Enxerto pediculado de omento maior como adjuvante no tratamento de osteomielite de tíbia em cão - relato de caso / [Greater omentum pedicle flap as adjunctive treatment of tibial osteomyelitis in a dog - case report]

A osteomielite é um desafio terapêutico em ortopedia, capaz de retardar ou mesmo impedir a consolidação óssea. O omento, há anos, tem sido empregado como alternativa em diferentes procedimentos cirúrgicos, por sua capacidade, entre outras, de angiogênese, sendo aplicado na ortopedia veterinária quando há o risco de não união óssea. Neste caso, um cão Fila Brasileiro foi submetido à realização de enxerto com retalho pediculado de omento maior, após osteomielite resistente presente em osteossíntese de fratura múltipla de tíbia aberta grau II. Durante 16 dias, manteve-se a comunicação do retalho, mas, diante do risco de peritonite, o pedículo foi seccionado. Numa sequência de intervenções cirúrgicas, após 89 dias, houve cicatrização óssea e remissão da osteomielite, mesmo na presença de bactérias multirresistentes. Neste relato, o omento foi efetivo como terapia adjuvante no tratamento da osteomielite e garantiu o retorno da função do membro.(AU)

Osteomyelitis is a therapeutic challenge in orthopedics, capable of delaying or even preventing bone healing. The omentum has been used in different surgical procedures as an alternative for its capacity, among others, of angiogenesis, being applied in veterinary orthopedics, when there is a risk of non-union of bone. In this case, a Brazilian row dog was submitted to grafting with pedicle flap of greater omentum, after resistant osteomyelitis present in open fracture osteosynthesis of open tibia grade II. For 16 days the communication of the flap was maintained, but at the risk of peritonitis, the pedicle was sectioned. In a sequence of surgical interventions, after 89 days, there was bone healing and remission of osteomyelitis, even in the presence of multi-resistant bacteria. In this report, the omentum was effective as adjuvant therapy in the treatment of osteomyelitis and guaranteed the return of limb function.(AU)

Precession-band variance missing from East Asian monsoon runoff.

Speleothem CaCO3 δ18O is a commonly employed paleomonsoon proxy. However, inferring local rainfall amount from speleothem δ18O can be complicated due to changing source water δ18O, temperature effects, and rainout over the moisture transport path. These complications are addressed using δ18O of planktonic foraminiferal CaCO3, offshore from the Yangtze River Valley (YRV). The advantage is that the effects of global seawater δ18O and local temperature changes can be quantitatively removed, yielding a record of local seawater δ18O, a proxy that responds primarily to dilution by local precipitation and runoff. Whereas YRV speleothem δ18O is dominated by precession-band (23 ky) cyclicity, local seawater δ18O is dominated by eccentricity (100 ky) and obliquity (41 ky) cycles, with almost no precession-scale variance. These results, consistent with records outside the YRV, suggest that East Asian monsoon rainfall is more sensitive to greenhouse gas and high-latitude ice sheet forcing than to direct insolation forcing.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells.

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 µM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Survivin, a target to modulate the radiosensitivity of Ewing's sarcoma.

BACKGROUND AND PURPOSE: Radiotherapy constitutes an essential element in the multimodal therapy of Ewing's sarcoma. Compared to other sarcomas, Ewing tumors normally show a good response to radiotherapy. However, there are consistently tumors with a radioresistant phenotype, and the underlying mechanisms are not known in detail. Here we investigated the association between survivin protein expression and the radiosensitivity of Ewing's sarcoma in vitro. MATERIAL AND METHODS: An siRNA-based knockdown approach was used to investigate the influence of survivin expression on cell proliferation, double-strand break (DSB) induction and repair, apoptosis and colony-forming ability in four Ewing's sarcoma cell lines with and without irradiation. RESULTS: Survivin protein and mRNA were upregulated in all cell lines tested in a dose-dependent manner. As a result of survivin knockdown, STA-ET-1 cells showed reduced cell proliferation, an increased number of radiation-induced DSBs, and reduced repair. Apoptosis was increased by knockdown alone and increased further in combination with irradiation. Colony formation was significantly reduced by survivin knockdown in combination with irradiation. CONCLUSION: Survivin is a radiation-inducible protein in Ewing's sarcoma and its down-regulation sensitizes cells toward irradiation. Survivin knockdown in combination with radiation inhibits cell proliferation, repair, and colony formation significantly and increases apoptosis more than each single treatment alone. This might open new perspectives in the radiation treatment of Ewing's sarcoma.

Desinserção proximal do músculo sartório na correção da luxação patelar medial graus III e IV, em cães / Sartorius muscle proximal release to repair grades III and IV medial patellar luxation in dogs

Six surgical corrections were performed with techniques of bone and soft tissue reconstruction, according to the individual presentation of each patient, in four Toy breed dogs, which had grades III and IV luxation, two of them bilaterally operated. During the recovery excessive medial tension associated to the Sartorius muscle was detected, which motivated its release at the iliac insertion. The animals had a functional recovery of the limb after 15 days, and complications or loss of function related to the release of the Sartorius muscle were not observed even after six months. It is believed that the proximal disinsertion of this muscle can be necessary and effective in some patients as a complementary technique to release medial tension in dogs with grades III and IV medial patellar luxation.

Denervação dos esfíncteres uretrais, via celiotomia pré-púbica sem necessidade de anestesia, em três gatos paraplégicos

O artigo não apresenta resumo.

Contribuição do distensor ósseo nos métodos fechados de redução de fraturas rádio-ulnares e tíbio-fibulares e de fixação dos fragmentos com pinos percutaneamente transfixados em cães e gatos

O artigo não apresenta resumo.

Avaliação pós-cirúrgica de fraturas rádio-ulnares, reduzidas pelo método fechado e estabilizadas com pinos percutaneamente transfixados, submetidas ou não a injeção local de medula óssea autógena em cães

O artigo não apresenta resumo.

Requirements of team effectiveness in neurosurgical rehabilitation.

The purpose of this article is to stress the importance of working with interdisciplinary teams in neurorehabilitation and describe requirements of team effectiveness. It is not sufficient to focus only on different impairments associated with brain injury and offer individuals a variety of therapy. The essential aspect in neurorehabilitation is the integration of disciplines and consistent goal setting to regard individual patient's needs. Interdisciplinary teams benefit from a leader qualified for neuroscience, neurorehabilitation, clinical neuropsychology and psychotherapy. A good structural organization of the team, notice of basic communication rules, understanding typical group dynamics and stressors of interdisciplinary teams, conflict management and a definite decision making increase productive interdisciplinary working and enable the team to continue to mature. Further empirical research is necessary to support the effectiveness of interdisciplinary teams as an important variable in the evaluation of rehabilitation outcome and quality control.

Gastric wall elasticity assessed by dynamic holographic endoscopy: ex vivo investigations in the porcine stomach.

BACKGROUND: Holographic interferometry is based on the superimposition of the holograms of different motional states of an object on a single holographic storage medium. It has been used to detect structural changes in prosthetic heart valves. The combination of holographic interferometry and endoscopic imaging were applied to assess disturbances of porcine stomach wall elasticity. METHODS: By connecting an electronic speckle pattern interferometry (ESPI) camera system (light source: continuous wave argon-ion laser, lambda = 514.5 nm) to different types of endoscopes, ex vivo experiments were performed on porcine stomachs to detect areas characterized by altered tissue elasticity. With linkage of the endoscopic ESPI camera complex to a fast image processing system, the method of double pulse exposure image subtraction was applied at a video frame rate of 12.5 Hz. RESULTS: The speckle correlation patterns resulting from gentle gastric wall deformation were analyzed in a series of experiments in 16 porcine stomachs. Interferograms of gastric wall areas without structural abnormalities exhibited concentric fringes, whereas fringe patterns corresponding to areas of reduced tissue elasticity were characterized by parallel lines. CONCLUSION: Applying the nondestructive method of dynamic holographic endoscopy, abnormalities of the gastric wall leading to diminished tissue elasticity can be distinguished reliably from surrounding healthy tissue.

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal.

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.

N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria.

The expression of mutants with deletions in the N-terminal signal-anchor sequence of cytochrome P450 2C2 and His-tag fusions was examined in Escherichia coli to determine the influence of N-terminal sequences on expression of the protein. Two mutants predicted to be translocated across the membrane inhibited bacterial growth. In other mutants, deletion of the N-terminal transmembrane domain (residues 2-20) reduced expression of functional P450 by about 75% and further deletion of the following linker sequence (residues 21-27) resulted in a modest further decrease. Expression of the mutant with residues 2-27 deleted contrasts with the lack of expression of functional protein if only the linker was deleted, which suggests that the linker sequence is critical for expression only if the protein is inserted into the membrane by the transmembrane domain. Fusion proteins of green fluorescent protein with full-length P450 2C2 and 2C2(Delta2-20) were predominantly membrane-associated in vivo as determined by fluorescence microscopy. Subcellular fractionation of bacteria expressing these proteins and extraction of the proteins from the membrane by high salt or alkaline buffer demonstrated that P450 2C2 was an integral membrane protein while 2C2(Delta2-20) was a peripheral membrane protein that associated with the membrane mainly by hydrophobic interactions. Residues 1-27 of P450 2C2 fused to green fluorescent protein resulted in a redistribution of fluorescence from cytosol to membrane, which, with the deletion studies, indicates that the P450 signal-anchor is both necessary and sufficient for normal membrane targeting and is the sole transmembrane domain of cytochrome P450 2C2 in bacteria. Addition of a His-tag at the N-terminus completely restored wild-type expression levels to the 2C2(Delta2-20) mutants in bacteria. In insect cells, functional 2C2(Delta2-20) was not expressed but an N-terminal His-tag also restored full expression. The increase in expression may be related to decreased association with the membrane mediated by the His-tag.

Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2.

In the final stages of genetic recombination, Holliday junction resolving enzymes transform the four-way DNA intermediate into two duplex DNA molecules by introducing pairs of staggered nicks flanking the junction. This fundamental process is apparently common to cells from all three domains of life. Two cellular resolving enzymes from extremely thermophilic representatives of both kingdoms of the domain Archaea, the euryarchaeon Pyrococcus furiosus and the crenarchaeon Sulfolobus solfataricus, have been described recently. Here we report for the first time the isolation, purification and characterization of Holliday junction cleaving enzymes (Hjc) from two archaeal viruses. Both viruses, SIRV1 and SIRV2, infect Sulfolobus islandicus. Their Hjcs both consist of 121 amino acid residues (aa) differing only by 18 aa. Both proteins bind selectively to synthetic Holliday-structure analogues with an apparent dissociation constant of 25 nM. In the presence of Mg(2+) the enzymes produce identical cleavage patterns near the junction. While S. islandicus shows optimal growth at about 80 degrees C, the nucleolytic activities of recombinant SIRV2 Hjc was highest between 45 degrees C and 70 degrees C. Based on their specificity for four-way DNA structures the enzymes may play a general role in genetic recombination, DNA repair and the resolution of replicative intermediates.

Functional analysis of the phenobarbital-responsive unit in rat CYP2B2.

An 163-bp fragment of the rat cytochrome P450 gene, CYP2B2 has been shown to contain sequences that mediate phenobarbital (PB) responsiveness of this gene. In studies on this rat gene and the orthologous mouse gene, Cyp2b10, the minimal fragment required for near full PB responsiveness has varied from about 50 to 80 bp depending on the gene used and the number of copies of the PB responsive sequences assessed. Since there is a single copy of the CYP genes in the genome, we have evaluated deletion and block mutations across an 84-bp region of the PB responsive unit (PBRU), by in situ transfection in rat liver using single copies of the PBRU sequences. From the 5' end, deletions to -2243 retained more than 50% responsiveness to PB compared to the 163-bp fragment. The fragment -2237 to -2155 retained less than 20% responsiveness even though it contained the nuclear receptor (NR)-1, NR-2, and NF-1 motifs which are present in the core of the PBRU. From the 3' end, deletions from -2170 to -2194 eliminated PB responsiveness indicating that the 74-bp sequence from -2243 to -2170 is able to mediate full PB responsiveness. Block mutations within the NR-1 and NF-1 regions reduced responsiveness most dramatically, but did not abolish it, and mutations 3' of the NF-1 site modestly reduced responsiveness. Protein binding was not affected by mutations in the NR-1 region as assessed by DNase I footprinting in vitro but mutations within the NR-2 region reduced binding to the NF-1 site. Mutations of the 5' half or the 3' half of the bipartite NF-1 site, resulted in loss of protection of the NF-1 site and new footprints to the 3' or 5' side, respectively, of the NF-1 site. These results indicate that sequences in addition to the NR-1 and -2 and the NF-1 sites are required for full responsiveness to PB and suggest that proteins which bind to these sites may interact.

Conformational flexibility in T4 endonuclease VII revealed by crystallography: implications for substrate binding and cleavage.

The structure of the N62D mutant of the junction-resolving endonuclease VII (EndoVII) from phage T4 has been refined at 1.3 A, and a second wild-type crystal form solved and refined at 2.8 A resolution. Comparison of the mutant with the wild-type protein structure in two different crystal environments reveals considerable conformational flexibility at the dimer level affecting the substrate-binding cleft, the dimerization interface and the orientation of the C-terminal domains. The opening of the DNA-binding cleft, the orientation of the C-terminal domains relative to the central dimerization domain as well as the relative positioning of helices in the dimerization interface appear to be sensitive to the crystal packing environment. The highly unexpected rearrangement within the extended hydrophobic interface does change the contact surface area but keeps the number of hydrophobic contacts about the same and will therefore not require significant energy input. The conformational flexibility most likely is of functional significance for the broad substrate specificity of EndoVII. Binding of sulphate ions in the mutant structure and their positions relative to the active-site metal ions and residues known to be essential for catalysis allows us to propose a possible catalytic mechanism. A comparison with the active-site geometries of other magnesium-dependent nucleases, among them the homing endonuclease I-PpoI and Serratia endonuclease, shows common features, suggesting related catalytic mechanisms.

Analyses of spontaneous mutations of cloned gene 49 of phage T4.

Holliday structure resolving enzyme endonuclease VII (endo VII) of phage T4 is highly toxic for E. coli when expressed outside of the phage infection environment. As a consequence, plasmids with a mutated gene 49, the gene which encodes for endo VII, can be easily isolated and characterised. We have isolated and characterised 400 survivors from independent transformations with a plasmid carrying gene 49 under the control of the T7 promoter. The majority had mutated gene 49 by IS10 insertions which almost exclusively mapped to a distinct site. When this site was mutated other insertion sites were observed as well as an increase in other mutational events including large deletions. Neither of the observed insertion sites mapped matched the consensus IS10 sequence completely. Additionally when the level of expression of gene 49 was altered the distribution of mutations was changed suggesting that other elements apart from the target sequence are necessary for determining IS10 insertion. The expression of gene 49 in E. coli provides a particularly useful tool for the analysis of mutational events.

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU.

Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.

Endoplasmic reticulum retention determinants in the transmembrane and linker domains of cytochrome P450 2C1.

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.

Cytochromes P450 2C1/2 and P450 2E1 are retained in the endoplasmic reticulum membrane by different mechanisms.

Cytochrome P450 (P450) 2C1/2 contains redundant endoplasmic reticulum (ER) retention signals and is excluded from the recycling pathway. Other P450s, such as P450 2E1, have been detected in the plasma membrane and Golgi apparatus. To examine whether the mechanisms of ER retention might differ for P450 2C1/2 and P450 2E1, chimeras of green flourescent protein and the full-length proteins, N-terminal signal/anchor sequences, or the cytoplasmic catalytic domains from these proteins have been expressed in COS1 cells. Chimeras with either the N-terminal signal/anchor sequence or the cytoplasmic domain of P450 2C1/2 were retained in the ER and the distribution was not altered by treatment with nocodazole. A chimera with full-length P450 2E1 was located in the ER, but in contrast to P450 2C1/2, treatment with nocodazole resulted in redistribution to a vesicular pattern, which suggested that this protein was retained in the ER by a retrieval mechanism. In support of this possibility, the P450 2E1 chimera, but not the P450 2C1/2 chimera, was included in transport vesicles generated in an in vitro budding assay. A chimera with only the N-terminal signal/anchor sequence of P450 2E1 fused to green fluorescent protein was located in the ER and nocodazole treatment altered its distribution, whereas a chimera with only the cytoplasmic domain of P450 2E1 was not efficiently retained in the ER and accumulated primarily in the Golgi region. These results demonstrate that the mechanisms for retention in the ER of two closely related members of the P450 superfamily are different and that the N-terminal signal/anchor sequence contains the dominant retention signal.