Food habits of wolves in central Italy based on stomach and intestine analyses

We investigated wolfCanis lupus Linnaeus, 1758 food habits in central Italy by examining stomach and/or intestine contents of 59 individuals. Road accident and illegal kills were main causes of the wolves’ death. Ungulates represented the bulk of the diet (55% in frequency), and among them wild boar was the most important prey, followed by domestic Caprinae. Food items of domestic origin accounted for about 1/3 of all the diet. Diet composition did not vary between stomachs and intestines in spite of the higher degree of digestion of the intestines’ contents. The frequent detection of numerous larvae of Diptera and/or necrophagous Coleoptera, let suppose the consumption of already dead animals, and suggests a general underestimate of the wolf’s scavenging behaviour in previous studies based on scat analyses.     

Single strand conformational polymorphism evaluation for the identification of TH3R alleles of the circumsporozoite protein gene of Plasmodium falciparum

Highly polymorphic regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are associated with cellular immune responses. One of these regions, the TH3R polymorfic region of the csp gene codes for known T-cell epitopes. The present study tested the use of SSCP to determine sequence variations of the TH3R regions of four clones of P. falciparum (3D7, HB3, Dd2 and K1) which are known to have different TH3R regions. Single-strand conformation polymorphism (SSCP) technique was performed on amplified products labelled with fluorescent primer (both strands) and electrophoresed in an automated sequencer. Various gel compositions and electrophoresis conditions were tested. Even if some electrophoretogram differences were observed between clones, they could not distinguish between the alleles.     

Low-temperature micronization of a peptide drug in fluid propellant: Case study cetrorelix

Aim of this study was to elaborate an efficient method for the micronization of the decapeptide cetrorelix (a GnRH-antagonist), in order to obtain a microsuspension as basis for other pharmaceutical preparations, such as e.g. inhalation aerosols. A modified pearl-mill coupled with a cryostat was used for the micronization of cetrorelix in fluid propellant and operated under different conditions. The obtained cetrorelix suspensions were analyzed for particle size distribution, purity of cetrorelix, and for metal contamination through abrasion from parts of the mill. The method allowed an effective micronization of cetrorelix. The mean particle size of the initial cetrorelix lyophilizate bulk ware was reduced from 52.5 μ (Volume Mean Diameter, VMD) down to 14.9, 6.1 and 3.1 μm, respectively, respectively. The HPLC analysis of all cetrorelix suspensions after micronization did not show signs of decomposition as compared to the initial product. The elementary analysis of the suspensions performed by inductively coupled plasma mass spectrometry revealed a negligible amount of contaminants in the suspension (Zr=max. 0.6 ppm; Fe, Cr, Ni, Ba, below limit of quantification, i.e.<0.14 ppm). The only appreciable contaminant. Aluminum (Al=1.1 ppm), was derived from the mechanical capping of aluminum canisters prior to analysis. The Zr determination in the suspension of 0.6 ppm, is still considered to be negligible as compared to the legally tolerated limit of air contamination. By low-temperature micronization in fluid propellant, fine drug suspensions of cetrorelix for pMDIs can be directly manufactured in one-step procedure without destruction of the peptide structure and without appreciable product contamination. Published: July 12, 2001.     

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus

The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.     

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.     

Study of the biological effects and DNA damage exerted by a new dipalladium-Hmtpo complex on human cancer cells

The new dipalladium complex [Pd(2)(mu-mtpo-N(3),N(4))(2)(phen)(2)](NO(3))(2) (where phen=1,10-phenantroline; Hmtpo=5,7-dihydro-7-oxo-5-methyl[1,2,4]triazolopyrimidine), (Pd(2)-Hmtpo, or complex I), interacts effectively with DNA plasmid (pBS), as studied by circular dichroism spectroscopy (CD), causing large helix distortions, altering the direction of the main DNA helix axis and producing unwinding of the DNA double helix. DNA damage induced by complex I was highly significant at 2.81 microM (ovarian carcinoma TG cell line), as assessed by comet assay, a dose at which all treated nuclei showed more than 30% DNA migration to the comet tail. DNA damage effect is a consequence of genotoxicity and not a false positive response caused by cytotoxicity. In vitro cytotoxic assay on the two human tumor cell lines TG and BT-20 (breast carcinoma), shows that doses of 0.47, 1.41 and 2.81 microM produce significant antiproliferative effects after 4 days of treatment compared with control. Complex I was highly cytotoxic at 2.81 microM causing an inhibition of viable cells of 65.5%. Cisplatin (cis-DDP) exhibits lower cytotoxic activity in TG cells than dipalladium complex (a cisplatin dose of 6.67 microM inhibits 30.3%) and does not cause migration of DNA to comet tail.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.