xanthigen® is a nutraceutical combination for weight management capable of increasing energy expenditure via uncoupling protein 1 (UCP-1) in white adipose tissue. It consists of brown seaweed Undaria pinnatifida extract, rich in the carotenoid fucoxanthin (FX) and pomegranate seed oil (PSO), rich in punicic acid. Xanthigen was screened to determine its genotoxicity and 90-days repeated oral toxicity. Genotoxicity was assessed with the Ames test (TA89, TA100, TA1535, TA1537, WP2), chromosomal aberration assay (Chinese hamster ovary cells) and mammalian micronucleus test (in mice). Xanthigen did not exhibit genotoxicity in any tested strain. Sub-chronic toxicity was evaluated with daily oral administration of 250, 500 and 1000 mg/kg/day doses of Xanthigen® to Sprague-Dawley rats over 90 days. No deaths and no deleterious effects were observed during the 90-day treatment, indicating an absence of sub-chronic toxicity and a no observed adverse effect level greater than 1000 mg/kg/day. A statistically significant decrease in bodyweight and food intake in Xanthigen® treated groups was attributed to the weight loss property of Xanthigen®. Overall, Xanthigen® shows no significant mutagenic or toxic effects.

Total synthesis of epothilone B.

[structure-see text] A convergent and stereoselective total synthesis of epothilone B (2) is described. The key steps are Normant reaction, Wadsworth-Emmons reaction of a methyl ketone 14 with the phosphonate reagent 7, diastereoselective aldol condensation of aldehyde 3 with enolate 4 to form the C6-C7 bond, and macrolactonization.

Synthesis and antibacterial activity of 2-substituted 6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids.

A series of 2-substituted 6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids was prepared and evaluated for antibacterial activity. The 6-fluoro-2-methyl-1-prenyl-1,4-dihydro-7-(3,5-dimethylpiperazinyl)-4-oxo-3-quinolinecarboxylic acid (14f) exhibited the most potent antibacterial activity against gram-positive bacteria among the total 32 derivatives. The synthetic strategies involve the use of well known keto ester condensation of benzoyl chloride and reductive cyclization of intermediates (4a-d) to afford 4-hydroxy-1,2-dihydro-2-oxo-quinoline derivatives (5a,b) or 1-hydroxy-1,4-dihydro-4-oxo-quinoline derivatives (6a,b).

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia.

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood-brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood-brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood-brain barrier-associated protein, occludin. Correspondingly, blood-brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood-brain barrier and white matter components.

Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94.

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Identification and sequencing of pineal-specific enhancing region in the mouse tryptophan hydroxylase promoter.

Pineal-specific expression of the tryptophan hydroxylase (TPH) gene has been demonstrated by a number of studies. However, little is known about the regulatory mechanism for pineal-specific expression of the TPH gene. To identify the cis-acting region responsible for pineal-specific expression of the TPH gene, we investigated a 6.1-kb 5'-flanking region of the mouse TPH gene using an immortalized pineal cell line (PGT-beta) derived from transgenic mice. By deletion analysis, it was demonstrated that the pineal-specific enhancing region resides approximately between -6.1 and -4.7 kb upstream from the transcription initiation site of the mouse TPH gene. Additionally, nucleotide sequence analysis of this region showed that the (AC/TG)22 repetitive sequence is located approximately -5.78 kb upstream of the mouse TPH gene, and several known tissue-specific cis-acting elements, such as Pit-1 and the pituitary specific element (PSE), have also been identified in the region. We believe that the analysis of the sequence and several cis-acting elements in the pineal-specific enhancing region of the mouse TPH promoter would enhance our understanding of the precise mechanism of pineal-specific expression.

Synthesis of 4-hydroxy-1-thiocoumarin derivatives-1: an efficient synthesis of thioflocoumafen.

An efficient procedure for the preparation of 4-hydroxy-3-{1,2,3,4-tetra-hydro-3-[4-(4-triflu-oromethylbenzyl oxy)phenyl]-1-naphthyl}thiocoumarin (thioflocoumafen, 1a and 1b) is described. The key step in the synthesis involves the condensation reaction of 3-(4-methoxyphenyl)-1-tetralol (2) with 4-hydroxy-1-thiocoumarin (3).

Efficacy of new platinum analog DPPE in an orthotopic nude mouse model of human colon cancer.

A surgical orthotopic implantation (S.O.I.) model of the human colon cancer cell line Co-3 in nude mice was treated with two doses of the new platinum analogs {Pt(cis-dach) (DPPE).2NO3} and {Pt(trans-dach)(DPPE).2NO3}. The analogs were evaluated for antimetastatic efficacy in comparison to two doses of cisplatinum. Unlike the untreated control group, there were no mesenteric lymph node metastases in the groups treated with the high or low doses of both forms of new DPPE platinum analogs as well as cisplatinum-treated group. However, much more body-weight loss occurred in the cisplatinum-treated group than the DPPE-treated groups. The results obtained with SOI animal model of colon cancer demonstrated both cis- and trans-forms of DPPE had as strong an inhibitory effect on metastasis as that of cisplatinum, but with much less toxicity. Thus the new platinum analogs appears to have promising clinical potential.

Response of primary rabbit kidney proximal tubule cells to estrogens.

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red-free medium. 17beta-estradiol was observed to stimulate growth at dosages as low as 10(-10) M. The growth stimulatory effects of 17beta-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10(-9) to 10(-8) M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17beta-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17beta-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17beta-estradiol. 17beta-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures.

Hypericin induces both differentiation and apoptosis in human promyelocytic leukemia HL-60 cells.

Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.

Role of 5' HoxD genes in chondrogenesis in vitro.

In the present study, we have examined by in situ hybridization, expression of five 5' HoxD cluster genes (D9, D10, D11, D12 and D13) during chondrogenesis of chick limb bud mesenchymal cells in vitro. After one day in culture, D9 and D13 gene expression was restricted to patches of mesenchymal cells, while expression of D10, Dll, and D12 gene was prominent in all mesenchymal cells. In 3-day cultures, D9 and D13 genes were expressed only in cartilage nodules, while D10, Dll, and D12 genes were expressed in both cartilage nodules and in all mesenchymal cells. These observations indicate two different patterns of expression; one for D9 and D13, and a different one for D10, Dll, and D12. These patterns of expression seem to correlate with patterns of cell proliferation and differentiation to chondrocytes. The role of these HoxD genes was further investigated by employing antisense S-oligomers. We found that oligodeoxynucleotides complementary to HoxD (D10-D 13) mRNAs were capable of inhibiting chondrogenesis. These data suggest that expression of HoxD genes is required for mesenchymal condensation, and differentiation to chondrocytes. This in turn implies that these HoxD genes aside from their role in the patterning of the developing skeletal elements might regulate down-stream factors necessary for cartilage differentiation as well.

Regulation of homeobox-containing genes during lens regeneration.

In this study, the expression of homeobox-containing genes was evaluated after lentectomy in the newt, which is competent for lens regeneration, and in the axolotl which is not. Such a comparison was designed to offer insights about possible regulation due to regenerative abilities. Six homeobox-containing genes were examined: NvHox A4, NvHox B1, NvHox 7, NvHox X, Nvmsx-1 and Xbr1. For all genes examined, it was found that soon after lentectomy in the newt there was a general down-regulation in the retina. This down-regulation varied among the Hox genes with NvHox 7 and NvHox B1 being virtually absent in the initial stages; their expression was re-established to the original levels after the reappearance of lens. The expression patterns, for NvHox 7 and NvHox B1 were the same when the lens was removed and then displaced. However, in axolotl, down-regulation was not observed. These data suggest that the observed regulation is related to the process of lens regeneration and provide the first molecular evidence that lens regeneration could be dependent on retina and underline the importance of this tissue in lens regeneration. Such patterns link expression of homeobox-containing genes and lens regeneration and can be now used to understand the underlying mechanisms of lens regeneration and transdifferentiation.

Antimetastatic activity of the new platinum analog [Pt(cis-dach) (DPPE).2NO3] in a metastatic model of human bladder cancer.

Surgical orthotopic implantation (SOI) of histologically intact human RT-4 bladder tumor tissue in nude mice resulted in local growth, invasion, regional extension and metastases as well as distant metastases to other organ sites and lymph nodes, thus mimicking the bladder cancer patient. This metastatic bladder tumor animal model was treated with two doses of new platinum analog ¿Pt(cis-dach)(DPPE).2NO3¿ for the evaluation of antimetastatic efficacy compared to two doses of cisplatinum. Unlike the untreated control group or the group treated with the low dose of cisplatinum, there were no metastases in either the high or low-dose platinum-analog-treated groups and the high-dose cisplatinum-treated group. The results obtained with this patient-like nude-mouse model of bladder cancer indicate that the new platinum analog appears to be a valuable lead compound with antimetastatic efficacy and clinical potential.

Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results.

Most methods for the diagnosis of hepatitis B virus (HBV) infection largely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The quality assurance program recently established in Europe reported that PCR-mediated HBV DNA detection methods used in many laboratories produced a high rate of false-positive and false-negative results. Thus, we attempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed method of releasing HBV DNA from virion by NaOH treatment of patient serum. Using four different primer sets specific to the HBV core region, we found that the sensitivity of first-round PCR can be improved by more than two orders of magnitude depending on the primers. The second round of PCR using nested primers was sensitive enough to detect up to 10(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient sera investigated in our laboratory, more than 60% of the tested samples gave positive results in the first-round PCR. The rate of positive results obtained using our experimental conditions is very high in comparison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% positive results. For diagnosis of HBV infection, we routinely used 1 microliter of patient serum, which was found to be optimum in our laboratory. Surprisingly, from 20% of our positive results, even serum diluted to 1/100 (0.01 microliter) produced a stronger signal than 1 microliter. This observation suggests that direct PCR amplification of HBV DNA released from serum by NaOH treatment has to be compensated by other DNA detection methods for correct quantitation. In order to eliminate the false positive signal resulting from the carry-over due to massive screening of a large number of samples, PCR reaction mixture containing 8-methoxypsoralen was exposed to ultraviolet light prior to thermal cycle amplification. This exercise did not decrease the sensitivity of the detection method, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-mediated HBV DNA detection methods to attain more reliable and easily applicable methods.

Antitumor activity and nephrotoxicity of a novel platinum complex, [1,3-bis(diphenylphosphino)propane](trans-1-dach)platinum(I I) dinitrate.

We have developed a new class of platinum complex [Pt(trans-l-dach)(1,3-bis(phosphino)propane)] dinitrate (KHPC-001) with potent antitumor activity and low nephrotoxicity, confirmed in vitro and compared in vivo with cisplatin, KHPC-001 or cisplatin was intraperitoneally injected on days 1, 5, and 9 into P388-bearing mice and the antitumor effects were compared. In vitro cytotoxicity, Pt accumulation, and DNA cross-link index were measured in P388 and LLC-PK1 cells after treatment with KHPC-001 or cisplatin. Twenty mg/kg (below one-tenth of LD50) of KHPC-001 had stronger antitumor effects than 2 mg/kg (about one-fifth of LD50) of cisplatin and cured 2 out of 6 mice without any toxicity. While the cytotoxicity of KHPC-001 and cisplatin were similar on P388 mouse leukemia cells, this new compound was much less cytotoxic to a kidney-derived line, LLC-PK1. This lower toxicity on the kidney cells was based on its low accumulation, causing less DNA crosslinking. KHPC-001 is a unique third-generation platinum complex with potent antitumor activity and low nephrotoxicity.

Interleukin-6 production in primary histoculture by normal human kidney and renal tumor tissues.

Interleukin-6 (IL-6) is a multifunctional cytokine with many biologic activities in vitro, including synergistic or antagonistic actions with one or more other cytokines. The role and induction parameters of IL-6 in renal cell carcinoma (RCC) are not fully understood. To understand the ability of RCC to produce IL-6, we determined the IL-6 concentration in the supernatant of histocultured human normal kidney, RCC, renal Wilms' tumor and renal oncocytoma. From these studies, we conclude that the kidney is one of the main sources of IL-6. Normal renal cortical tissues and renal tumors can produce IL-6 in histoculture without stimulation. Thus histoculture supports the long-term production of IL-6, potentially allowing many important studies of this cytokine in the normal and malignant kidney.

Conservation of fibroblast growth factor function in lens regeneration.

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.

Efficacy of the platinum analog [Pt(cis-dach)(DPPE)-2NO3] on histocultured human patient bladder tumors and cancer cell lines.

Cisplatinum is currently used as a front line agent in many important tumors, but its dose-limiting nephrotoxicity prevents potential efficacy. There is therefore great interest in developing new platinum agents that have less toxicity. We have synthesized new platinum analogues containing DACH as a carrier ligand and DPPE as a leaving group. Previously we showed that these new platinum complexes have much less nephrotoxicity than cisplatinum. In the present study, the efficacy of one new platinum complex was evaluated with human patient bladder tumor specimens in three-dimensional histoculture as well as with monolayer cultures of cancer cell lines. The efficacy end points used were glucose consumption and thymidine incorporation on the histocultured specimens and MTT reduction on monolayer cell cultures. Our results showed that the new platinum complex was more effective at high concentration (10(-3) M) but less effective at low concentration (10(-4) M) compared to cisplatinum on histocultured bladder tumor specimens. The compound demonstrated higher efficacy than cisplatinum on P-388, and L-1210 leukemic cell lines. The new analog demonstrated similar efficacy to cisplatinum on the MKN-45 human stomach cancer cell line. The PC-14 human lung cancer cell line, MH1C1 rat hepatoma cell line, NIH-OV3, SKOV-3 ovarian cancer cell lines were as sensitive to the new analog as to cisplatinum at high concentrations of the new platinum analogue. The cisplatinum-resistant M-14 melanoma cell line was not sensitive to either the new analog or cisplatinum. Based on these results, this novel platinum compound appears to be a valuable lead compound with high efficacy and low nephrotoxicity.

9-cis retinoic acid antagonizes the stimulatory effect of 1,25 dihydroxyvitamin D3 on chondrogenesis of chick limb bud mesenchymal cells: interactions of their receptors.

Retinoids or vitamin D have been found to profoundly affect pattern formation and chondrogenesis in the developing limb. These substances mediate their actions through their nuclear receptors. In the present investigation, we present data showing that 9-cis RA, the ligand for RXR can stimulate chondrogenesis of chick limb bud mesenchymal cells, however, in combination, it antagonizes the stimulatory effect of vitamin D in the same system. The receptors for 9-cis RA (RXR) and vitamin D (VDR) were also shown to be present in the mesenchymal cells and to form heterodimers. These results implicate these receptors in cartilage differentiation during limb development.

cAMP induces phosphorylation of a 40-kDa nuclear protein which is distinct from CREB during chondrogenesis of chick limb bud mesenchymal cells in vitro.

We examined the effect of cAMP on the phosphorylation of intracellular proteins in cultured chondroblasts to understand the stimulatory role of intracellular cAMP in chondrogenesis of chick limb bud mesenchymal cells. A 40-kDa protein was remarkably phosphorylated by cAMP and the phosphorylation was completely blocked by an inhibitor of cAMP-dependent protein kinase. The phosphorylation of the 40-kDa protein was maximum at early stage of chondrogenesis (i.e., 24 hr of culture) which is consistent with the changes in the level of intracellular cAMP. The 40-kDa phosphoprotein was exclusively located in the nuclear parts of chondroblast but distinct from cAMP response element binding protein.