Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions.     

Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.     

New sensible method to quantize the intestinal absorption of receptor ligands

In recent years, special attention has been paid to the A₃ adenosine receptor (A₃AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A₃AR agonist reported so far, named AR 170 (N⁶-methyl-2-phenylethynyl-5′-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A₃AR could presuppose good topical anti-inflammatory effects of this compound.     

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A₁, A_2A and A₃ adenosine receptors and in adenylyl cyclase experiments for their potency at the human A_2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N ⁶ position of 9-propyladenine significantly increased binding affinity at the human A₁ and A₃ adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N ⁶ position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A_2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A_2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Combined use of biallelic and microsatellite Y-chromosome polymorphisms to infer affinities among African populations.

To define Y-chromosome haplotypes, we studied seven biallelic polymorphic sites. We combined data with those from four dinucleotide-repeat polymorphisms, to establish Y-chromosome compound superhaplotypes. Eight biallelic haplotypes that matched the dendrogram proposed by other investigators were identified in 762 Y chromosomes from 25 African populations. For each biallelic site, coalescence time of lineages carrying the derived allele was estimated and compared with previous estimates. The "ancestral" haplotype (haplotype 1A) was observed among Ethiopians, "Khoisan" (!Kung and Khwe), and populations from northern Cameroon. Microsatellite distributions within this haplotype showed that the Khoisan haplotypes 1A are widely divergent from those of the other two groups. Populations from northern Africa and northern Cameroon share a haplotype (i.e., 1C), which is not observed in other African populations but represents a major Eurasian cluster. Haplotypes 1C of northern Cameroon are clearly distinct from those of Europe, whereas haplotypes 1C of northern African are well intermingled with those of the other two groups. Apportionment of diversity for the Y-chromosomal biallelic haplotypes was calculated after populations were clustered into different configurations. Despite some correspondence between language affiliation and genetic similarity, geographic proximity seems to be a better predictor of genetic affinity.     

Gene amplification and cold adaptation of pepsin in Antarctic fish. A possible strategy for food digestion at low temperature

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.