Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Comparative studies on the mechanism of cytotoxic action of novel platinum II complexes with pyrazole ligands

In search for new platinum-based anticancer drugs, four cisplatin analogues, which contain pyrazole rings as non-leaving ligands, have been synthesized: cis-PtCl(2)(3,5-DM HMPz)(2), cis-PtCl(2)(Pz)(2), cis-PtCl(2)(ClMPz)(2), and cis-PtCl(2)(HMPz)(2), where Pz=pyrazole, H=hydroxyl, M=methyl. We tested their cytotoxicity, apoptosis induction ability, DNA damaging and modification properties comparing them in respect to the parent compound. The cytotoxic activity of these platinum pyrazole complexes toward the murine leukemia cell line was 2.9-3.8 times lower than actvity of cisplatin. The tested compounds varied in their mechanism of action by producing different DNA lesions. The most interesting compound seems to be the complex with chloromethyl groups at N1 of pyrazole rings, which exhibited the highest ability to form bifunctional adducts with DNA in vitro.     

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-α) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-α, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-α (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-α in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-α and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-α, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-α may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates. J. Cell. Physiol. 176:506–515, 1998. © 1998 Wiley-Liss, Inc.     

Sperm deliver a new second messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.     

Leg movements of stick insects walking with five legs on a treadwheel and with one leg on a motor-driven belt

Stick insects walking with five legs on a self-propelled treadwheel and with the left hindleg (L3) on a motor-driven belt may move the "belt" leg L3 and the "wheel" legs with different frequencies. When L3 made less steps than L2, that step of L2, which was performed during the swing phase of L3, is prolonged. The time interval between the end of swing phase of L3 and the onset of the following swing phase of L2 was remarkably constant. When L3 made more steps than L2, that step of L3, which was performed during the swing phase of L2, is prolonged. Again, the time interval between the end of swing phase of L3 preceding a L2 swing phase and the onset of the L2 swing phase was relatively constant. For both kinds of walking situations phase response curves were drawn. They show that two types of coordinating channels exist: An anteriorly directed type is more dependent on absolute time than on phase. A posteriorly directed type is phase-dependent. Both inhibit the transition from stance to swing for some time. The results are compared with the existing coordination models.     

Do different disparity proxies converge on a common signal? Insights from the cranial morphometrics and evolutionary history of Pterosauria (Diapsida: Archosauria)

Disparity, or morphological diversity, is often quantified by evolutionary biologists investigating the macroevolutionary history of clades over geological timescales. Disparity is typically quantified using proxies for morphology, such as measurements, discrete anatomical characters, or geometric morphometrics. If different proxies produce differing results, then the accurate quantification of disparity in deep time may be problematic. However, despite this, few studies have attempted to examine disparity of a single clade using multiple morphological proxies. Here, as a case study for this question, we examine the disparity of the volant Mesozoic fossil reptile clade Pterosauria, an intensively studied group that achieved substantial morphological, ecological and taxonomic diversity during their 145+ million-year evolutionary history. We characterize broadscale patterns of cranial morphological disparity for pterosaurs for the first time using landmark-based geometric morphometrics and make comparisons to calculations of pterosaur disparity based on alternative metrics. Landmark-based disparity calculations suggest that monofenestratan pterosaurs were more diverse cranially than basal non-monofenestratan pterosaurs (at least when the aberrant anurognathids are excluded), and that peak cranial disparity may have occurred in the Early Cretaceous, relatively late in pterosaur evolution. Significantly, our cranial disparity results are broadly congruent with those based on whole skeleton discrete character and limb proportion data sets, indicating that these divergent approaches document a consistent pattern of pterosaur morphological evolution. Therefore, pterosaurs provide an exemplar case demonstrating that different proxies for morphological form can converge on the same disparity signal, which is encouraging because often only one such proxy is available for extinct clades represented by fossils. Furthermore, mapping phylogeny into cranial morphospace demonstrates that pterosaur cranial morphology is significantly correlated with, and potentially constrained by, phylogenetic relationships.     

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca²⁺ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca²⁺ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca²⁺ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca²⁺ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.