Rhox homeobox gene cluster: recent duplication of three family members

We recently reported the discovery of a homeobox gene cluster on the mouse X chromosome, Rhox, whose 12 members are selectively expressed in specific cell types in reproductive organs. Here we report the existence of 20 additional Rhox homeobox genes in this gene cluster. Most of the newly identified Rhox paralogs retain the same order and relative orientation as three of the originally described Rhox genes, suggesting that they arose from recent duplications of this trimer unit. Many of these new Rhox family members are expressed in the testis and placenta. Analysis of synonymous and nonsynonymous substitutions in their homeodomain region suggests that these new Rhox paralogs duplicated so recently that their encoded proteins have not yet acquired distinct DNA-binding specificities. The existence of these new Rhox genes provides an opportunity to examine the initial stages of gene cluster evolution.     

The food contaminant semicarbazide acts as an endocrine disrupter: Evidence from an integrated in vivo/in vitro approach

Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75, 140 mg/kg bw pro die during the juvenile period. Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM 40 and 75 mg/kg and delayed at 140 mg/kg, the latter effect probably due to the significantly decreased body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose–response was not evidenced. Testosterone catabolism was altered in a gender-related way, aromatase activity was increased in treated males at 75 and 140 mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo–in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis.     

New Coumarinyl Ethers in Daphne oleoides Schreb. Collected from Sardinia Island

The phytochemical analysis of the ethanolic extract obtained from D. oleoides collected from Sardinia Island allowed the isolation of several new constituents for the species ( 3 , 8 , and 9 ) together with two new coumarinyl ethers ( 1 and 2 ) besides the chemotaxonomic markers of the Daphne genus ( 4 – 7 and 10 ) which are also known to possess interesting biological activities. The structure of the new compounds were elucidated by extensive spectroscopic and spectrometric analyses. The identification of these compounds gives an experimental evidence of the variability in the secondary metabolites pattern owned by populations growing in restricted area in respect to populations not confined by geographical barrier.     

Antimicrotubular and cytotoxic activity of geiparvarin analogues, alone and in combination with paclitaxel

Geiparvarin is an antiproliferative compound isolated from the leaves of Geijera parviflora, and may represent a new drug which targets tubulin. To better explore the potential use of this agent, we investigated the antimicrotubular and cytotoxic effects of new synthetic aromatic derivatives of geiparvarin. These drugs inhibited polymerization of microtubular protein, particularly when the assembly was induced by paclitaxel. The microtubular network organization of fibroblasts was altered more effectively by some drugs. Normal microtubule architecture completely disappeared when the cells were treated simultaneously with drugs and paclitaxel: microtubules depolymerized or were reorganized into bundles, in a similar but more disarrayed fashion than that observed after treatment with paclitaxel alone. Cytotoxicity studies showed a dose-dependent effect, whereas combined administration of drugs and paclitaxel increased cytotoxicity, more effectively in paclitaxel versus derivatives administration alone.     

Microtubule-interfering activity of parthenolide

Parthenolide is an active sesquiterpene lactone present in a variety of medicinal herbs, well known as anti-inflammatory drug. It has recently been proposed as a chemotherapeutic drug, but the pharmacological pathways of its action have not yet been fully elucidated. Firstly, we explored whether the anticancer properties of parthenolide may be related to a tubulin/microtubule-interfering activity. We additionally compared bioactivities of parthenolide with those checked after combined treatments with paclitaxel in human breast cancer MCF-7 cells. Parthenolide exerted in vitro stimulatory activity on tubulin assembly, by inducing the formation of well-organized microtubule polymers. Light microscopy detections showed that parthenolide-induced alterations of either microtubule network and nuclear morphology happened only after combined exposures to paclitaxel. In addition, the growth of MCF-7 cells was significantly inhibited by parthenolide, which enhanced paclitaxel effectiveness. In conclusion, the antimicrotubular and antiproliferative effects of parthenolide, well known microtubule-stabilizing anticancer agent, may influence paclitaxel activity. The tubulin/microtubule system may represent a novel molecular target for parthenolide, to be utilized in developing new combinational anticancer strategies.     

Comparative studies on biological activity of certain microtubule-interacting taxanes

We have compared the nature of interaction of certain taxanes with microtubular protein, and the mechanism of action underlying cytotoxic activity. Taxanes induced tubulin assembly in vitro, but only taxanes bearing side chain were capable of inducing the formation of stable tubulin polymers. Electron microscopy detections showed that taxane-induced polymers are structurally similar to microtubules formed by paclitaxel, with differences in length. Otherwise, light microscopy views have shown that intracellular microtubule network is deeply reorganized by taxanes into short fibers, unlike paclitaxel-bundled microtubules. Taxanes inhibited the growth of various human tumor cell lines, but cell cycle analysis did not always indicate a block in the G2/M phase. These agents alter some apoptotic signal transduction pathways, probably by a mechanism distinct from microtubule interaction. Briefly, the effectiveness of taxanes is closely related to their chemical structure, and depends on their interaction with microtubular protein. By virtue of this mechanism, some of these taxanes may provide usefulness for therapeutic improvements.     

Nucleotide sequence of cDNA coding for saporin-6, a type-1 ribosome-inactivating protein from Saponaria officinalis

We have isolated and sequenced partial cDNA clones that encode SO-6, a ribosome-inactivating protein from Saponaria officinalis. A cDNA library was constructed from the leaves of this plant and screened with synthetic oligonucleotide probes representing various portions of the protein. The deduced amino acid sequence shows the signal peptide and a coding region virtually accounting for the entire amino acid sequence of SO-6. The sequence reveals regions of similarity to other ribosome-inactivating proteins, especially in a region of the molecule where critical amino acid residues might participate in the active site.