Nickel-Catalyzed Synthesis of Stereochemically Defined Enamides via Bi- and Tricomponent Coupling Reaction.

The stereoselective synthesis of (E)-trisubstituted tertiary enamides is documented via site-selective Ni-catalyzed ß-arylation of allenamides with boronic acids in high yields (up to 89%). The nucleophilic character of the "organo-Ni" intermediates is further exploited to implement a one-pot tricomponent procedure involving the final allylation of aldehydes (yields up to 93%). Mechanistic insights and efficiency on a gram scale process were also documented.

Synthesis, characterization, crystal structure and preliminary reactivity behaviour of new heteropolytopic ligands based on the 1,3,5-triazine spacer and pyrazolyl, tris-pyrazolylmethyl and tris-pyrazolylethoxy bonding fragments.

Four new potentially polytopic nitrogen donor ligands based on the 1,3,5-triazine fragment, L(1)-L(4) (L(1) = 2-chloro-4,6-di(1H-pyrazol-1-yl)-1,3,5-triazine, L(2) = N,N'-bis(4,6-di(1H-pyrazol-1-yl)-1,3,5-triazin-2-yl)ethane-1,2-diamine, L(3) = 2,4,6-tris(tri(1H-pyrazol-1-yl)methyl)-1,3,5-triazine, and L(4) = 2,4,6-tris(2,2,2-tri(1H-pyrazol-1-yl)ethoxy)-1,3,5-triazine) have been synthesized and characterized. The X-ray crystal structure of L(3) confirms that its molecular nature consists of a 1,3,5-triazine ring bearing three tripodal tris(pyrazolyl) arms. L(1), L(2), and L(4) react with Cu(I), Cu(II), Pd(II) and Ag(I) salts yielding mono-, di-, and oligonuclear derivatives: [Cu(L(1))(Cy(3)P)]ClO(4), [{Ag(2)(L(2))}(CF(3)SO(3))(2)]·H(2)O, [Cu(2)(L(2))(NO(3))(2)](NO(3))(2)·H(2)O, [Cu(2)(L(2))(CH(3)COO)(2)](CH(3)COO)(2)·3H(2)O, [Pd(2)(L(2))(Cl)(4)]·2H(2)O, [Ru(L(2))(Cl)(OH)]·CH(3)OH, [Ag(3)(L(4))(2)](CF(3)SO(3))(3) and [Ag(3)(L(4))(2)](BF(4))(3). The interaction of L(3) with Ag(I), Cu(II), Zn(II) and Ru(II) complexes unexpectedly produced the hydrolysis of the ligand with formation, in all cases, of tris(pyrazolyl)methane (TPM) derivatives. In detail, the already known [Ag(TPM)(2)](CF(3)SO(3)) and [Cu(TPM)(2)](NO(3))(2), as well as the new [Zn(TPM)(2)](CF(3)SO(3))(2) and [Ru(TMP)(p-cymene)]Cl(OH)·2H(2)O complexes have been isolated. Single-crystal XRD determinations on the latter derivatives confirm their formulation, evidencing, for the Ru(II) complex, an interesting supramolecular arrangement of the anions and crystallization water molecules.

Implications of antioxidant enzymes in human gastric neoplasms.

The present study is the first to evaluate the expression and activity of MnSOD, Cu/ZnSOD and catalase in human gastric samples, since ROS play a significant role in the pathogenesis of different forms of malignancy inducing mutations and various diseases such as gastric cancer. Biopsies and surgical samples from 53 patients (male/female 22/31, mean age 56.5+/-15.8 years) consisted of 15 healthy, 12 autoimmune atrophic gastritis, 10 Helicobacter pylori (HP) infection, 8 HP-negative chronic gastritis (CG) and 8 adenocarcinoma cases. Enzyme activity and expression were evaluated by spectrophotometry and immunoblotting after specific extraction in phosphate buffer. We found that MnSOD activity was increased in adenocarcinoma, CG and HP tissues (p<0.05-0.001), while Cu/ZnSOD was significantly lower in adenocarcinoma and HP tissues (p<0.001) when compared to the healthy control. MnSOD and Cu/ZnSOD were expressed to a significantly higher degree in adenocarcinoma and HP tissues (p<0.05 and <0.001 respectively) and to a significantly lower degree in CG tissues with respect to the healthy patients (p<0.05 and <0.001). A significant decrease in CAT activity in adenocarcinoma and HP tissues was observed (p<0.01 and <0.05). Gastric human neoplasms showed significant changes in antioxidant enzymes, that represent the first line in antioxidant protection against radical attack. The difficulties in correlating the antioxidant enzyme with the neoplasms was related to the complexity of the biochemical pathways that regulate the cellular redox balance. Our results are important in enhancing the understanding of the role that these enzymes play in the promotion/suppression of the carcinogenesis cascade in human gastric mucosa.

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[a]P.

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.

Chloramphenicol influence on antioxidant enzymes with preliminary approach on microsomal CYP1A immunopositive-protein in Chamelea gallina.

Chloramphenicol (CA) is a largely used antibiotic and it is an inhibitor of protein synthesis that also induces ROS production. In this work there were investigated activities and expressions in the Adriatic bivalve Chamelea gallina of some antioxidant and detoxification proteins like superoxide dismutase (Mn-SOD, Cu/Zn-SOD), catalase (CAT) and Cytochrome P450 (CYP1A). Clams exposed to 5mgl(-1) of chloramphenicol were sampled 2, 4 and 8 days after treatment (CA2, CA4 and CA8). SODs, CAT, and CYP1A activity and/or expression were detected in pooled digestive glands by Western blotting and by spectrophotometrical analysis. Enzymes activities increase during the entire antibiotic exposure. With respect to the control Cu/Zn-SOD expression increases, while Mn-SOD expression decreases significantly after 4 days. Two CYP1A immunopositive-proteins (57.7 and 59.8kDa) were detected. The lower band significantly decreases in CA8, the upper one also in CA4 condition. High levels of Mn-SOD, CAT activity and Cu/Zn-SOD expression, indicate intense ROS production while Mn-SOD expression inhibition might be ascribable to mitochondrial alterations due to CA and indirectly to ROS. CYP1A1 action determines H2O2 production that would contribute to a CYP1A1 gene promoter down regulation, a response to oxidative stress with the antioxidant enzymes activation as a final result. This study highlights the close association, in C. gallina, in presence of chloramphenicol, between SOD/CAT and CYP system, and it appear particularly interesting to the lack of similar researches on mollusc species.

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis.

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical-pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.

Trace metals in arcid clam Scapharca inaequivalvis: effects of molluscan extracts on bioluminescent bacteria.

The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the "bioactive" metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

Leptin does not seem to influence glucose uptake by bovine mammary explants.

Leptin, a protein produced and secreted by adipocytes, is know to regulate food intake and whole-body energy metabolism, but knowledge about its possible effect in bovine mammary gland is scarce. Leptin may be involved in the regulation of glucose transport even though this effect at the tissue level remains controversial. Once uptaken by the mammary gland, glucose is utilised in several ways but the majority, about 60-70%, is drained for lactose synthesis. This study was aimed at investigating the effect of leptin on glucose regulation in bovine mammary gland. We have examined the effects of leptin on the expression of GLUT1 mRNA, pyruvate kinase (PK) as well as glucose-6-phosphate dehydrogenase (G6PDH) activity. Treatment of mammary gland explants with recombinant leptin did not influence glucose assimilation, pathway transport (GLUT1 mRNA) and glucose metabolism (PK and G6PDH) in this tissue. The results from this study seem to exclude an involvement of leptin in glucose uptake and metabolism in bovine mammary gland.

Heterobimetallic, cubane-like Mo(3)S(4)M' cluster cores containing the noble metals M' = Ru, Os, Rh, Ir. Unprecedented tri(mu-carbonyl) bridge between ruthenium atoms in [(eta(5)-Cp')(3)Mo(3)S(4)Ru)2(mu-CO)3]2+.

Reaction of the methylcyclopentadienyl (Cp') cluster compound [(eta(5)-Cp')(3)Mo(3)S(4)][pts] (pts = p-toluenesulfonate) with noble metal alkene complexes resulted in the formation of four new heterobimetallic cubane-like Mo(3)S(4)M' cluster cores (M' = Ru, Os, Rh, Ir). Thus, reaction with [(1,5-cod)Ru(CO)(3)] or [(1,3-cod)Os(CO)(3)] (cod = cyclooctadiene) afforded [(eta(5)-Cp')(3)Mo(3)S(4)M'(CO)(2)][pts] (M' = Ru: [1][pts]; M' = Os: [2][pts]). When [1][pts] was kept in CH(2)Cl(2)/pentane solution, partial loss of carbonyl ligands occurred and the carbonyl-bridged dicubane cluster [((eta(5)-Cp')(3)Mo(3)S(4)Ru)(2)(mu-CO)(3)][pts](2) was isolated. An X-ray crystal structure revealed the presence of the hitherto unobserved Ru(mu-CO)(3)Ru structural element. The formation of cluster compounds containing Mo(3)S(4)Rh and Mo(3)S(4)Ir cores was achieved in boiling methanol by reacting [(eta(5)-Cp')(3)Mo(3)S(4)][pts] with [M'Cl(cyclooctene)(2)](2) (M' = Rh, Ir) in the presence of PPh(3). In this way [(eta(5)-Cp')(3)Mo(3)S(4)M'Cl(PPh(3))][pts] (M' = Rh, Ir) could be isolated. An alternative route to the Mo(3)S(4)Rh cluster core was found in the reaction of [(eta(5)-Cp')(3)Mo(3)S(4)][pts] with [RhCl(1,5-cod)](2), which yielded [(eta(5)-Cp')(3)Mo(3)S(4)Rh(cod)][pts](2) ([7][pts](2)). Substitution of the cod ligand in [7][pts](2) by 1,3-bis(diphenylphosphanyl)propane (dppp) gave [(eta(5)-Cp')(3)Mo(3)S(4)Rh(dppp)][pts](2).

Effects of bilateral ablation or 6-OHDA lesion of the frontal cortex upon the behaviors induced by apomorphine and amphetamine in adult cats.

The main aim of this study was to compare the effects of bilateral 6-OHDA lesion of the prefrontal, frontal and media cerebral cortex, with those of suction ablation of the same areas, upon apomorphine and amphetamine evoked behaviors. Twenty five cats were distributed in four groups: 1) 8 cats received bilateral, injection of 6-OHDA (32 micrograms per hemisphere), and the behaviors elicited by separate injection of apomorphine (2.0 mg/kg s.c.) and amphetamine (2.5 mg/kg s.c.) before and after the lesion were compared; 2) 3 cats received a unilateral injection of 6-OHDA into the cited areas and dopamine concentration on the lesioned site and the contralateral non-lesioned side were compared; 3) 8 cats had bilateral suction ablation of the cited cortical areas and the results of apomorphine and amphetamine evoked behaviors before and after ablation were compared, and 4) the effects of solvent injections and sham operations were studied in 6 additional cats. 6-OHDA lesion had scanty effects on the behaviors evoked by apomorphine and amphetamine. Only two (fear like behavior and lack of food motivation) out of 9 behaviors elicited by apomorphine were modified, while two of five behaviors elicited by amphetamine (immobility and food motivation) showed a change. On the other hand, the ablation procedure had a much wider effect on the behaviors elicited by the dopaminergic agonist drugs. All 5 behaviors evoked by amphetamine were modified, while only 4 of nine behaviors induced by apomorphine did change. These results show the importance of the interplay between dopamine and the target tissue that receive the dopaminergic fibers in the production of the behaviors evoked by parenteral injection of apomorphine and amphetamine.

[Multiple sclerosis in health care in our country: electrophysiological, immunological and clinical analysis]. / La esclerosis multiple en nuestro medio asistencial: análisis clínico, electrofisiológico e inmunológico.

Among 70 patients referred for suspected multiple sclerosis (MS) a total of 40 met criteria for definite (17), likely (12) or possible (11) diagnosis of the disease. Visual evoked potentials provided evidence of CNS lesion in 13 cases. Abnormalities of somatosensory evoked potentials, particularly on stimulation of the posterior tibial nerve, were present in 90% of patients. Brainstem auditory evoked potentials were of little use, contributing to diagnosis of one subclinical lesion; they were normal in 14 cases with suspected brainstem involvement. A definite diagnosis of MS was made in 4 cases by study of oligoclonal bands. The most frequently involved sites were the optic nerve and the brainstem, followed by the spinal cord. Hemispheral location was demonstrated in only 3 cases. These findings of the first study on MS in our country suggest a different pattern for demyelinating diseases in Chile.

Familial Creutzfeldt-Jakob disease in Chile.

Of the 87 cases of Creutzfeldt-Jakob disease (CJD) ascertained in Chile since 1931, 39 are familial accounting for 45% of all cases, and 25% of the 51 definite cases. There are 11 affected families with an average of 3.5 affected members per family, and a rate of occurrence consistent with autosomal dominant transmission. There is no evidence for maternal lineage, and age at death is not significantly different from that of sporadic cases. About half of the cases died around the same age, suggesting some form of vertical transmission. Three pairs of affected members in 3 different families died at the same time, possibly indicating common exposure to CJD agent. The study of absolute death intervals and temporal and spatial separations between affected members suggests minimum incubation periods ranging from 2 to 37 years, assuming case-to-case transmission. CJD occurring in a woman related by marriage to one of the affected families strongly argues for horizontal transmission. The high proportion of familial CJD observed in Chile is probably the result of both a genetically determined susceptibility to the CJD virus, and a high degree of case ascertainment. However, the present study leaves unanswered the mode of transmission of the agent within the affected families.