Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability.

The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.

[Cerebral cryptococcosis and immune reconstitution inflammatory syndrome. Case report]. / Criptococosis cerebral, voriconazol, niveles plasmáticos y síndrome de reconstitución inmune. Reporte de un caso.

We report a 45-year-old male with AIDS who had a Cryptococcus neoformans central nervous system infection. He was treated with amphotericin B deoxycholate subsequently changed to voriconazole due to systemic toxicity of the former. Plasma levels of voriconazole were insufficient with a standard dose (0.7 µg/mL), therefore, the dose was increased thereafter to reach appropriate levels (4.5 µg/mL). Anti-retroviral therapy was started five weeks after voriconazole initiation with non-interacting drugs and he was discharged after a favorable evolution. He was re-admitted three months later due to seizures; a brain magnetic resonance showed new sub-cortical nodules. After excluding alternative causes and demonstrating fungal eradication, an immune reconstitution inflammatory syndrome (IRIS) event was suspected and treated with a short course of steroids. His evolution was satisfactory.

Powdered coconut water as a storage medium to preserve the viability of periodontal ligament cells: a laboratory study.

AIM: To investigate the ability of newly developed powdered coconut water formulas (ACP) with different osmolarities to maintain the viability of periodontal ligament (PDL) cells over time compared with other solutions. METHODOLOGY: Dogs teeth were extracted and stored for two periods, 3 h or 24 h, in the following media: long-shelf life CW (CW), pH-adjusted long-shelf life CW (pH-CW) and powdered CW that was pH and osmolality adjusted (ACP-404-I, 250 mOsm kg-1 H2 O; pH 7.0; ACP-404-II, 372 mOsm kg-1 H2 O; pH 7.0; ACP-404-III, 300 mOsm kg-1 H2 O; pH 7.4). The positive control group (Pc) corresponded to immediate measurement after tooth extraction, and two negative controls (Nc) corresponded to 3 h and 24 h of dry time. PDL cells were extracted, and cell viability analysed by Trypan blue exclusion. Data were analysed statistically using two-way anova followed by the Tukey test and one-way anova followed by the Dunnett test (P < 0.05). RESULTS: At 3 h and 24 h, ACP-404-I had a performance similar to those of ACP-404-II and pH-CW, with significantly higher (P = 0.004) percentages of viable cells than ACP-404-III and CW. The positive control group had a significantly higher (P = 0.002) percentage of viable cells than the negative control groups, CW and ACP-404-III, irrespective of the period evaluated. CONCLUSION: Powdered coconut water formulas, ACP-404-I and ACP-404-II, preserved viability for up to 24 h.

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12 nm protein with interacting hemes.

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c(7)-type domains (A, B, C and D) with homology to triheme cytochromes c(7). In cytochromes c(7) all three hemes are bis-His coordinated, whereas in c(7)-type domains the last heme is His-Met coordinated. The full-length GSU1996 has a 12nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a "nanowire" of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c(7)-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c(7)-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c(7)-type domains from one multiheme protein were compared.

Current practice in regional anaesthesia in South America: An online survey. / Práctica actual de la anestesia regional en América del Sur: encuesta en línea.

OBJECTIVE: A survey was conducted in order to obtain a profile of the practice of regional anesthesia in South America, and determine the limitations of its use. METHODS: After institutional ethics committee approval, a link to an online questionnaire was sent by e-mail to anaesthesiologists in Argentina, Bolivia, Chile, Colombia, Panamá, Paraguay, Perú, and Uruguay. The questionnaire was processed anonymously. RESULTS: A total of 1,260 completed questionnaires were received. The results showed that 97.6% of the anaesthesiologists that responded used regional anaesthesia in clinical practice, 66.9% performed peripheral nerve block (PNB) regularly, 21.6% used continuous PNB techniques, and 4.6% used stimulating catheters. The primary source of training was residency programs. As regards PNB, the most common performed were interscalene (52.3%), axillary (45.1%), femoral (43.2%), and ankle block (43%). As regards the localisation technique employed, 16% used paraesthesia, 44.2% used a peripheral nerve stimulator, and 18.1% ultrasound guidance. CONCLUSIONS: Regional anaesthesia and PNB are commonly used among South American anaesthesiologists. Considering that each country has its own profile for use, this profile should guide training in clinical practice, especially in residency programs.

Homotropic and heterotropic cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris.

The thermodynamic parameters which govern the homotropic (e-/e-) and heterotropic (e-/H+) cooperativity in the tetrahaem cytochrome c3 isolated from Desulfovibrio vulgaris (Hildenborough) were determined, using the paramagnetic shifts of haem methyl groups in the NMR spectra of intermediate oxidized states at different pH levels. A model is put forward to explain how the network of positive and negative cooperativities between the four haems and acid/base group(s) enables the protein to achieve a proton-assisted 2e- step.

Carbon-13 NMR studies of the influence of axial ligand orientation on haem electronic structure.

Three-quarters of the carbon-13 resonances of nuclei attached to the four haems of Desulfovibrio vulgaris ferricytochrome c3 are assigned. Preliminary analysis of their Fermi contact interactions shows that the shifts are directly related to the orientation of both of the axial histidine ligands in each case and the approach can therefore be used to obtain structural information in other cytochromes with bis-histidinyl coordination. The implications for the control of redox potential in cytochromes are discussed.

Office paper platform for bioelectrochromic detection of electrochemically active bacteria using tungsten trioxide nanoprobes.

Electrochemically active bacteria (EAB) have the capability to transfer electrons to cell exterior, a feature that is currently explored for important applications in bioremediation and biotechnology fields. However, the number of isolated and characterized EAB species is still very limited regarding their abundance in nature. Colorimetric detection has emerged recently as an attractive mean for fast identification and characterization of analytes based on the use of electrochromic materials. In this work, WO3 nanoparticles were synthesized by microwave assisted hydrothermal synthesis and used to impregnate non-treated regular office paper substrates. This allowed the production of a paper-based colorimetric sensor able to detect EAB in a simple, rapid, reliable, inexpensive and eco-friendly method. The developed platform was then tested with Geobacter sulfurreducens, as a proof of concept. G. sulfurreducens cells were detected at latent phase with an RGB ratio of 1.10 ± 0.04, and a response time of two hours.

Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR.

Using 2D-NMR the four haems of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 within the X-ray structure were fully cross assigned according to their redox potential. The strategy used was based on a complete network of chemical exchange connectivities between the NMR signals obtained for all oxidation levels to the corresponding ones in the fully reduced spectrum [1992, Eur. J. Biochem., in press]. This unequivocal cross-assignment disagrees with earlier results obtained for the similar protein from Desulfovibrio vulgaris (Miyazaki F.).

NMR structure of the haem core of a novel tetrahaem cytochrome isolated from Shewanella frigidimarina: identification of the haem-specific axial ligands and order of oxidation.

The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.

To what extent do premenstrual and interictal dysphoric disorder overlap? Significance for therapy.

BACKGROUND: Premenstrual dysphoria has shown a significant relationship to epilepsy, and its symptoms correspond to those of the interictal dysphoric disorder. The established treatment for interictal dysphoric disorder is explored as an effective treatment for premenstrual dysphoria. METHODS: Women with premenstrual dysphoria (the majority with epilepsy) were evaluated and treated systematically. RESULTS: Premenstrual disorder responded well to the combination of antidepressant and antiepileptic medication. CONCLUSION: Concordance of symptomatology between interictal and premenstrual dysphoric disorder may extend to treatment. LIMITATIONS: The treatment needs to be evaluated in a large series of women without epilepsy. CLINICAL RELEVANCE: A novel understanding and treatment of premenstrual dysphoria is suggested.

Comparação da qualidade seminal de caprinos das raças Canindé e Alpina Britânica no Nordeste brasileiro / Comparison of the seminal quality of Canindé and British Alpine goats in the Brazilian Northeast

O estudo objetivou avaliar a qualidade seminal de caprinos das raças Canindé (autóctone) e Alpina Britânica (exótica) no Nordeste brasileiro. O experimento foi realizado nos períodos de julho a setembro dos anos de 2015 e 2016. As coletas seminais foram realizadas com auxílio de vagina artificial, de machos das raças Canindé (n = 4) e Alpina Britânica (n = 7). Após a coleta, o sêmen foi avaliado quanto a: volume (ml), concentração (sptz/ml), motilidade (%) e vigor (1-5). Em seguida, diluído em ACP-101c e criopreservado em máquina TK3000TM. Posteriormente, foram analisados os parâmetros cinéticos, através do software SCA®, e a atividade mitocondrial. No sêmen fresco, as duas raças apresentaram valores de motilidade e concentração dentro do preconizado para a espécie. Entretanto, os valores de volume e circunferência escrotal foram superiores na raça Alpina Britânica (0,73 ml ± 0,32; 27,35 cm ± 2,09) do que na raça Canindé (0,36 ml ± 0,07; 23,25 cm ± 0,95) (p < 0,05). Já no sêmen descongelado, os parâmetros motilidade total - MT (36,96% ± 11,16 vs. 20,50% ± 14,15), motilidade progressiva - MP (26,67% ± 11,13 vs. 11,29% ± 9,42), vigor (2,91 ± 0,90 vs. 1,88 ± 0,67), velocidade curvilinear - VCL (78,12 µm/s ± 12,41 vs. 59,28 µm/s ± 15,57), velocidade linear - VSL (49,23 µm/s ± 9,87 vs. 29,9 µm/s ± 9,48), velocidade média da trajetória - VAP (66,08 µm/s ± 12,77 vs. 45,70 µm/s ± 12,20), linearidade - LIN (62,97% ± 6,96 vs. 49,54%±9,50), retilinearidade - STR (74,63% ± 6,44 vs. 65,77% ± 8,92), e oscilação - WOB (84,32% ± 5,94 vs. 74,42% ± 7,31) foram superiores para a raça Canindé em detrimento à Alpina Britânica (p < 0,05). Portanto, o sêmen fresco das duas raças podem ser utilizados em biotécnicas reprodutivas. Já o sêmen pós-descongelação da raça Canindé apresentou melhor qualidade, provavelmente pelos animais estarem mais adaptados às condições adversas da região Nordeste, sendo recomendado para programas de inseminação artificial.(AU)

The objective of this study was to evaluate the sperm quality of Canindé (native) and British Alpine (exotic) goats in the Northeast of Brazil. The experiment was carried out from July to September of the years 2015 and 2016. Sperm collections of Canindé (n = 4) and British Alpine (n = 7) males were performed using artificial vagina. After collection, the sperm was evaluated for volume (ml), concentration (sptz/ml), motility (%), and vigor (1-5). Then diluted in ACP-101c and cryopreserved in TK3000TM machine. Subsequently, kinetic parameters were analyzed through SCA TM software and mitochondrial activity. In fresh sperm, the two races presented values of motility and concentration within the recommended for the specie. However, volume and scrotal circumference values were higher in the British Alpine breed (0.73 mL ± 0.32; 27.35 cm ± 2.09) than in the Canindé breed (0.36 mL ± 0.07; 23 , 25 cm ± 0.95) (p <0.05). In the thawed sperm, the parameters total motility - TM (36.96% ± 11.16 vs. 20.50% ± 14.15), progressive motility - PM (26.67% ± 11.13 vs. 11.29 % ± 9.42), vigor (2.91 ± 0.90 vs. 1.88 ± 0.67), curvilinear velocity - VCL (78.12 µm/s ± 12.41 vs. 59.28 µm/s ± 15.57), linear velocity - VSL (49.23 µm/s ± 9.87 vs. 29.9 µm/s ± 9.48), mean velocity of the trajectory - VAP (66.08 µm/s ± 12.77 vs. 45.70 µm/s ± 12.20), linearity - LIN (62.97% ± 6.96 vs. 49.54% ± 9.50), rectilinearity - STR (74.63% ± 6.44 vs. 65.77% ± 8.92), and oscillation -WOB (84.32% ± 5.94 vs. 74.42% ± 7.31) were higher for Canindé breed than for British Alpine ( p < 0.05). Therefore, fresh sperm from both breeds can be used in reproductive biotechniques. On the other hand, the post-thawed sperm of the Canindé breed showed better quality, probably because the animals were more adapted to the adverse conditions of the Northeast region and are recommended for artificial insemination programs.(AU)

Criptococosis cerebral, voriconazol, niveles plasmáticos y síndrome de reconstitución inmune: reporte de un caso / Cerebral cryptococcosis and immune reconstitution inflammatory syndrome: case report

We report a 45-year-old male with AIDS who had a Cryptococcus neoformans central nervous system infection. He was treated with amphotericin B deoxycholate subsequently changed to voriconazole due to systemic toxicity of the former. Plasma levels of voriconazole were insufficient with a standard dose (0.7 μg/mL), therefore, the dose was increased thereafter to reach appropriate levels (4.5 μg/mL). Anti-retroviral therapy was started five weeks after voriconazole initiation with non-interacting drugs and he was discharged after a favorable evolution. He was re-admitted three months later due to seizures; a brain magnetic resonance showed new sub-cortical nodules. After excluding alternative causes and demonstrating fungal eradication, an immune reconstitution inflammatory syndrome (IRIS) event was suspected and treated with a short course of steroids. His evolution was satisfactory.

Influencia del entrenamiento visual en el control postural de pacientes con accidente cerebrovascular crónico: estudio piloto aleatorizado controlado / Influence of visual training on the postural control of patients with chronic stroke: A randomised-controlled pilot trial

Antecedentes y objetivos: El accidente cerebrovascular supone una de las primeras causas de discapacidad física de la población adulta. Los afectados pueden presentar alteraciones motoras y sensitivas, así como alteración del control postural o equilibrio y, consecuentemente, un elevado riesgo de caída. Conociendo la relación entre el control postural y los sistemas sensoriales somatosensorial, vestibular y visual, este estudio se centra en la influencia del sistema visual. El objetivo es averiguar la efectividad del entrenamiento visual en la rehabilitación del equilibrio en pacientes con accidente cerebrovascular crónico. Material y métodos: Se ha realizado un estudio piloto aleatorizado simple ciego (estudio experimental, longitudinal y prospectivo). El total de participantes fue de 12 (accidente cerebrovascular > 1 año), aleatoriamente distribuidos en 2 grupos. Los grupos realizaron terapia del equilibrio orientada a tarea (30 min) y, adicionalmente, el grupo experimental realizó entrenamiento visual (15 min). Ambos grupos completaron 5 sesiones en 3 semanas. El control postural se ha valorado con la Escala de Equilibrio de Berg, prueba Timed Up and Go y monitorización del centro de presión. Resultados: Relativo a todas las variables, se ha observado mayor rango de evolución positiva en el grupo experimental en comparación con el grupo de control. Sin embargo, los únicos resultados estadísticamente significativos fueron relativos a la simetría del centro de presión corporal (p = 0,05 y p = 0,01). Conclusiones: El entrenamiento visual parece ser útil en la rehabilitación del control postural de individuos con accidente cerebrovascular crónico. Son necesarias futuras investigaciones para confirmar su efectividad

Background and objectives: Stroke is one of the first causes of physical disability in the adult population. Those affected may have motor and sensory disturbances, as well as altered postural control, and consequently high risk of falls. Knowing the relationship between postural control and somatosensory, vestibular and visual systems, this study focuses on the influence of the visual system. The aim of this study is to determine whether visual training increases the effectiveness of balance rehabilitation in patients with chronic stroke. Material and methods: A single blind randomised pilot study (prospective longitudinal experimental study) was conducted on a total of 12 participants (Stroke > 1 year) randomly distributed into two groups. The groups performed task-oriented balance therapy (30 minutes), in addition the experimental group performed visual training (15 minutes) in the same session. Both groups completed 5 sessions in 3 weeks. Postural control was assessed using the Berg Balance Scale, Timed Up and Go test, and pressure centre monitoring. Results: A greater range of positive progress was observed in all variables in the experimental group compared to the control group. However, the only statistically significant results were related to the symmetry of the centre of body pressure (P = .05 and P = .01). Conclusions: Visual training seems to be useful in postural control rehabilitation of individuals with chronic stroke. Further research is needed to confirm its effectiveness

Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: implications for signal transduction.

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (-156 mV and -251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.

Práctica actual de la anestesia regional en América del Sur: encuesta en línea / Current practice in regional anaesthesia in South America: an online survey

Objetivo. El objetivo de este estudio fue describir el perfil de utilización y las limitaciones del uso de la anestesia regional en países de América del Sur. Métodos. Tras la aprobación del comité de ética institucional, se envió un enlace a una encuesta en línea por e-mail a anestesiólogos de Argentina, Bolivia, Chile, Colombia, Panamá, Paraguay, Perú y Uruguay. La encuesta se procesó de forma anónima. Resultados. Se recibieron un total de 1.260 cuestionarios completos. El 97,6% de los anestesiólogos encuestados refiere utilizar anestesia regional en su práctica clínica habitual, el 66,9% realiza bloqueos de nervio periférico (BNP) con regularidad, el 21,6% usa técnicas de BNP continuo y un 4,6% usa catéteres estimulantes. La principal fuente de formación fueron los programas de residencia. En cuanto a los BNP más usados, los más comunes son el interescalénico (52,3%), el axilar (45,1%), el femoral (43,2%) y el de tobillo (43%). En cuanto a la técnica de neurolocalización, el 16% utiliza parestesia, el 44,2% estimulador de nervio periférico y un 18,1% ultrasonografía. Conclusiones. La anestesia regional y los BNP son de uso común entre los anestesiólogos de América del Sur. Cada país tiene su propio perfil de utilización y sus propias limitaciones para su uso. Dicho perfil debe ser tomado en cuenta a la hora de planificar las distintas formas de entrenamiento, en especial los programas de residencia (AU)

Objective. A survey was conducted in order to obtain a profile of the practice of regional anesthesia in South America, and determine the limitations of its use. Methods. After institutional ethics committee approval, a link to an online questionnaire was sent by e-mail to anaesthesiologists in Argentina, Bolivia, Chile, Colombia, Panamá, Paraguay, Perú, and Uruguay. The questionnaire was processed anonymously. Results. A total of 1,260 completed questionnaires were received. The results showed that 97.6% of the anaesthesiologists that responded used regional anaesthesia in clinical practice, 66.9% performed peripheral nerve block (PNB) regularly, 21.6% used continuous PNB techniques, and 4.6% used stimulating catheters. The primary source of training was residency programs. As regards PNB, the most common performed were interscalene (52.3%), axillary (45.1%), femoral (43.2%), and ankle block (43%). As regards the localisation technique employed, 16% used paraesthesia, 44.2% used a peripheral nerve stimulator, and 18.1% ultrasound guidance. Conclusions. Regional anaesthesia and PNB are commonly used among South American anaesthesiologists. Considering that each country has its own profile for use, this profile should guide training in clinical practice, especially in residency programs (AU)

Avaliação de espermatozoides caprinos congelados em meio à base de água de coco em pó (ACP-101®) ou TRIS / Evaluation of goat spermatozoa frozen in media based on powder coconut water media based (ACP-101®) or TRIS

Compararam-se as características cinéticas e morfológicas de espermatozoides caprinos congelados nos meios à base de ACP-101® e TRIS. Os diluentes utilizados foram: ACP-101® (+ 2,5 por cento gema ovo + 7 por cento glicerol) e TRIS (+ 20 por cento gema ovo + 6,8 por cento glicerol). Quarenta e oito ejaculados de quatro bodes foram coletados, avaliados, divididos em duas alíquotas e diluídos nos meios ACP-101® e TRIS, respectivamente, posteriormente congelados e, após 30 dias, descongelados. A avaliação da motilidade espermática por computador foi realizada aos 5, 60 e 120 minutos pós-descongelação. As características de motilidade espermática analisadas foram: motilidade total (MT) ( por cento) e progressiva (MP) ( por cento), velocidades média do trajeto do espermatozoide (VAP) (µm/s) e linear (VSL) (µm/s) e população de espermatozoides rápidos (ER) ( por cento). As avaliações de morfologia espermática quantificaram a porcentagem de espermatozoides normais (N) e as alterações da cabeça (AC), da peça intermediária (API) e do flagelo (AF), aos cinco e 120 minutos pós-descongelação. O diluente TRIS apresentou resultados cinéticos mais elevados que o ACP-101® aos 60 e 120 minutos pós-descongelação. As AC aos 120 minutos pós-descongelação foram mais altas nos espermatozoides congelados em ACP-101®. Conclui-se que o diluente TRIS promoveu maior viabilidade in vitro dos espermatozoides caprinos pós-descongelação.

The aim of the work was to compare kinetic and morphologic characteristics of goat sperm frozen in diluent media based on ACP-101® and TRIS. The employed diluents were: ACP-101® (+ 2.5 percent egg yolk + 7 percent glycerol) and TRIS (+ 20 percent egg yolk + 6.8 percent glycerol). Forty eight ejaculates from four bucks were collected, assessed and divided into two aliquots and diluted into the experimental treatments ACP-101® and TRIS. The samples were frozen and after 30 days, thawed. The computer assisted spermatic motility evaluations were placed into 5, 60 and 120 minutes post-thawing. The motion parameters assessed were: total motility (MT) ( percent), progressive motility (MP) ( percent), average path velocity (VAP) (µm/s), straight linear velocity (VSL) (µm/s), and population of rapid spermatozoa (ER) ( percent). The morphologic parameters: normal spermatozoa (N), head alteration (AC), intermediary piece alteration (API), tail alteration (AF) were evaluated at 5 and 120 min post-thawing. The media based on TRIS showed kinetic results significantly superior to ACP-101® at 60 and 120min. After 120min post-thawing the AC was higher in frozen sperm in media based on ACP-101®. The TRIS media promoted better goat spermatozoa in vitro viability post-thawing.

Use of paramagnetic NMR probes for structural analysis in cytochrome c3 from Desulfovibrio vulgaris.

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) (c3DvH) is determined by means of a novel procedure. In this method the 13C chemical shifts of the nuclei directly bound to the haems are used to determine the in-plane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of e(g) symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. V. (1995) Biochim. Biophys. Acta 1246, 24-281. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c3DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c3DvH haem substituents were fitted considering both the pseudocontact and contact shifts of each haem simultaneously. The dipolar shifts calculated by this method were tested against the observed dipolar shifts for some amino acid residues strategically placed in the protein and also for the haem propionate groups. The effect of considering the calculated dipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at different pH values was also analysed. The several tests applied to the calculated dipolar shifts have shown that the method is extremely useful for predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structures of paramagnetic proteins and hence to probe subtle structural rearrangements around the haem pocket.