The sequence variability of the ribosomal internal transcribed spacer (ITS) region, which comprises the 5.8 gene and the flanking regions ITS1 and ITS2, was investigated in the arbuscular mycorrhizal fungus Gigaspora margarita. DNA analysis of a multispore preparation and three single spores led to the identification of 11 slightly different sequences (three variants within a single spore), indicating substantial intersporal and intrasporal genetic variability (up to 9% sequence divergence). The sequence variations inside a single spore may be higher than that observed between spores. Even so, primers designed on the ITS1 and ITS2 regions identified Gi. margarita isolates and detected the endophyte during colonization.

The phylogeny of the Afro-Asian Lorisoidea is controversial. While postcranial data attest strongly to the monophyly of the Lorisidae, most molecular analyses portray them as paraphyletic and group the Galagidae alternately with the Asian or African lorisids. One of the problems that has bedevilled phylogenetic analysis of the group in the past is the limited number of taxa sampled for both ingroup families. We present the results of a series of phylogenetic analyses based on 635 base pairs (bp) from two mitochondrial genes (12S and 16S rRNA) with and without 36 craniodental characters, for 11 galagid and five lorisid taxa. The outgroup was the gray mouse lemur (Microcebus murinus). Analyses of the molecular data included maximum parsimony (MP), neighbor joining (NJ), maximum likelihood (ML), and Bayesian methods. The model-based analyses and the combined "molecules+morphology" analyses supported monophyly of the Lorisidae and Galagidae. The lorisids form two geographically defined clades. We find no support for the taxonomy of Galagidae as proposed recently by Groves [Primate Taxonomy, Washington, DC: Smithsonian Institution Press. 350 p, 2001]. The taxonomy of Nash et al. [International Journal of Primatology 10:57-80, 1989] is supported by the combined "molecules+morphology" analysis; however, the model-based analyses suggest that Galagoides may be an assemblage of species united by plesiomorphic craniodental characters.

The formaldehyde (FA) genotoxic potential in occupationally exposed individuals is conflicting. A relevant indoor-air FA pollution was found in hospitals and scientific institutions where FA is used as a bactericide and tissue preservative. In the present study, we evaluated the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes from workers in pathology wards who have been exposed to FA, compared with a group of unexposed subjects. The subjects were also analyzed for the GSTM1 and GSTT1 metabolic gene polymorphisms. The exposed subjects showed a significant increase in the frequency of CA per cell and in the percentage of cells with aberrations compared to control subjects. The different GST genotypes did not affect the level of cytogenetic damage since CA frequencies were not statistically different between the GST "null" genotypes and the GST "positives". The generalized linear models showed that the number of CAs and cells with CAs increased with age, but, independent of age, it was significantly higher in the experimental rather than in the control group. Cubic-spline regression confirmed the linear relationship between CAs and age, but it provided evidence for a non-linear relationship between CAs and the number of years of FA exposure. Similar results were observed when the model included the number of cells with CAs as dependent variables. Our results demonstrate that air FA induces CAs even consequently to low levels of daily exposure, indicating an increased risk of genetic damage for workers exposed to this air pollutant.

Chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) frequencies were estimated in peripheral lymphocytes from 21 radiology technicians, and from 21 nonexposed control subjects. We exclusively considered individuals who neither smoke nor consume drugs or alcohol for a period of at least two years prior to the analysis. Significant differences were found between exposed and controls in terms of SCEs and CAs frequencies. Technicians showed a significant higher number of high-frequency individuals (HFIs) with respect to the control group. Nevertheless, the mean frequency of SCEs observed among technician HFIs did not significantly differ with respect to that observed among control HFIs. Vice versa, the nonHFIs belonging to technicians group showed a statistically higher difference in the SCEs/NSM value with respect to the non-HFIs belonging to control group. Since the differences in the SCEs frequencies between the two groups are due to non-HFIs, our results seem to indicate a general genotoxic effect of the IR, not affected by HFIs. Among technicians, the level of chromosome damage correlated neither with years of radiation exposure nor with the age of the subjects. Vice versa, in the control group, a positive correlation was found between the number of SCEs and age. In both samples the gender status did not influence the frequencies of CAs and SCEs. Our results suggest that chronic long-term exposure to low doses of ionizing radiation could increase the CAs and SCEs frequencies. This study reinforces the relevance of the biomonitoring of hospital workers chronically exposed to ionizing radiation.

With the aim of further investigating the molecular evolution of beta defensin genes, after having analysed beta defensin 1 (DEFB1) in humans and several nonhuman primate species, we have studied the evolution of the beta defensin 2 gene (DEFB2), which codifies for a peptide with antimicrobial and chemoattractant activity, in humans and 16 primate species. We have found evidence of positive selection during the evolution of orthologous DEFB2 genes at two points on a phylogenetic tree relating these primates: during the divergence of the platyrrhines from the catarrhines and during the divergence of the Cercopithecidae from the Hylobatidae, Great Apes and humans. Furthermore, amino acid variations in Old World Monkeys seem to centre either on residues that are involved in oligomerisation in the human molecule, or that are conserved (40-80%) in beta-defensins in general. It is thus likely that these variations affect the biological function of the molecules and suggest that their synthesis and functional analysis might reveal interesting new information as to their role in innate immunity.

Numerous phylogenetic hypotheses have been advanced for the Malagasy lemuriform radiation, drawing on data from morphology, physiology, behaviour and molecular genetics. Almost all possible relationships have been proposed, ,md most nodes have been contested. We present a phylogenetic analysis, using several analytical methods, of a partial sequence from the 12s rRNA mitochondrial gene. This gene codes for the small ribosomal subunit, and functional constraints require that the secondary structure of the molecule is strongly conserved, which in idurn exerts constraints on the primary sequence structure. Although previous studies have suggested a very wide range of phylogenetic applicability for this molecule, our results indicate that it is most useful in strepsirrhine primates J'or estimating relationships among genera within families and among relatively recently diverged families (mean sequence divergence about 11Vo). Relationships among families separated by larger genetic distances (>12Vo divergence; e.g. Cheirogaleidae, Daubentoniidae, Megaladapidae) are difficult to resolve consistently. Our data show strong support for an Indridae-Lemuridae sister group and for monophyly of the Lemuridae with Varecia as the sister to all other lemurids. They also support, albeit less strongly, sister group relationships between Lemur and Hapalemur within the Lemuridae and between PmpLthecus and Avahi in the Indridae.

Lemuroid phylogeny is a source of lively debate among primatologists. Reconstructions based on morphological, physiological, behavioural and molecular data have yielded a diverse array of tree topologies with few nodes in common. In the last decade, molecular phylogenetic studies have grown in popularity, and a wide range of sequences has been brought to bear on the problem, but consensus has remained elusive. We present an analysis based on a composite molecular data set of approx. 6,400 bp assembled from the National Center for Biotechnology Information (NCBI) database, including both mitochondrial and nuclear genes, and diverse analytical methods. Our analysis consolidates some of the nodes that were insecure in previous reconstructions, but is still equivocal on the placement of some taxa. We conducted a similar analysis of a composite data set of approx. 3,600 bp to investigate the controversial relationships within the family Lemuridae. Here our analysis was more successful; only the position of Eulemur coronatus remained uncertain.

Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the pufferfish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 kDa and 40 kDa, and other faint bands with apparent molecular masses around 70 kDa, 57 kDa and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of quantitative Real-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other non-mammalian vertebrates.