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Escherichia coli is one of the most important pathogens associated with puerperal metritis in dairy cows. Phage therapy has emerged as attractive therapeutic alternative in the control of multidrug-resistant pathogens. In this study, we present the complete genome sequence of vB_EcoM_ECO1230-10 (referred in this manuscript as ECO1230-10), a lytic E. coli phage isolated from dairy farms' manure systems. Ultrastructure of the phage revealed that ECO1230-10 belongs to Myoviridae family. The genome consisted of 41,666 bases with a G+C content of 53.37% and 56 predicted ORFs. It displays circular topology in a modular organization seen in phages, as demonstrated by the presence of gene modules, which is typical among members of this family. Based on similarity to genes from other related phages, the genome of ECO1230-10 was modularly clustered in tail-baseplate-head morphogenesis, DNA packaging, host cell lysis, and DNA replication and metabolism modules. Lysogeny control module was not detected in the ECO1230-10 genome. Comparative analyses showed that ECO1230-10 presents phylogenetic relationship with an Enterobacteria phage of the Myoviridae family, but it is almost in a taxonomic group of its own. The genome sequence of ECO1230-10, which is notably active against pathogenic E. coli isolated from uterine secretion of postpartum dairy cows, should provide new insights into the biology and evolution of this therapeutic candidate and possibly new strategies to control metritic-causing E. coli., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The title compound, [RuBr(C(10)H(8)N(2))(C(6)H(12)S(3))]PF(6) or [RuBr(bpy)([9]aneS(3))]PF(6) ([9]aneS(3) is 1,4,7-trithia-cyclo-nonane and bpy is 2,2'-bipyridine), exhibits a very similar octahedral coordination geometry for the Ru(2+) atom to that of its [RuCl(bpy)([9]aneS(3))](+) analogue, with only the chloride ligand being substituted by a bromide ligand. The presence of a PF(6) (-) anion (alongside with the coordinated bromide ligand) promotes the existence of an extensive network of weak C-H⋯X (X = F, Br) inter-actions.

At present, many bacterial species are validly known as etiological agents of dairy cattle metritis, yet the vast uncultured fraction has received no attention so far. The purpose of this study was to use culture-independent methods to describe and compare the uterine bacterial composition in healthy and metritic postpartum Holstein dairy cows. Both group-specific 16S ribosomal DNA PCR-denaturing gradient gel electrophoresis (DGGE) and clone library sequencing of broad-range 16S ribosomal DNA PCR revealed differences in the bacterial communities comparing healthy and metritic cows. Bacterial diversity in healthy and metritic uteri was greater and more complex than described previously by traditional culture methods. Sequences were assigned to 5 major groups (Gammaproteobacteria, Firmicutes, Fusobacteria, Bacteroidetes, and Tenericutes) and to uncultured bacteria. Additionally, DGGE suggested the presence of Actinobacteria. Most clone sequences in the metritic status libraries were affiliated with the phylum Fusobacteria. Many components, especially from other phyla, have not previously been isolated from cases of metritis. In the clone libraries from the healthy status dairy cows, Gammaproteobacteria was the most prominent group and most sequences showed high identity with Mannheimia varigena, Pasteurella hemolytica, and members of the phylum Tenericutes. Our data showed that the uterine bacterial community in postpartum dairy cows differed considerably between healthy and metritic cows and described the occurrence of a previously unrecognized extent of this diversity in the bovine intrauterine microbiota., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Paresthesia as a result of mechanical trauma is one of the most frequent sensory disturbances of the inferior alveolar nerve. This case report describes surgical treatment for paresthesia caused by a compressive phenomenon within the mandibular canal. The cause of the compression, a broken instrument left in the patient's mouth during previous endodontic therapy, was identified during routine radiography and computed tomography. Once the foreign object was removed by surgery, the paresthesia resolved quickly. This case highlights the potential for an iatrogenic mechanical cause of paresthesia.

Unlabelled: In order to properly assess patients with primary headache, one needs to follow the cases up longitudinally. In Brazil, there were no studies using this methodology published after the publication of the latest issue of the International Classification of Headaches in 2004 - ICHD-2. This is especially important when we consider that it was only after such publication that we had the criteria used to classify some types of headaches which evolve with daily, or almost daily, spells, and which are very common in tertiary health care centers., Objective: To assess the frequency of headache types in a tertiary health care center, in a longitudinal fashion., Method: We assessed 95 consecutive patients. These patients were diagnosed and classified according to the ICDH-2. The subjects were followed up for 18 months, they were treated and reassessed., Results: Most of the individuals had more than one type of headache. Among those with episodic migraine in 2007, 6 developed chronic migraine in 2008, producing an incidence rate of 7.2%. Among those with chronic migraine in 2007, 9 remitted, producing a remission rate of 75%. In 2007, 24 individuals abused analgesic agents and 17 no longer showed abuse criteria in 2008 - when 7 new cases were found., Conclusion: The diagnosis of migraine remained stable. On the other hand, treatment brought about a reduction in the frequency of headaches caused by excessive use of analgesic, although the frequency of daily chronic headache was almost unaltered.

In the title compound, [RuCl(C(10)H(8)N(2))(C(6)H(12)S(3))]NO(3)·H(2)O or [RuCl(bpy)([9]aneS(3))]NO(3)·H(2)O, ([9]aneS(3) is 1,4,7-tri-thia-cyclo-nonane and bpy is 2,2'-bipyridine), the Ru(II) cation has a slightly distorted octa-hedral environment composed of three facially coordinated S atoms from ([9]aneS(3)), two N atoms from bpy and a chloride anion. The nitrate counter-ion and the water mol-ecule of crystallization are engaged in O-H⋯O hydrogen-bonding inter-actions, leading to a supra-molecular chain running parallel to the c axis.

Arcanobacterium pyogenes is considered the most significant bacterium involved in the pathogenesis of metritis in cattle. Infections caused by antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to present an overview of antimicrobial resistance in A. pyogenes isolated from the uteruses of postpartum Holstein dairy cows and to identify virulence factors. Seventy-two A. pyogenes isolates were phenotypically characterized for antimicrobial resistance to amoxicillin, ampicillin, ceftiofur, chloramphenicol, florfenicol, oxytetracycline, penicillin, spectinomycin, streptomycin and tetracycline by the broth microdilution method. Presence of virulence factor genes of A. pyogenes was investigated. Isolates exhibited resistance to all antimicrobial agents tested; high levels of resistance were found to amoxicillin (56.9%); ampicillin (86.1%), chloramphenicol (100%), florfenicol (59.7%), oxytetracycline (54.2%), penicillin (86.1%) and tetracycline (50%). Of all isolates, 69 (95.8%) were resistant to at least 2 of the antimicrobial agents tested and multidrug resistance (resistant to at least 3 antimicrobials) was observed in 64 (88.9%) of the A. pyogenes isolates. The major multidrug resistance profile was found for chloramphenicol-ampicillin-penicillin-florfenicol-amoxicillin-tetracycline, which was observed in 21 (29.2%) multidrug resistant isolates. No isolate was resistant to all nine antimicrobial agents tested but four isolates (5.6%) were resistant to eight antimicrobials. The information highlights the need for prudent use of specific antimicrobial agents. All four virulence factor genes occurred in isolates from normal puerperium and clinical metritis; however, the fimA gene was present in significantly higher frequency in isolates from metritis cows., ((c) 2010 Elsevier B.V. All rights reserved.)

Unlabelled: Acoustic radiation is the sound energy escape from a bone vibrator that may be detected by air conduction mechanisms. The presence of acoustic radiation may result in an unreal bone conduction threshold, promoting an unreal air/bone gap in the high frequencies., Aim: aim to analyze the acoustic radiation effect on the extension of air/bone gap at 2,000, 3,000 and 4,000 Hz., Materials and Method: our clinical and experimental study had a sample of 101 individuals, who matched inclusion criteria: to have an air/bone gap higher than 10 dB in the frequencies of 2,000; 3,000 and 4,000 Hz. All of them had their bone conduction threshold measured in two conditions: open ear canal and closed ear canal., Results: we found that major differences between the two conditions evaluated occurred at the 4,000 Hz; data analysis showed significant difference in the extension for the air/bone gap; analysis of the number of cases of mixed hearing loss that changed to sensorineural was significant too., Conclusion: These studies concluded that when the MAE is occluded, the acoustic radiation phenomenon is controlled or avoided, enabling bone measures at the frequencies of 3,000 and 4,000Hz to be more accurate.

Depressive disorders, including major depression, are serious and disabling, whose mechanisms are not clearly understood. Since life stressors contribute in some fashion to depression, chronic variable stress (CVS) has been used as an animal model of depression. In the present study we evaluated some parameters of oxidative stress [thiobarbituric acid reactive substances (TBARS), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)], and inflammatory markers (interleukin 6, C reactive protein, tumor necrosis factor-alpha and nitrites), as well as the activity of butyrylcholinesterase in blood of rats subjected to chronic stress. Homocysteine and folate levels also were measured. Stressed animals were submitted to different mild stressors for 40 days. After CVS, a reduction in weight gain was observed in the stressed group, as well as an increase in immobility time in the forced swimming test as compared with controls. Stressed animals presented a significant increase on TBARS and SOD/CAT ratio, but stress did not alter GPx activity and any inflammatory parameters studied. CVS caused a significant inhibition on serum butyrylcholinesterase activity. Stressed rats had higher plasmatic levels of homocysteine without differences in folate levels. Although it is difficult to extrapolate our findings to the human condition, the alterations observed in this work may be useful to help to understand, at least in part, the pathophysiology of depressive disorders.

To evaluate the effects of potassium chromate on mice sperm cells after a short-term exposure, male ICR-CD1 mice were administered with 5 or 10mgK(2)CrO(4)/bw for 4 consecutive days. One group of mice was sacrificed at day 5, starting from the beginning of the experiment and another group was sacrificed at day 35. Testis and epididymis histology was evaluated by light microscopy and testicular cells populations were evaluated by flow cytometry (FCM). Spermatozoa were collected from the epididymis and their morphology and several functional parameters (density, motility, viability, mitochondrial function, acrosome integrity) were evaluated. Furthermore, DNA fragmentation and chromatin status of sperm cells were assessed at both experimental periods. Besides a reduction in seminiferous tubules diameter, exposure to potassium chromate did not induce further histopathological changes in mice testis or epididymis. These results were supported by the analysis of testicular cellular subpopulations by FCM. Concerning spermatozoa morphology, an increase in the percentage of multiple abnormalities and a decrease in the percentage of normal spermatozoa were found at days 5 and 35, respectively. Although spermatozoa mitochondrial function or viability was not affected, its motility was significantly reduced by potassium chromate exposure at both experimental periods. A decrease in acrosome integrity was found in mice injected with 10mgK(2)CrO(4)/bw after 35 days. Exposure to potassium chromate did not affect either DNA fragmentation or chromatin susceptibility to acid denaturation of sperm cells. In this work, we were able to show the effects of potassium chromate on spermatozoa physiological parameters such as motility, morphology and acrosome status and also demonstrate that the doses tested did not induce DNA damage to sperm cells after one spermatogenic cycle.

Background: the Random Gap Detection Test (RGDT) evaluates temporal resolution threshold. There are doubts as to whether performance in this task remains unchanged with the aging process. At the same time, there is a concern about how much the difficulties of communication experienced by elderly individuals are related to the deterioration of temporal resolution., Aim: to determine auditory temporal resolution threshold in elderly individuals with normal peripheral hearing or symmetric mild sensorineural hearing loss, and to correlate findings with gender, age, audiometric findings and scores obtained in the Self - Assessment of Communication (SAC) questionnaire., Methods: 63 elderly individuals, aged between 60 and 80 years (53 women and 10 men), were submitted to the RGDT and the SAC., Results: statistical analysis of the relationship between gender and the RGDT indicated that the performance of elderly females was statistically poorer when compared to elderly males. Age and audiometric configuration did not correlate to performance in the RDGT and in the SAC. The results indicate that in the SAC both genders presented no significant complaints about communication difficulties regardless of the outcome obtained in the RGDT or audiometric configuration., Conclusion: the average temporal resolution threshold for women was 104.81ms. Considering gender, females did not present correlations between age and audiometric configuration, not only when considering the RGDT results but also when analyzing the SAC results.

In the present study we evaluated the effect of acute and chronic homocysteine administrations on glutamate uptake in parietal cortex of rats. The immunocontent of glial glutamate transporter (GLAST) and sodium-dependent glutamate/aspartate transporter (GLT-1) in the same cerebral structure was also investigated. For acute treatment, neonate or young rats received a single injection of homocysteine or saline (control) and were sacrificed 1, 8, 12 h, 7 or 30 days later. For chronic treatment, homocysteine was administered to rats twice a day at 8 h interval from their 6th to their 28th days old; controls and treated rats were sacrificed 12 h, 1, 7 or 30 days after the last injection. Results show that acute hyperhomocysteinemia caused a reduction on glutamate uptake in parietal cortex of neonate and young rats, and that 12h after homocysteine administration the glutamate uptake returned to normal levels in young rats, but not in neonate. Chronic hyperhomocysteinemia reduced glutamate uptake, and GLAST and GLT-1 immunocontent. According to our results, it seems reasonable to postulate that the reduction on glutamate uptake in cerebral cortex of rats caused by homocysteine may be mediated by the reduction of GLAST and GLT-1 immunocontent, leading to increased extracellular glutamate concentrations, promoting excitotoxicity., (Copyright 2009 ISDN. Published by Elsevier Ltd. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) are an attractive source for generation of cells with beta-cell properties. Previous studies have demonstrated the ability of prolactin to induce an increase in beta-cell mass and maturation, which suggests beneficial effects of its use in MSC differentiation protocols., Objective: To evaluate the expression of endocrine differentiation markers in rat MSCs treated in vitro with prolactin., Methods: Mesenchymal stem cells from bone marrow of Wistar rats were isolated, expanded, and characterized. Differentiation of MSCs was induced in medium containing 23 mmol/L of glucose, and nicotinamide, 2-mercaptoethanol, and exendin-4, in the presence or absence of 500 ng/mL of rat recombinant prolactin. Expression of endocrine markers and prolactin receptor genes was evaluated using real-time polymerase chain reaction, and compared between culture stages and presence vs absence of prolactin in the culture medium. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was also evaluated at immunofluorescence microscopy., Results: Isolated cells were mostly MSCs, as confirmed at fluorescent-activated cell sorting and cytochemistry. Pax6, Ngn-3, Isl1, NeuroD1, Nkx2.2, and Nkx6.1 exhibited varied expression during culture stages. The long form of the prolactin receptor messenger RNA was induced in prolactin-treated cultures (P < .05). The somatostatin gene was induced in early stages of differentiation (P < .05), and its expression was induced by prolactin, as confirmed using immunofluorescence., Conclusion: Culture of rat bone marrow MSCs in differentiation medium induces expression of pancreatic endocrine-specific genes, and somatostatin and prolactin receptor expression was also induced by prolactin., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child., Objective: To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype., Methods: Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence., Results: Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed., Conclusions: The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The objective of this study was to isolate bacteriophages from environmental samples of 2 large commercial dairy farms using Escherichia coli isolated from the uteri of postpartum Holstein dairy cows as hosts. A total of 11 bacteriophage preparations were isolated from manure systems of commercial dairy farms and characterized for in vitro antimicrobial activity. In addition, a total of 57 E. coli uterine isolates from 5 dairy cows were phylogenetically grouped by triplex PCR. Each E. coli bacterial host from the uterus was inoculated with their respective bacteriophage preparation at several different multiplicities of infections (MOI) to determine minimum inhibitory MOI. The effect of a single dose (MOI=10(2)) of bacteriophage on the growth curve of all 57 E. coli isolates was assessed using a microplate technique. Furthermore, genetic diversity within and between the different bacteriophage preparations was assessed by bacteriophage purification followed by DNA extraction, restriction, and agarose gel electrophoresis. Phylogenetic grouping based on triplex PCR showed that all isolates of E. coli belonged to phylogroup B1. Bacterial growth was completely inhibited at considerably low MOI, and the effect of a single dose (MOI=10(2)) of bacteriophage preparations on the growth curve of all 57 E. coli isolates showed that all bacteriophage preparations significantly decreased the growth rate of the isolates. Bacteriophage preparation 1230-10 had the greatest antimicrobial activity and completely inhibited the growth of 71.7% (n=57) of the isolates. The combined action of bacteriophage preparations 1230-10, 6375-10, 2540-4, and 6547-2, each at MOI=10(2), had the broadest spectrum of action and completely inhibited the growth (final optical density at 600 nm

The use of pathogenic-specific antimicrobials, as proposed by bacteriophage therapy, is expected to reduce the incidence of resistance development. Eighty Escherichia coli isolated from uteri of Holstein dairy cows were phenotypically characterized for antimicrobial resistance to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline by broth microdilution method. The lytic activity of a bacteriophage cocktail against all isolates was performed by a similar method. Additionally, the effect of different concentrations of antimicrobials and multiplicities of infections (MOI) of the bacteriophage cocktail on E. coli growth curve was measured. Isolates exhibited resistance to ampicillin (33.7%), ceftiofur (1.2%), chloramphenicol (100%), and florfenicol (100%). All strains were resistant to at least 2 of the antimicrobial agents tested; multidrug resistance (>or=3 of 7 antimicrobials tested) was observed in 35% of E. coli isolates. The major multidrug resistance profile was found for ampicillin-chloramphenicol-florfenicol, which was observed in more than 96.4% of the multidrug-resistant isolates. The bacteriophage cocktail preparation showed strong antimicrobial activity against multidrug-resistant E. coli. Multiplicity of infection as low as 10(-4) affected the growth of the E. coli isolates. The ratio of 10 bacteriophage particles per bacterial cell (MOI=10(1)) was efficient in inhibiting at least 50% of all isolates. Higher MOI should be tested in future in vitro studies to establish ratios that completely inhibit bacterial growth during longer periods. All isolates resistant to florfenicol were resistant to chloramphenicol and, because florfenicol was recently introduced into veterinary clinics, this finding suggests that the selection pressure of chloramphenicol, as well as other antimicrobials, may still play a relevant role in the emergence and dissemination of florfenicol resistance in E. coli. The bacteriophage cocktail had a notable capacity to inhibit the in vitro growth of E. coli isolates, and it may be an attractive alternative to conventional treatment of metritis by reducing E. coli in uteri of postpartum dairy cows., (Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The complex Ru([9]aneS(3))(gly)Cl (gly = glycine) was obtained from the reaction of the precursor Ru([9]aneS(3))dmsoCl(2) with glycine and encapsulated into native beta-CD, a hydroxypropylated derivative HPbetaCD, and the methylated cyclodextrins TRIMEB and CRYSMEB. All four inclusion compounds were obtained with a 1:1 host:guest stoichiometry and characterised in the solid-state by powder X-ray diffraction, thermogravimetric analysis (TGA), and (13)C{(1)H} CP/MAS NMR and FTIR spectroscopies. The cytostatic and antiproliferative activity of the complex Ru([9]aneS(3))(gly)Cl and its four CD inclusion compounds was tested on the human osteosarcoma MG-63 cell line and the results compared to the inhibitory effect exerted by the pure cyclodextrins.

In the present study, we first investigated the effect of single homocysteine administration on consolidation of short- and long-term memories of inhibitory avoidance task in Wistar rats. We also measured brain-derived neurotrophic factor levels in the hippocampus and parietal cortex of rats. The influence of pretreatment with folic acid on behavioral and biochemical effects elicited by homocysteine was also studied. Wistar rats were subjected to a folic acid or saline pretreatment from their 22(nd) to 28(th) day of life; 12 h later they were submitted to a single administration of homocysteine or saline. For motor activity and memory evaluation we performed open-field and inhibitory avoidance tasks. Hippocampus and parietal cortex were obtained for brain-derived neurotrophic factor immunocontent determination. Results showed that homocysteine impaired short- and long-term memories and reduced brain-derived neurotrophic factor levels in the hippocampus. Pretreatment with folic acid prevented both the memory deficit and the reduction in the brain-derived neurotrophic factor immunocontent induced by homocysteine injection. Further studies are required to determine the entire mechanism by which folic acid acts and its potential therapeutic use for memory impairment prevention in homocystinuric patients.

The complex [Ru([9]aneS(3))phenCl]Cl (phen = 1,10-phenanthroline) and its synthetic precursor [Ru([9]aneS(3))dmsoCl(2)] were immobilized in permethylated beta-cyclodextrin (TRIMEB). A new crystalline structure of the precursor, obtained from a batch ethanol solution at low temperature (4 degrees C), is fully described from single-crystal X-ray diffraction data. [Ru([9]aneS(3))phenCl]Cl was also encapsulated in native beta-cyclodextrin for comparison with the TRIMEB compound. All three compounds were obtained with a 1:1 host:guest stoichiometry and were studied by powder X-ray diffraction (including synchrotron radiation data), thermogravimetric analysis (TGA), (13)C{(1)H} CP/MAS NMR and FTIR spectroscopies. The bacterial growth inhibitory action of the complex [Ru([9]aneS(3))phenCl]Cl and its two cyclodextrin compounds was tested on Gram-negative (Salmonella, Escherichia) and Gram-positive strains (Bacillus, Listeria, Enterococcus and Staphilococcus) and results show a positive effect of cyclodextrin immobilization on the antimicrobial properties.

Unlabelled: The workings of the auditory pathway of patients with tinnitus and normal hearing can be associated with an auditory efferent pathway dysfunction at the level of the superior olivary complex. Otoacoustic emission suppression with contralateral noise can represent an alternative to its evaluation., Aim: to investigate Transient Otoacoustic Emission suppression in normal hearing adults with and without tinnitus and to compare the two groups., Study Design: cross-sectional contemporary cohort., Materials and Methods: we assessed 40 female individuals between 18 and 59 years of age, 20 without tinnitus and 20 with it. We studied the TOAE suppression with a contralateral white noise at 50 dBSPL., Results: TOAE amplitude was lower in the group with tinnitus. There was no difference between the groups with and without tinnitus in terms of TOAE suppression, except in the frequency of 1000 Hz in the left ear in the tinnitus group., Conclusion: the afferent system assessment may contribute to the topographic diagnosis of tinnitus; however, we still need further studies to establish the proper methodology and normative values to carry out these tests.

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelial cells to the basement membrane. HD components include the alpha6beta4 integrin, plectin, and BPAGs (bullous pemphigoid antigens). HD disassembly in keratinocytes is necessary for cells to migrate and can be induced by EGF through beta4 integrin phosphorylation. We have identified a novel phosphorylation site on the beta4 integrin: S(1424). Preventing phosphorylation by mutating S-->A(1424) results in increased incorporation of beta4 into HDs and resistance to EGF-induced disassembly. In contrast, mutating S-->D(1424) (mimicking phosphorylation) partially mobilizes beta4 from HDs and potentiates the disassembly effects of other phosphorylation sites. In contrast to previously described sites that are phosphorylated upon growth factor stimulation, S(1424) already exhibits high constitutive phosphorylation, suggesting additional functions. Constitutive phosphorylation of S(1424) is distinctively enriched at the trailing edge of migrating keratinocytes where HDs are disassembled. Although most of this S(1424)-phosphorylated beta4 is found dissociated from HDs, a substantial amount can be associated with HDs near the cell margins, colocalizing with plectin but always excluding BPAGs, suggesting that phospho-S(1424) might be a mechanism to dissociate beta4 from BPAGs. S(1424) phosphorylation is PKC dependent. These data suggest an important role for S(1424) in the gradual disassembly of HDs induced by cell retraction.

The aim of the present study was to describe the biological aspects of larvae of Argas miniatus at 27+/-1 masculineC and 80+/-5% and in environmental condition. Domestic fowls with age lower than one week were infested with approximately 700 larvae of A. miniatus 15 days posthatching. The period of larval fixation varied from 3 to 7 days in four repetitions. Approximately 90% of larvae were recovered in the 4th and 5th day after attachment. The mean weight increased approximately 81.37 times the initial weight of the unfed larvae. The mean larval mortality in controlled conditions and laboratory environment were 6.35% and 13.97%, respectively. Molting period varied from 4 to 9 days in controlled condition, while in the environment conditions the interval varied from 5 to 15 days. The larval longevity was 120 days, in both conditions. Unfed larvae maintained at 27+/-1 masculineC and 80+/-5% and environment condition were capable to attach from 6 to 60 days and 8 to 45 days, respectively.

The aim of this study was to demonstrate, through nested PCR (nPCR) method, the occurrence of congenital babesiosis in two foals born of carrier mares. All mares were positive for T. equi based in visualization of intraerytrocytic parasites in blood smears, in indirect fluorescent antibody test (IFAT) and nPCR reactions. Just one mare was nPCR-positive for B. caballi. After the birth, all foals presented nPCR-positive for T. equi, while just one foal presented nPCR-positive for B. caballi. The present study prove the occurrence of congenital babesiosis in new-born foals, however, new studies are necessary to elucidate if the infection occur for transplacental transmission or in parturition moment.

The tumor suppressor tuberin, encoded by the Tuberous Sclerosis Complex (TSC) gene TSC2, negatively regulates the mammalian target of rapamycin (mTOR) pathway, which plays a key role in the control of cell growth and proliferation. In addition to naturally occurring mutations, several kinases including Akt, RSK1, and ERK are known to phosphorylate and inactivate tuberin. We demonstrate a novel mechanism of tuberin inactivation through ubiquitination by Pam, a putative RING finger-containing E3 ubiquitin (Ub) ligase in mammalian cells. We show that Pam associates with E2 ubiquitin-conjugating enzymes, and tuberin can be ubiquitinated by Pam through its RING finger domain. Tuberin ubiquitination is independent of its phosphorylation by Akt, RSK1, and ERK kinases. Pam is also self-ubiquitinated through its RING finger domain. Moreover, the TSC1 protein hamartin, which forms a heterodimer with tuberin, protects tuberin from ubiquitination by Pam. However, TSC1 fails to protect a disease-associated missense mutant of TSC2 from ubiquitination by Pam. Furthermore, Pam knockdown by RNA interference (RNAi) in rat primary neurons elevates the level of tuberin, and subsequently inhibits the mTOR pathway. Our results provide novel evidence that Pam can function as an E3 Ub ligase toward tuberin and regulate mTOR signaling, suggesting that Pam can in turn regulate cell growth and proliferation as well as neuronal function through the TSC/mTOR pathway in mammalian cells.

We recently reported that ruthenium complexes, with general structure [mu-bidppz(bipy)4Ru2](4+) (B) or [mu-bidppz(phen)4Ru2](4+) (P) (bidppz=11,11'-bi(dipyrido[3,2- a:2',3'-c]phenazinyl)), show extreme kinetic selectivity for long AT tracts over mixed-sequence calf thymus DNA (ct-DNA), a selectivity that also varies markedly with the size (between B and P) and sense of chirality of the complex. Earlier studies, exploiting the great increase in luminescence intensity when the compound intercalates, have yielded complex kinetics indicating the presence of both first- and second-order processes. Even with a homogeneous DNA sequence, such as poly(dAdT)2, the luminescence kinetics generally requires more than a single exponential for a satisfactory fit. We here reveal that at least part of the complexity is a result of the extreme sensitivity of the effective quantum yield of the complexes, so that the luminescence trajectories also reflect subtle variations in the environment and binding geometry that the complex is sampling on its path to its final binding site. By monitoring the rearrangement process using circular dichroism (CD), we show that threading of both enantiomers of B and P into poly(dAdT)2 is effectively a monoexponential process, as expected if the compounds are not affecting each other during the intercalation process. Thus, the complex luminescence trajectories may be explained by slow relaxations in the binding geometry (DNA conformation) and associated changes in the environment of the entering complexes. To further disentangle the intriguing features of the threading intercalation kinetics, and how they may depend on the flexibility and size of the ruthenium complexes, we have also designed and studied two new ruthenium complexes, [mu-dtpf(phen)4Ru2](4+) (F) (dtpf=4,5,9,12,16,17,21,25-octaaza-23 H-ditriphenyleno[2,3-b:2,3-h]fluorene), in which the bridging ligand is made totally rigid, and [mu-bidppz([12]aneS4) 2Ru2](4+) (S), which has less bulky, nonaromatic ancillary ligands. The threading of F into poly(dAdT)2, also found to be a monoexponential process, is about 3 times slower than for P, indicating that the flexibility of the bridging ligand is an important factor for the intercalation rate. Surprisingly, in contrast to all other compounds, S requires two exponentials to fit its binding kinetics as monitored by CD. Also surprisingly, in view of the smaller steric bulk, even the fastest phase is roughly 2 times slower for S than for B and P. Thus, not only the size of the ancillary ligand but also other properties that can influence the energy landscape of the threading path are rate-determining factors. With mixed-sequence ct-DNA, threading of B and that of P are both multiphasic processes when monitored with CD as well as with luminescence. The rate constants for threading into ct-DNA show much larger variations between complexes than for poly(dAdT)2, confirming earlier results based on luminescence data.

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.

Purpose: To evaluate 20 cases of nonmetastatic synovial sarcoma of the extremities regarding prognostic factors, and to propose a histologic grading system with prognostic significance., Methods: The cases of 20 patients (14 females and 6 males) with nonmetastatic synovial sarcomas of the extremities treated between 1985 and 1998, were retrospectively evaluated regarding prognostic factors. A histologic grading system with prognostic significance is proposed., Results: The mean follow-up period was 48.4 months (range, 16-116 months). There was local recurrence in 3 cases (15%), microscopic surgical margin being the only prognostic factor identified. Seven patients (35%) died of the disease in a mean postoperative period of 31.7 months (range, 16-53 months), all with pulmonary or brain metastasis. The survival rate was 65% in 48.4 months of follow-up., Conclusion: The unfavorable prognostic factors identified regarding survival were high histologic grade, tumors proximal to the knee or elbow, and spontaneous tumor necrosis over 25%. Local recurrence did not have influence on survival in this study. The presence of mast cells appears to have a positive influence on survival, although statistical significance was not reached (P = 0.07). The oncologic and functional result was good in 6 cases (30%), regular in 7 (35%), and poor in 7 cases (35%).

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Purpose: To evaluate the impact of chemotherapy and surgery on the outcome of osteosarcoma (OS) of the extremities and to identify prognostic factors in Brazilian patients., Patients and Methods: A total of 225 patients with metastatic and nonmetastatic OS of the extremities were enrolled and assessed in two consecutive studies designed and implemented by the Brazilian Osteosarcoma Treatment Group., Results: The 5-year survival and event-free survival rates for the 209 assessable patients were 50.1% and 39%, respectively; for the 178 patients with nonmetastatic disease at diagnosis, the rates were 60.5% and 45.5%, respectively. The multivariate analysis showed that the following variables were associated with a shorter survival: metastases at diagnosis (P < .001), necrosis grades 1 and 2 (P = .046), and tumor size (P = .0071)., Conclusion: The overall 5- and 10-year survival rates were lower than the rates reported in North American and European trials. A pattern of advanced disease at diagnosis was often present, with a high proportion of patients having metastases (20.8%) and large tumor size (42.9%). However, these features were not necessarily associated with longer duration of prediagnostic symptoms. These findings were considered in the strategic planning of the current Brazilian cooperative study, with the aim of improving survival and quality of life of a large number of patients with OS.

We recently identified Pam (for protein associated with c-Myc), as a binding partner for the tuberous sclerosis complex (TSC) protein tuberin in brain. The highly conserved Pam homologs in Drosophila and C. elegans are neuron-specific proteins that regulate synaptic growth. The Pam gene contains 83 exons and encodes a 4,641-amino-acid polypeptide with a predicted molecular weight of approximately 510 kDa. In a previous study, we demonstrated that Pam is expressed as two forms, approximately 450 kDa in rat embryonic and a approximately 350 kDa in rat adult brain. Here we have extended that work to show the approximately 450 kDa form is expressed in rat embryonic kidney, heart, and lung and in rat cell lines, and the approximately 350 kDa form is expressed in adult rat tissues as well as in human and mouse brain and human and mouse cell lines. To understand the size difference, we investigated alternative splicing of Pam in brain and detected six isoforms in the Myc-binding region resulting from splicing of exon 53, and three new exons, 52A, 56, and 56A. We also demonstrate that the presence of exon 52A in Pam significantly enhances binding to Myc, suggesting functional importance of this alternative splicing. The presence of Pam in many cellular compartments, its spliced variants, as well as its multiple binding partners, including tuberin, make it a complex, yet intriguing protein in the nervous system., (Copyright 2005 Wiley-Liss, Inc.)

Background: Cr(V) species are formed during the intracellular reduction of Cr(VI), a ubiquitous environmental pollutant. In this study, the acute toxicity of a physiologically stable Cr(V) compound, [CrV-BT](2-) (BT = bis(hydroxyethyl)aminotris(hydroxymethyl)methane) was investigated in the male reproductive system of sexually mature 60-day-old male ICR-CD1 mice., Methods: Eight-week-old animals were subcutaneously injected daily with a dose of ca 8 micromol of Cr/mouse, during 5 days. The control group was injected with 0.5 mL of BT buffer. Testis and epididymis morphology was evaluated using light and transmission electron microscopy. Epididymal sperm counts, motility and acrosome integrity were also assayed using standard methods., Results: Seminiferous epithelium abnormalities were detected in the CrV-BT experimental group, including intraepithelial vacuolation, and remarkable degeneration of Sertoli cells, spermatocytes and spermatids. The premature release of germ cells into the tubular lumen was also evident. Histological evaluation of epididymal compartments revealed apparently normal features. However, the epididymal epithelium presented vacuolation. [CrV-BT](2-) induced a reduction in sperm acrosome integrity. However, sperm motility and density were not significantly affected., Conclusion: This in vivo study using a Cr(V) compound, provides evidence for the potential reproductive hazards caused on male reproductive system by species containing chromium in intermediate oxidation states.

Background: The study of the relationship of the contralateral acoustic reflex with the auditory skill of closure., Aim: To analyze the identification of a speech signal in the presence of competitive sounds in subjects with absence of contralateral acoustic reflex., Method: Application of the synthetic sentence identification (SSI) test under the conditions of competitive contralateral message (SSI-CCM), with the signal-to-noise ratio of 0 and -40dB, and ipsilateral competitive message (SSI-ICM), with the signal-to-noise ratio of 0, -10, -15 and -20dB, in 43 young adults (group A = 21 subjects with contralateral acoustic reflex present in all of the investigated frequencies, and group B = 22 subjects with contralateral reflex absent at the frequency of 500Hz, or in all of the investigated frequencies, or still in some of the investigated frequencies necessarily including 500Hz), of both gender, with no hearing, otologic or learning disabilities., Results: The acoustic reflex threshold was above 100dB NA in 59% of the individuals in group B and in 14% of the individuals in group A. All subjects performed according to the normal pattern suggested in the specialized literature for the SSI test. The performance of group B in the SSI-ICM test was inferior to that of group A for all the signal-to-noise ratios used, although the difference was not statistically significant. Group B, which presented an acoustic reflex threshold higher than 100dB NA or the absence of the acoustic reflex, was also the group that presented the worse performance in the SSI test., Conclusion: The absence of the contralateral acoustic reflex seems to interfere in the identification of the speech signal in the presence of competitive noises.

1. We have investigated the effect of tityus gamma (TiTX gamma) scorpion toxin on the release of [3H]dopamine in rat brain prefrontal cortical slices. The stimulatory effect of TiTX gamma on the release of [3H]dopamine was dose/time-dependent with an EC50 of 0.01 microM. 2. Tetrodotoxin blocked the TiTX gamma-induced release of [3H]dopamine, indicating the dependency for Na+ channels. 3. EGTA had no effect on the TiTX gamma-induced release of [3H]dopamine, indicating the process is independent of extracellular calcium. Release of [3H]dopamine evoked by TiTX gamma was inhibited by 57% by BAPTA, a chelator of intracellular calcium. 4. Xestospongin and 2-APB, putative blockers of IP3-sensitive release of intracellular calcium stores, caused an equal and significant inhibition of 24% of the TiTX gamma-induced release of [3H]dopamine, while the slight inhibition evoked by dantrolene, a putative blocker of ryanodine-sensitive calcium store was not significant. 5. Nomifensine and ascorbic acid, blockers of dopamine transporter (DAT), caused an inhibition of 27 and 29%, respectively, on the toxin-induced release of [3H]dopamine suggesting that most of the TiTX gamma-induced release of dopamine is not due to the reversal of Na+ gradient. 6. In conclusion the majority of the TiTX gamma-induced release of [3H]dopamine is exocytotic and mobilizes calcium from the intracellular IP3-sensitive calcium stores.

Transport of macromolecules from the interstitial testis tissue to cells at the adlumenal compartment of the seminiferous epithelium occurs naturally through Sertoli cells. In previous studies we have shown that Cr(V) intoxication disturbed spermatogenesis in mice. To test if Sertoli cells are affected by chromium, a well proved carcinogen, the uptake and the horseradish peroxidase transport ability of isolated seminiferous tubules of mice administered with a chromium(V) compound, have been studied. Male CD-R mice were exposed daily for 5 days to [CrV-BT]2- through subcutaneous injection and comparisons were made with groups of vehicle-treated mice. Using an in vitro assay we demonstrated that the seminiferous tubules were able to uptake and transport the tracer, in a much faster way than controls, mainly via intercellular and transcellular pathways, providing evidence that this functional role of Sertoli cells is affected by the Cr(V) compound. These findings might improve the knowledge on the toxicity mechanisms of chromium.

A Zn,Al layered double hydroxide (LDH) with Zn/Al = 1.45 and containing nitrate anions was prepared by coprecipitation and characterized by powder X-ray diffraction, Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS), thermogravimetric analysis, FTIR and FT Raman spectroscopy, and (27)Al MAS NMR spectroscopy. Three Zn...O and four Zn...M (M = Zn, Al) shells could be fitted to the low-temperature (40 K) EXAFS spectrum, in accordance with a model for an ordered cationic sheet. The nitrate anions were easily exchanged by 2,2'-bipyridine-5,5'-dicarboxylate anions, resulting in an increase in the basal spacing from 9 to 18 A. The basal spacing of the pillared derivative indicates that the anions are arranged with their longest dimension nearly perpendicular to the host layers. This material exhibits a high encapsulating ability, as evidenced by its interaction with a dichloromethane solution of the dioxomolybdenum(VI) complex MoO(2)Cl(2)(THF)(2). A material with a metal loading of 11.2 wt % was obtained. Molybdenum K-edge EXAFS analysis could not substantiate the formation of a supported complex of the type MoO(2)Cl(2)(N-N) but instead indicated the formation of unidentate-bridged entities of the type [O(2)Mo-O-MoO(2)] with a metal-metal separation of 3.29 A. The molybdenum-containing LDH was active as a catalyst for the liquid-phase epoxidation of cis-cyclooctene, 1-octene, and trans-2-octene using tert-butyl hydroperoxide as the oxygen source, yielding the corresponding epoxides as the only products. For reactions carried out with no additional solvent (other than n-decane) or in the presence of 1,2-dichloroethane, the solid catalyst could be recycled with no major loss of activity. Other tests confirmed that the systems functioned as true heterogeneous catalysts.

Tuberous sclerosis complex is caused by mutations in tumor suppressor genes TSC1 or TSC2 and is characterized by the presence of hamartomas in many organs. Although tuberous sclerosis complex is a tumor suppressor gene syndrome with classic "second hits" detectable in renal tumors, conventional genetic analysis has not revealed somatic inactivation of the second allele in the majority of human brain lesions. We demonstrate a novel mechanism of post-translational inactivation of the TSC2 protein, tuberin, by physiologically inappropriate phosphorylation, which is specific to tuberous sclerosis complex-associated brain lesions. Additional analysis shows that tissue specificity is due to abnormal activation of the Akt and mitogen-activated protein kinase pathways in brain but not in renal tumors. These results have widespread implications for understanding the tissue specificity of tumor suppressor gene phenotypes.

We studied the influence of metabolites of gamma-aminobutyric shunt of the tricarboxylic acid cycle on the activities of aconitate hydratase (EC.4.2.1.3) as well as NAD- and NADP-specific isocitrate dehydrogenases (EC.1.1.1.41 and EC.1.1.1.42, respectively) using purified enzyme preparations from pea leaves (Pisum sativum L.) and maize scutellum (Zea mays L.). gamma-Aminobutyric acid and succinate proved to have no significant effect on these enzymes, while 0.1-0.2 mM glutamate considerably activated NADP isocitrate dehydrogenase from the both sources, particularly, at unsaturating concentration of the substrate. Succinic semialdehyde stimulated the activities of aconitate hydratase and NADP isocitrate dehydrogenase. The obtained data points to a similar pattern of the effect of intermediates of gamma-aminobutyric shunt on the studied enzymatic activities for both photosynthetic tissues (pea leaves) and those with acidifying, transport, and digestive functions (maize scutellum). However, the absence of pronounced control effects of most metabolites on the studied enzymes allows us to assign them to a relatively inert pool of metabolites.

Low-molecular weight GTP-binding proteins (LMWGPs) of the Ras superfamily are believed to play a role in Schistosoma mansoni female development and egg production. Here we describe the characterization of a novel S. mansoni gene (SMRHO1), highly homologous to Rho-type LMWGPs from several other organisms and encoding a polypeptide with 193 amino acids and an estimated molecular mass of 21.8 kDa. SMRHO1 complemented a Saccharomyces cerevisiae rho1 null mutant strain even in restrictive temperature and calcium concentration, in contrast with the human RHOA GTPase that was not able to provide complementation in such conditions. Comparison of the amino acid sequence of the alpha3-helix loop7 regions of the two proteins allowed the identification of the proline 96 and threonine 100 amino acid residues of human RHOA as the most probable determinants of the complementation differences. We generated SMRHO1 mutants (smrho1(E97P), smrho1(L101T) and smrho1(E97P,) L101T) by site directed mutagenesis and reproduced the conditional lethality phenotype at high temperature, providing strong evidence that the related amino acid positions (Gln(101) and Ile(105)) in the Rho1 GTPase are indeed important for regulation of the cell wall synthesis performed by this protein in yeast. The observation that specific amino acid positions seem to be important for the different functions performed by the Rho GTPases leads to the idea that SMRHO1 might be a useful target in the development of new anti-schistosomiasis drugs, although it does share high sequence homology with the human RhoA GTPase.

Chromium toxicity is strongly dependent on its oxidation state. Cr(VI) is carcinogenic and mutagenic, although its in vivo and in vitro toxic effects are related to its intracellular fate. Inside the cells, Cr(VI) is rapidly reduced to stable Cr(III). As Cr(V) and Cr(IV) species have been reported to be formed in the Cr(VI) reduction pathways, Cr(VI)-induced damage is thought, at least in part, to arise from these hypervalent species. The study of Cr(VI) reduction mechanisms and the characterization of the effects of each reactive intermediate constitute important steps towards a better understanding of chromium toxicity. The purpose of this work is to enlarge the scope of Cr(VI)-induced alterations in mouse to other chromium species. Our studies have led to the in situ preparation of a new Cr(V) complex, 1Cr(V)-BT](2-), a stable compound at neutral pH, which mimics Cr(VI) reduction intermediates. The effect of Cr(V) on the histology of mice liver is assessed and compared with similar Cr(VI) assays. Liver toxicity was examined after single administrations of Cr(VI) or [Cr(V)-BT](2-) to mice. Both compounds produced reversible hepatic damage in a time-dependent manner. However, Cr(V) toxic effects have proved to be more rapid than with Cr(VI), permitting the role of Cr(VI) intermediates formed during intracellular chromium reduction to be highlighted.

Aim: The functional integrity of the blood-testis barrier (BTB) in male mice exposed to Cr(V) was studied in order to clarify the mechanism underlying testicular injury., Methods: Adult male mice were subcutaneously injected repeated doses of 8.02 micromol (0.5 ml) of Cr/mouse.day for 5 days. Animals receiving a similar volume of bis(hydroxyethyl)-aminotris(hydroxymethyl)methane buffer (BT) were used as controls. The animals were sacrificed on day 6 and small fragments of seminiferous tubules, approximately 8-10 mm length, were incised and sutured at both ends. They were exposed in vitro to horseradish peroxidase-containing culture medium for 10 minutes. Tissues were then fixed and processed for ultrastructural studies., Results: Controls and Cr(V)-treated group resulted in the uptake of the tracer by Sertoli cells. However, the major finding consisted in the permeability of the BTB only in the Cr(V)-group, as evidenced by the presence of the tracer within the junctions between the neighbouring Sertoli cells., Conclusion: The BTB is disrupted in mice submitted to Cr(V). The permeability of the BTB is a crucial feature to be investigated for the understanding of lesions within the seminiferous tubule.

The synthesis and properties of 3 new ligand-bridged bimetallic complexes, 1(2+), 2(2+), and 3(2+), containing [RuCl([9]aneS(3))](+) metal centers are reported. Each complex was bridged by a different ditopic ligand. 1(2+) is bridged by 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (bptz), while 2(2+) and 3(2+) are bridged by 2,3-bis(2-pyridyl)pyrazine (dpp) and 2,2'-bipyrimidine (bpym), respectively. The Ru([II]) isovalent states of these complexes have been investigated using a variety of techniques. In the case of 3(2+), X-ray crystallography studies show preferential crystallization of an anti form with respect to coordinated chloride ligands (crystal data for [3][Cl(2)].4H(2)O: C(20)H(38)Cl(4)N(4)O(4)Ru(2)S(6), monoclinic, space group P2(1)/a, a = 10.929(14), b = 13.514(17), c = 11.299(16) A, beta = 90.52(1), V = 1669 A(3), Z = 2). UV/vis spectroscopy shows that spectra of these complexes are dominated by intraligand (pi-->pi) and metal-to-ligand Ru(d)-->L(pi) charge transfer transitions. Electrochemical studies reveal that metal-metal interactions are sufficiently intense to generate the Ru(III)/Ru(II) mixed valence [[RuCl([9]aneS(3))(2)](L-L)](3+) state, where L-L = individual bridging ligands. Although the 1(3+), 2(3+), and 3(3+) mixed valence states were EPR silent at room temperature and 77 K, isotropic solution spectra were observed for the electrochemically generated radical cations 1(+), 2(+), and 3(+), with 1(+) displaying well-resolved hyperfine coupling to bridging ligand nitrogens. Using UV/vis/NIR spectroelectrochemistry, we investigated optical properties of the mixed valence complexes. All three showed intervalence charge transfer (IVCT) bands that are much more intense than electrochemical data indicate. Indeed, a comparison of IVCT data for 1(3+) with an analogous structure containing [(NH3)(3)Ru](2+) metal centers shows that the IVCT in the new complex is an order of magnitude more intense. It is concluded that although the new complexes show relatively weak electrostatic interactions, they possess large resonance energies.

Electrospray ionization mass spectrometry (ESIMS) was used to characterize a series of new ruthenium(II) complexes with several nitrogen heterocycles and a common ligand: the crown thioether 1,4,7-trithiacyclononane, [9]aneS(3). ESIMS allows the easy identification of the [Ru(II)Cl([9]aneS(3))Y]X complexes, where Y is a bidentate nitrogen heterocycle and X is Cl(-) or PF(6)(-), through the formation of two diagnostic ions by fragmentation of the common ligand [9]aneS(3). Structures for these ions and mechanisms for their gas-phase formation are proposed based on data from product ion spectra., (Copyright 2001 John Wiley & Sons, Ltd.)

The complexes [Ru([9]aneS(3))(dppz)Cl]Cl 1 and [Ru([12]aneS(4))(dppz)]Cl(2)2 ([9]aneS(3) = 1,4,7- trithiaciclononane and [12]aneS(4) = 1,4,7,10-tetrathiaciclododecane) were synthesised and fully characterised. These complexes belong to a small family of dipyridophenazine complexes with non-polypyridyl ancillary ligands . Interaction studies of these complexes with CT-DNA (UV/Vis titrations, steady-state emission and thermal denaturation) revealed their high affinity for DNA . Intercalation constants determined by UV/Vis titrations are of the same order of magnitude (10(6)) as other dppz metallointercalators, namely [Ru(II)(bpy)(2)dppz]S(2+). Differences between l and2 were identified by steady-state emission and thermal denaturation studies . Emission results are in accordance with structural data, which indicate how geometric distortions and different donor and/or acceptor ligand abilities affect luminescence. The possibility of noncovalent interactions between ancillary ligands and nucleobases by van der Waals contacts and H-bridges is discussed . Furthermore, complex l undergoes aquation under intra-cellular conditions and an equilibrium with the aquated form l' is attained . This behaviour may increase the diversity of available interaction modes.

Cr(VI), the highest oxidation state for chromium, is a carcinogenic and mutagenic agent. In vivo and in vitro Cr(VI) toxic effects are related to its intracellular fate. Once inside the cell it is reduced to stable Cr(III) by cysteine, glutathione and ascorbic acid. Additionally, as Cr(V) and/or Cr(IV) intermediates have been reported in Cr(VI) reactions with biological reductants, chromium damage is thought to originate from these chemical species. This work investigated the morphology of splenic cells after short-term exposure to Cr(VI). A dose of 30 mg of K2CrO4/kg body weight was administered to mice and the effects were studied 24 and 48 h after the injections. Histological results revealed a time-dependency effect of Cr(VI) on splenic cells. Changes included enlargement of the capsule and depletion of the red pulp cells, accompanied by an increase in macrophages, 24 h after injection. Partial restoration of red pulp was noted after 48 h.

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.

Neurotoxins can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45Ca2+ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45Ca2+ influx and they should be useful in studies involving Ca(2+)-dependent processes.

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor.

A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.