Inhibin/activin subunits alpha, beta-A and beta-B are differentially expressed in normal human endometrium throughout the menstrual cycle

Inhibins are dimeric glycoproteins composed of an alpha () subunit and one of two possible beta (-) subunits (A or B). The aims of this study were to assess the frequency and tissue distribution patterns of the inhibin subunits in normal human endometrium. Samples from human endometrium from proliferative phase (PP; n=32), early secretory phase (ES; n=10) and late secretory phase (LS; n=12) were obtained. Immunohistochemistry, immunofluorescence and a statistical analysis were performed. All three inhibin subunits were expressed by normal endometrium by immunohistochemistry and immunofluorescence. Inhibin- was primarily detected in glandular epithelial cells, while inhibin- subunits were additionally localised in stromal tissue. Inhibin- staining reaction increased significantly between PP and ES (P<0.05), PP and LS (P<0.01), and ES and LS (P<0.02). Inhibin-A and -B were significant higher in LS than PP (P<0.05) and LS than ES (P<0.05). All three inhibin subunits were expressed by human endometrium varying across the menstrual cycle. This suggests substantial functions in human implantation of inhibin- subunit, while stromal expression of the subunits could be important in the paracrine signalling for adequate endometrial maturation. The distinct expression in human endometrial tissue suggests a synthesis of inhibins into the lumen and a predominant secretion of activins into the stroma.I. Mylonas and U. Jeschke contributed equally to this work     

Molecular phylogeny of the Greek legless skink Ophiomorus punctatissimus (Squamata: Scincidae): the impact of the mid-Aegean trench in its phylogeography

Sequence data derived from three mitochondrial markers (cytochrome b, 16S rRNA and 12S rRNA genes) were used to infer the evolutionary history of several insular and mainland populations of the Greek legless skink (Ophiomorus punctatissimus), covering most of its distributional range. All phylogenetic analyses produced topologically identical trees that revealed a well-resolved phylogeny. These trees support two O. punctatissimus clades, which are geographically separated (west and east of the mid-Aegean trench). The assumption of a clock-like evolution could not be rejected, and thus a local clock was calibrated for the O. punctatissimus lineages. The non-overlapping geographic distributions of the major clades suggest a spatial and temporal sequence of diversification that coincides with paleogeographic separations during the geological history of the Aegean region. It seems that O. punctatissimus is an old eastern Mediterranean species that has been differentiating in this region at least from middle Miocene. It is possible that the ancestral form of O. punctatissimus invaded the Aegean region from Anatolia before the complete formation of the mid-Aegean trench, when the Aegean was still a uniform landmass, while other vicariant events have led to its present distribution.     

Testing for nestedness in the terrestrial isopods and snails of Kyklades islands (Aegean archipelago, Greece)

Most insular communities exhibit nestedness, with the species assemblages of the more depauperate islands constituting subsets of those of the richer. Several methods for the estimation and evaluation of nestedness have been developed during the last fifteen years. In this paper we use two of the more recent and elaborate methods, namely the ‘temperature’ method of Atmar and Patterson and the ‘departures’ method of Lomolino, in order to investigate patterns of nestedness in the distribution of two well studied and speciose animal groups, terrestrial isopods and land snails, in the Kyklades archipelago (Aegean Sea. Greece) that lies between two continental regions, Significant nestedness is present in both species assemblages and, surprisingly, each method gives almost identical levels of nestedness for the two animal groups. Isolation has been found to be more important in producing nestedness in both groups than area, which does not seem to be an important explanatory factor. However, the role of isolation in this case is better understood under an historical perspective, taking into account the complex palaeogeography of the region and the differential departmentalisation of distinct island groups. Additionally, certain metrics of habitat diversity that were included in the analysis were the best explanatory factors of nestedness, indicating a more complex causal pattern that also involves extinction. Since the two methods used are based on different assumptions and have different scopes, their results do not converge. The ‘temperature’ method finds the maximum possible nestedness in an island sorting which does not necessarily lead to plausible biogeographical explanations, while the ‘departures’ method, although more useful in detecting causality, fails to fully evaluate levels of nestedness. Nevertheless, both methods are valuable tools in the exploration of interesting distributional patterns, when this effort is accompanied by a good understanding of historical, ecological and idiosyncratic properties of each particular data set.     

Molecular phylogeny of the extinct pleistocene dwarf elephant Palaeoloxodon antiquus falconeri from Tilos Island,Dodekanisa, Greece

A partial sequence of cytochrome b (228 bp) gene of mitochondrial DNA was successfully determined from rib bones of the dwarf elephant Palaeoloxodon antiquus falconeri BUSK, which were excavated from Charkadio cave of the island of Tilos, Dodekanisa, Greece. This is the first report of DNA sequence of a dwarf elephant. The sequences were used to examine the phylogenetic relationships among Elephantidae. Phylogenetic trees reconstructed by the neighbor-joining and maximum parsimony methods provided identical topologies. The results support the "Palaeoloxodon–Elephas" clade, which is consistent with previous morphological reports according to which Palaeoloxodon is more closely related to Elephas than to Loxodonta or Mammuthus.     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.Matrix metalloproteinases (MMP)² are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1–3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I–III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5–12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13–19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20–22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23–25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26, 27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29–31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two domains, which are connected by a short linker (14 and 20 amino acids, respectively). It is difficult to envisage that rigid and compact molecules of this type can interact with triple helical collagen in a way that can lead to first unwinding and then cleavage of individual filaments. It has been recently suggested that such concerted action could occur much more easily if the two domains could enjoy at least a partial conformational independence (9). Slight differences in the reciprocal orientation of the CAT and HPX domains of MMP-1 in the presence (29) and absence (30, 31) of the prodomain were indeed taken as a hint that the two domains could experience relative mobility (29).Two recent solution studies have shown that conformational independence is indeed occurring in gelatinase MMP-9 (33) and elastase MMP-12 (34), whereas the x-ray structure of the latter (34) is only slightly less compact than those of MMP-1 (29–31) and MMP-2 (32). Among MMPs, MMP-9 features an exceptionally long linker (68 amino acid) (33, 35), which in fact constitutes a small domain by itself (the O-glycosylated domain) (33), and therefore, this inspiring observation can hardly be taken as evidence that conformational freedom is a general characteristic of the two-domain MMPs. MMP-12 features a much more normal 16-amino acid linker, thereby making more probable a general functional role for this conformational freedom (34). However, both MMP-9 and MMP-12 retain their full catalytic activity against their substrates even when deprived of the HPX domain (9). Therefore, the question remains of whether conformational freedom is also a required characteristic for those MMPs that are only active as full-length proteins, i.e. collagenases. Interestingly, the three collagenases (MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all MMPs. Demonstrating or negating the presence of conformational freedom in one of these collagenases would therefore constitute a significant step forward to formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution (34) have shown that a combination of NMR relaxation studies and small angle x-ray scattering (SAXS) is enough to show the presence and the extent of the relative conformational freedom of the two domains of MMPs. Here we apply the same strategy to full-length MMP-1 and show that sizable conformational freedom is indeed experienced even by this prototypical collagenase, although somewhat less pronounced than that observed for MMP-12.     

Synthesis, molecular structure determination, and antitumor activity of platinum(II) and palladium(II) complexes of 2-substituted benzimidazole

The complexes of 2-aminomethyl benzimidazole, 2-(beta-aminoethyl)benzimidazole, and 2-(alpha-aminoethy-l)benzimidazole with Pt(II) and Pd(II) have been prepared. The molecular structure of the free ligands and their complexes were studied by IR and 1H NMR. It was concluded that the substituted benzimidazole derivatives behave as bidentate ligands, being bound to the metal atoms via the nitrogen of the -N = group and the amino group of the side chain of the benzimidazole ring. The metal complexes were tested for antineoplastic activity both in cultures of neoplastic cells (MEL-745, K-562, Colon 205, IMP-32, SK-N-SH) and in vivo in rodents bearing L-1210 leukemia. The antiproliferative activity of these agents was compared to that of cis-platin.     

Oestrus synchronization in sheep with progesterone-impregnated (MAP) intravaginal sponges and a prostagland in analogue

A double injection of PGF2α at two different dosage levels, 5 mg and 10 mg, eleven days apart, were compared with a combination of MAP-sponges and 5 mg or 10 mg PGF2α.The combination of MAP-sponges plus 10 mg PGF2α gave the best syn=chronization results (93,4%), as well as the best conception rate (84,9%) when ewes were inseminated on a fixed time basis 68 and 80 hours after treatment.