Structure-activity relationship study of ProxyPhos chemosensors for the detection of proximal phosphorylation and other phosphate species.

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PPi and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn2+-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn2+ centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn2+-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, Pi and PPi. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PPi, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

Characterization and application studies of ProxyPhos, a chemosensor for the detection of proximally phosphorylated peptides and proteins in aqueous solutions.

Proximal phosphorylation on proteins appears to have functional significance and has been associated with several diseases, including Alzheimer's and cancer. While much remains to be learned about the role of proximal phosphorylation in biological systems, no simple and/or affordable technique is available for its detection. To this end, we have previously developed a ProxyPhos chemosensor, which detects proximally phosphorylated peptides and proteins over mono- and non-phosphorylated motifs in aqueous solutions. In this follow-up work, we performed extensive characterization of peptide and protein ProxyPhos assay conditions to achieve enhanced detection, and further explored the selectivity of ProxyPhos, and its potential off-targets. As a result of characterization studies, selective sensing of proximally phosphorylated over mono-phosphorylated peptides and proteins was achieved. Moreover, studies demonstrated that ProxyPhos was compatible with the detection of all commonly phosphorylated residues (i.e. tyrosine, serine and threonine residues). Under optimized conditions, ProxyPhos efficiently discriminated between peptides derived from the activated (proximally phosphorylated, disease-relevant) and inactive (mono-phosphorylated) forms of JAK2, SYK and MAPK1 kinases. In addition, ProxyPhos can be used to probe phosphatase activity on peptides and proteins via detecting changes in proximal phosphorylation, demonstrating immediate utility of this chemosensing system.

Seminal vesicle cyst and ipsilateral renal agenesis.

A case of seminal vesicle cyst with ipsilateral agenesis is presented. The embryologic events, clinical symptoms, and diagnosis are discussed, emphasizing the value of sonography and computer tomography in identifying the cyst. Surgical resection is the treatment of choice.

Toxoplasma gondii in free-ranging wild small felids from Brazil: molecular detection and genotypic characterization.

Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals.

First isolation and genotyping of Toxoplasma gondii from bats (Mammalia: Chiroptera).

There are currently no reports on the isolation and molecular examination of Toxoplasma gondii from bats. Here, we report the isolation and genotypic characterisation of two T. gondii isolates from bats. A total of 369 bats from different municipalities in São Paulo state, southeastern Brazil, were captured and euthanised, and collected tissues (heart and pectoral muscle) were processed for each bat or in pools of two or three bats and bioassayed in mice (a total of 283 bioassays). Eleven PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) markers were used to genotype positive samples: SAG1, SAG2 (5'-3'SAG2 and alt. SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, CS3 and Apico. The parasite was isolated from two bats from São Paulo city: an insectivorous bat, the velvety free-tailed bat Molossus molossus, and a hematophagous bat, the common vampire bat Desmodus rotundus. Isolates were designated TgBatBr1 and TgBatBr2, respectively. The genotype of the isolate from M. molossus (TgBatBr1) has been previously described in an isolate from a capybara from São Paulo state, and the genotype from the D. rotundus isolate (TgBatBr2) has already been identified in isolates from cats, chickens, capybaras, sheep, a rodent and a common rabbit from different Brazilian states, suggesting that this may be a common T. gondii lineage circulating in some Brazilian regions. Isolation of T. gondii from a hematophagous species is striking. This study reveals that bats can share the same isolates that are found in domesticated and wild terrestrial animals. This is the first report of the isolation and genotyping of T. gondii in chiropterans.

Performance of dairy goats fed whole sugarcane.

Five lactating goats were used in a 5x5 Latin square experiment to determine the effects of feeding whole sugarcane (WSC) on intake, total tract nutrient digestibilities, milk yield and milk composition. Goats were fed diets containing 0, 100, 200, 300, and 400 g kg(-1) WSC and 400, 300, 200, 100, and 0 g kg(-1) tifton hay (TH). Intake of dry matter and neutral detergent fiber (NDF) decreased linearly (p<0.05) as the level of WSC in the diet increased. Total tract nutrient digestibilities were not influenced by WSC inclusion except for the digestibility of NDF which decreased linearly (p<0.05) as the level of WSC in the diet increased. Inclusion of WSC linearly (p<0.05) decreased milk yield without affecting milk composition. It was concluded that WSC had a lower feeding values than TH for lactating goats.

Screening for Toxoplasma gondii in aborted bovine fetuses in Brazil / Pesquisa de Toxoplasma gondii em fetos bovinos abortados no Brasil

The aim of this study was to determine if Toxoplasma gondii was present in aborted bovine fetuses in Brazil. Histopathology of 105 cases with suspected infectious abortion, analyzed during the period from 2006 to 2008 at Centro de Pesquisa e Desenvolvimento de Sanidade Animal of Instituto Biológico, São Paulo, showed 75 cases with indications of abortion due to apicomplexan protozoa. These cases were submitted to PCR for verification of the laboratory diagnosis. Fetal DNA was extracted from central nervous system, heart, liver, muscle, and/or placenta samples to obtain a 529 bp DNA fragment. T. gondii DNA was not detected in any of the bovine fetuses analyzed, suggesting that it may not be a frequent cause of bovine abortion.

Pesquisa de toxoplasma gondii em fetos bovinos abortados no Brasil. O objetivo do presente trabalho foi avaliar a presença de Toxoplasma gondii em fetos bovinos abortados no Brasil. Com base em laudos histopatológicos, de um total de 105 casos com suspeita de aborto infeccioso, recebidos no período de 2006 a 2008, no Centro de Pesquisa e Desenvolvimento de Sanidade Animal do Instituto Biológico, São Paulo, 75 casos foram sugestivos de abortamento por protozoário Apicomplexa e foram submetidos à técnica de PCR para confirmação do diagnóstico laboratorial. O DNA foi extraído a partir de amostras de sistema nervoso central, coração, músculo, fígado e/ou placenta dos fetos para obtenção de um fragmento de DNA de 529pb. Não foi detectada a presença de DNA de T. gondii em qualquer dos fetos bovinos analisados, não sendo este agente infeccioso uma causa frequente de abortamentos.

Granulomatous meningomyeloencephalitis in a calf caused by Mycobacterium bovis / Meningomieloencefalite granulomatosa em um bezerro, causada por Mycobacterium bovis

Bovine tuberculosis is a chronic disease caused by Mycobacterium bovis, characterized by respiratory disorders, emaciation and lymphadenomegaly. Neurological signs associated with granulomatous lesions in the central nervous system are rarely seen. In Brazil, the National Program for Control and Eradication of bovine tuberculosis has begun in 2001, and notification of the disease is compulsory. A case of tuberculous meningomyeloencephalitis is reported in a dairy calf less than one year old presenting neurological signs (irritability, aggressiveness, weakness, dizziness and recumbency) which progressed to death in three days. Brain, spinal cord, and lung tissue samples were examined histopathologically. The spinal cord showed granulomatous meningomyelitis with Langhans giant cells in the meninges and perivascular mononuclear cuffing in the white and gray matter. There were a few granulomas in the cerebral cortex with a central area of necrosis surrounded by mononuclear infiltrate. Mononuclear perivascular cuffing, satellitosis, hyperemia, and severe granulomatous meningitis with Langhans giant cells were observed in the neuropile. Samples of brain were inoculated into Petragnani and Stonebrink media. M. bovis was isolated and identified by PCR with JB21-JB22 primers. Considering the 2,888 samples of bovine CNS sent for surveillance of neurological syndrome and negative for Rabies, this was the only case of tuberculous meningomyeloencephalitis (0,035%) diagnosed from 2004 to 2008 by the Laboratory of Anatomopathology of Instituto Biológico. Although tuberculous meningomyeloencephalitis is not so frequent, it's important to diagnose M. bovis as a causative agent of bovine neurological disorders. In accordance with Brazilian regulations, sanitary measures in the infected herd (test-and-slaughter) should be adopted.

A tuberculose bovina é uma doença crônica causada por Mycobacterium bovis, caracterizada por desordens respiratórias, emaciação e linfadenomegalia. Em raros casos, sinais neurológicos associados com lesões granulomatosas no sistema nervoso central são observados. O Brasil iniciou o Programa Nacional para Controle e Erradicação dessa doença em 2001, tornando a notificação compulsória. Um caso de meningomieloencefalite tuberculosa é relatado em uma bezerra leiteira com um ano de idade, que apresentou sinais neurológicos (irritabilidade e agressividade, fraqueza e tontura, decúbito) que progrediram para morte em três dias. A medula espinhal apresentou meningomielite granulomatosa, com células gigantes tipo Langhans nas meninges e manguito perivascular mononuclear na substância branca e cinzenta. Havia alguns granulomas no córtex cerebral, com área central de necrose circundada por infiltrado mononuclear. No neurópilo observou-se manguito perivascular mononuclear, satelitose, hiperemia e severa meningite granulomatosa com células gigantes tipo Langhans. Amostras de cérebro foram inoculadas nos meios de cultivo Petragnani e Stonebrink e M. bovis foi isolado e identificado por PCR com primers JB21-JB22. Considerando as 2.888 amostras de SNC bovino enviadas para a vigilância epidemiológica de síndrome neurológica e negativas para raiva, este foi o único caso (0,035%) de meningomieloencefalite tuberculosa diagnosticado pelo Laboratório de Anatomia Patológica do Instituto Biológico no período de 2004 a 2008. Embora a meningomieloencefalite tuberculosa não seja tão freqüente, é importante incluir a infecção por M. bovis no diagnóstico diferencial de lesões do sistema nervoso central de bovinos. De acordo com a legislação brasileira, medidas sanitárias no rebanho infectado (teste e abate) devem ser adotadas.

Central alpha2-receptor mechanisms contribute to enhanced renal responses during ketamine-xylazine anesthesia.

We have recently developed an experimental approach to study central opioid control of renal function in anesthetized rats. This model system uses the intravenous infusion of the alpha2-agonist xylazine to enhance basal levels of urine flow rate and urinary sodium excretion in ketamine-anesthetized rats. This study examined the contribution of central and peripheral alpha2-adrenergic receptor mechanisms in mediating the enhanced renal excretory responses produced by xylazine. In ketamine-anesthetized rats, the enhanced levels of urine flow rate and urinary sodium excretion produced by the intravenous infusion of xylazine were reversed by the intravenous bolus injection of the alpha2-adrenoceptor antagonist yohimbine but not by the alpha1-adrenoceptor antagonist terazosin. In separate animals the intracerebroventricular administration of yohimbine only reduced urine flow rate by approximately 50% but did not alter urinary sodium excretion. The decrease in urine flow rate produced by intracerebroventricular yohimbine was reversed by the intravenous injection of a selective V2-vasopressin receptor antagonist. In a separate group of ketamine- and xylazine-anesthetized rats, the bilateral microinjection of yohimbine into the hypothalamic paraventricular nucleus (PVN) also significantly decreased urine flow rate by 54% without altering urinary sodium excretion. The microinjection of the beta-adrenoceptor antagonist propranolol into the PVN did not alter either renal excretory parameter. These results suggest that during intravenous infusion, xylazine increases urine flow rate by activating alpha2-adrenergic receptors in the PVN, which in turn decrease vasopressin release. The ability of alpha-adrenergic mechanisms in the PVN to selectively influence the renal handling of water, but not sodium, may contribute to the reported dissociation of the natriuretic and diuretic responses of alpha2-adrenoceptor agonists.

Clostridium perfringens types A and D associated with enterotoxemia in an 18-month-old goat

Postmortem examination of a Boer buck that died peracutely revealed bowel and liver diffusely congested and edematous. Kidney was apparently edematous. Clostridium perfringens type A was isolated from bowel and type D from kidney. Microscopic examination revealed large areas of necrosis in the renal cortex and medulla (pulpy kidney disease), hyperemia and centrilobular necrosis of the liver, necrosis of the small-intestine wall, pulmonary edema and congestion, intense hyperemia of the cerebellum, hyperemia and edema of the brain.