When morphometry meets genetics: inferring the phylogeography of Carabus solieri using Fourier analyses of pronotum and male genitalia

Population differentiation is a crucial step in the speciation process and is therefore a central subject in studies of microevolution. Assessing divergence and inferring its dynamics in space and time generally require a wide array of markers. Until now however, most studies of population structure are based on molecular markers and those concerning morphological traits are more scarce. In the present work, we studied morphological differentiation among populations of the ground beetle Carabus solieri, and tested its congruence with genetic population structure. The shape of pronotum and aedeagus was assessed using Dual Axis Fourier Shape Analysis. manova on Fourier coefficients revealed highly significant morphological variation between populations and a similar geographical pattern of differentiation for both structures. On the whole, morphological and genetic patterns were also found to be congruent. Our analysis confirms the phylogeographical scenario proposing that two entities of C. solieri differentiated during the last glaciation events before recolonizing the actual range of the species. It also indicates a large introgression between the two differentiated entities in the centre of the range.     

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula)

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.     

Novel peptide inhibitors for Grb2 SH2 domain and their detection by surface plasmon resonance.

One of the critical intracellular signal transduction pathways involves the binding of the Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factor receptors, such as epidermal growth factor receptor (EGFR) and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for the development of potential anticancer agents. As a continuation of our earlier work, herein we report the design and synthesis of new peptide analogs, and their inhibitory effect on the Grb2 SH2 domain using surface plasmon resonance (SPR) technology. These novel agents do not contain phosphotyrosine or phosphotyrosine mimics. Binding interactions between these peptides and the Grb2 SH2 domain were measured and analyzed using a BIAcore X instrument, which provides detailed information on the real-time detection of the binding interaction. The results of this study should provide important information for the further development of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain.     

The non-genomic effects on Na+/H+-exchange 1 by progesterone and 20alpha-hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.     

Orally active microtubule-targeting agent,MPT0B271, for the treatment of human non-small cell lung cancer,alone and in combination with erlotinib

Microtubule-binding agents, such as taxanes and vinca alkaloids, are used in the treatment of cancer. The limitations of these treatments, such as resistance to therapy and the need for intravenous administration, have encouraged the development of new agents. MPT0B271 (N-[1-(4-Methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-1-oxy-isonicotinamide), an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. Tubulin polymerization assay and immunofluorescence experiment showed that MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels. MPT0B271 reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of P-glycoprotein (P-gp), as determined by flow cytometric analysis of rhodamine-123 (Rh-123) dye efflux and the calcein acetoxymethyl ester (calcein AM) assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by the up-regulation of cyclin B1, p-Thr161 Cdc2/p34, serine/threonine kinases polo-like kinase 1, aurora kinase A and B and the downregulation of Cdc25C and p-Tyr15 Cdc2/p34 protein levels. The appearance of MPM2 and the nuclear translocation of cyclin B1 denoted M phase arrest in MPT0B271-treated cells. Moreover, MPT0B271 induced cell apoptosis in a concentration-dependent manner; it also reduced the expression of Bcl-2, Bcl-xL, and Mcl-1 and increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP). Finally, this study demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the human non-small cell lung cancer A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers.     

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen(4OHTAM) and two steroidal antiestrogens, ICI182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the Δ4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the ‘pure’ steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcx_L, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.     

TGF-�� induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid �� plaques in Alzheimer's disease

The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer''s disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.