Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Venom Complexity in a Pitviper Produced by Facultative Parthenogenesis.

Facultative parthenogenesis (FP) is asexual reproduction in plant and animal species that would otherwise reproduce sexually. This process in vertebrates typically results from automictic development (likely terminal fusion) and is phylogenetically widespread. In squamate reptiles and chondrichthyan fishes, FP has been reported to occur in nature and can result in the production of reproductively viable offspring; suggesting that it is of ecological and evolutionary significance. However, terminal fusion automixis is believed to result in near genome-wide reductions in heterozygosity; thus, FP seems likely to affect key phenotypic characters, yet this remains almost completely unstudied. Snake venom is a complex phenotypic character primarily used to subjugate prey and is thus tightly linked to individual fitness. Surprisingly, the composition and function of venom produced by a parthenogenetic pitviper exhibits a high degree of similarity to that of its mother and conspecifics from the same population. Therefore, the apparent loss of allelic diversity caused by FP appears unlikely to have a significant impact on the prey-capturing ability of this snake. Accordingly, the pitviper offspring produced by FP retained complex phenotypic characteristics associated with fitness. This result reinforces the potential ecological and evolutionary importance of FP and questions our understanding of the inheritance of venom-associated genes.

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction.

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF-P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF-P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF-P1, 1.25 ± 0.14 × 10(9) spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria.

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

Protein composition of seminal plasma in fractionated stallion ejaculates.

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH-2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion's own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse-phase liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry. The area-under-the-curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS-PAGE and densitometry. No significant differences emerged between fractions in the AUC-values of the Horse Seminal Protein-1 (HSP-1) and HSP-2 peaks, or the peak containing HSP-3 and HSP-4 (HSP-3/4). Levels of HSP-1, HSP-2 and HSP-3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60-70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.

Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow.

Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min, with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events.

The physiological roles of the boar ejaculate.

During ejaculation in the boar, sperm cohorts emitted in epididymal cauda fluid are sequentially exposed and resuspended in different mixtures of accessory sex gland secretion. This paper reviews the relevance of such unevenly composed fractions of seminal plasma (SP) in vivo on sperm transport and sperm function and how this knowledge could benefit boar semen processing for artificial insemination (AI). The firstly ejaculated spermatozoa (first 10 ml of the sperm-rich fraction, SRF [P1]) remain mainly exposed to epididymal cauda fluid and its specific proteins i.e. various lipocalins, including the fertility-related prostaglandin D synthase; than to prostatic and initial vesicular gland secretions. P1-spermatozoa are hence exposed to less bicarbonate, zinc or fructose and mainly to PSP-I spermadhesin; than if they were in the rest of the SRF and the post-SRF (P2). Since the P1-SP is less destabilizing for sperm membrane and chromatin, P1-spermatozoa sustain most in vitro procedures, including cryopreservation, the best. Moreover, ejaculated firstly, the P1-spermatozoa seem also those deposited by the boar as a vanguard cohort, thus becoming overrepresented in the oviductal sperm reservoir (SR). This vanguard SR-entry occurs before the endometrial signalling of SP components (as PSP-I/PSP-II and cytokines) causes a massive influx of the innate defensive PMNs to cleanse the uterus from eventual pathogens, superfluous spermatozoa and the allogeneic SP. The SP also conditions the mucosal immunity of the female genital tract, to tolerate the SR-spermatozoa and the semi-allogeneic conceptus. These in vivo gathered data can be extrapolated into procedures for handling boar spermatozoa in vitro for AI and other biotechnologies, including simplified cryopreservation.

Distinct effects of boar seminal plasma fractions exhibiting different protein profiles on the functionality of highly diluted boar spermatozoa.

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 x 10(6) spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4-6 contained low numbers of spermatozoa (<500 x 10(6)/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1-3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1-3 appear to promote sperm survival, whereas fractions 4-6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks.

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.

Pancreatitis autoinmune: pseudotumor inflamatorio, afectación multifocal, hipertensión portal y evolución a largo plazo / No disponible

La pancreatitis autoinmune es una enfermedad recientementecaracterizada y que en la actualidad constituye un reto diagnósticoespecialmente su diferenciación con el cáncer de páncreas. Suevolución a largo plazo es poco conocida, presentándose un casoestudiado a lo largo de 14 años y mostrando su evolución clínica,bioquímica y morfológica.Paciente mujer de 54 años que debuta con un cuadro de ictericiaobstructiva y molestias abdominales inespecíficas y constataciónen la TAC de un aumento de la cabeza del páncreas, todoello sugestivo de neoplasia de páncreas. Fue intervenida evidenciándoseun aumento difuso de todo el páncreas descartándosemalignidad intraoperatoriamente, realizando únicamente colecistectomíay coledocoduodenostomía, quedando diagnosticada entoncescomo pancreatitis crónica. Durante los años posterioresfueron apareciendo diferentes procesos autoinmunes como asma,sialoadenitis y colangitis esclerosante secundaria, así como episodiosrecurrentes de ictericia e insuficiencia pancreática endocrinay exocrina. La aparición de estas complicaciones y la detección deniveles séricos elevados de IgG4 y de anticuerpos antianhidrasacarbónica II condujo a la reevaluación de la histología inicial concluyendofinalmente con el diagnóstico de pancreatitis autoinmuneal evidenciarse una infiltración linfocitaria y plasmacitariaIgG4+, así como fibrosis y flebitis obliterativa. En los últimos añosse ha añadido a las anteriores complicaciones una fibrosis retroperitonealcon hipertensión portal, varices esofágicas y esplenomegalia

Autoimmune pancreatitis is a recently characterized diseasethat still constitutes a diagnostic challenge, especially regarding differentialdiagnosis from neoplasia. Long-term outcome is poorlyknown. We herein report a case of a patient with autoimmunepancreatitis and 14 years of follow-up, and show its clinical, biochemical,and morphological characteristics.A 54-year-old female presented with obstructive jaundice andabdominal tenderness, as well as a mass at the pancreatic head ona CT scan, suggestive of pancreatic neoplasia. Surgery showed anincrease of the whole pancreas, malignancy was intraoperativelyruled out, and a cholecystectomy and choledochoduodenostomywere carried out. The diagnosis was chronic pancreatitis. Over thefollowing years different autoimmune complications developed, includingasthma, salivary gland swelling, and sclerosing cholangitis,as well as recurrent episodes of jaundice, and exocrine and endocrinepancreatic failure. The development of these complicationscombined with the demonstration of high serum levels ofIgG4 and carbonic anhydrase II led to a re-evaluation of the initialhistology of the pancreas, leading to a final diagnosis of autoimmunepancreatitis: IgG4+ lymphoplasmacytic infiltrates, fibrosis,and obliterative phlebitis. New complications developed during thelast few years: retroperitoneal fibrosis with portal hypertension,esophageal varices, and splenomegaly

Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa.

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

[Autoimmune pancreatitis: inflammatory pseudotumor, multifocal fibrosclerosis, portal hypertension, and long-term outcome]. / Pancreatitis autoinmune: pseudotumor inflamatorio, afectación multifocal, hipertensión portal y evolución a largo plazo.

Autoimmune pancreatitis is a recently characterized disease that still constitutes a diagnostic challenge, especially regarding differential diagnosis from neoplasia. Long-term outcome is poorly known. We herein report a case of a patient with autoimmune pancreatitis and 14 years of follow-up, and show its clinical, biochemical, and morphological characteristics. A 54-year-old female presented with obstructive jaundice and abdominal tenderness, as well as a mass at the pancreatic head on a CT scan, suggestive of pancreatic neoplasia. Surgery showed an increase of the whole pancreas, malignancy was intraoperatively ruled out, and a cholecystectomy and choledochoduodenostomy were carried out. The diagnosis was chronic pancreatitis. Over the following years different autoimmune complications developed, including asthma, salivary gland swelling, and sclerosing cholangitis, as well as recurrent episodes of jaundice, and exocrine and endocrine pancreatic failure. The development of these complications combined with the demonstration of high serum levels of IgG4 and carbonic anhydrase II led to a re-evaluation of the initial histology of the pancreas, leading to a final diagnosis of autoimmune pancreatitis: IgG4+ lymphoplasmacytic infiltrates, fibrosis, and obliterative phlebitis. New complications developed during the last few years: retroperitoneal fibrosis with portal hypertension, esophageal varices, and splenomegaly.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Localization and expression of spermadhesin PSP-I/PSP-II subunits in the reproductive organs of the boar.

The epithelial localization and expression of the spermadhesin PSP-I and PSP-II subunits were determined in the testis, ductus epididymes (caput, corpus and cauda), seminal vesicles and bulbourethral glands of mature boars, using immunohistochemical, western blotting and RT-PCR methods. Immunohistochemistry showed positive labelling for PSP-I and PSP-II antibodies in the epithelium of seminal vesicles in all males tested. Positive immunolabelling, but with variable intensity, was also present in the epididymal epithelium (caput, corpus and cauda), although varying largely among segments and boars. Immunoreactivity was nearly or completely absent in the seminiferous epithelium and the bulbourethral gland, although SDS-PAGE and western blotting revealed the presence of PSP-I and PSP-II immunoreactive bands in all the tissue extracts, including the testis and the bulbourethral gland. mRNA amplification by RT-PCR using primers specific for PSP-I and PSP-II showed a trend similar to that observed for western blotting, i.e. intensity variation between tissues (even between segments of the same epididymis) and among boars. Our results indicate that the seminal vesicles are the main source of PSP-I and PSP-II spermadhesins, although epididymal segments, testis and the bulbourethral gland also participate in the expression of both proteins.

Improving the fertilizing ability of sex sorted boar spermatozoa.

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 x g/5 min) just after sorting and stored 16-18 h at 17 degrees C (groups Cc and Hc) at 6 x 10(6)sperm/mL. The second aliquot was directly stored at 17 degrees C for 16-18 degrees C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 x 10(6)sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 x 10(6) sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16-18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower (p<0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer (p<0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.

Síndrome miasteniforme de Eaton-Lambert. Revisión y presentación de un caso / Lambert-Eaton myasthenic syndrome. Review and a case report

El síndrome miasteniforme de Eaton-Lambert (SMEL) es un trastorno autoimmune presináptico de la transmisión neuromuscular, que se manifiesta clínicamente por debilidad muscular proximal, principalmente de los miembros inferiores, disfunción autosómica y reflejos osteotendinosos disminuidos. Presentamos el caso clínico de un paciente diagnosticado inicialmente de una polineuropatía crónica diabética, que tras la realización de un nuevo estudio electrofisiológico es diagnosticado de SMEL. Se revisan los principales aspectos sobre la enfermedad expuestos en la literatura médica, con particular énfasis en la importancia que tiene un correcto estudio electrofisiológico para el diagnóstico y el papel de la rehabilitación en el tratamiento de estos pacientes

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune presynaptic disorder of neuromuscular transmission, clinically manifest by proximal muscle weakness, principally involving the lower limbs, autonomic dysfunction and depressed deep tendon reflexes. We present the clinical case of a patient initially diagnosed of chronic diabetic polyneuropathy, who was diagnosed of LEMS after new electrophysiological testing. We review the main aspects on the disease presented in the literature, with special emphasis on the importance of an appropriate electrophysiological study to confirm diagnosis and the role of the rehabilitation in the treatment of these patients

Isolation and characterization of a new agglutinin from the red marine alga Hypnea cervicornis J. Agardh.

The biochemical characterization of a new lectin (Hypnea cervicornis agglutinin or HCA) isolated from the Brazilian red alga H. cervicornis is reported. The haemagglutinating activity of the lectin was only inhibited by the glycoprotein porcine stomach mucin at a minimum inhibitory concentration of 19 microg x mL(-1). No haemagglutination inhibition was detected after the addition of simple sugars. The MALDI-TOF molecular masses of native and reduced and carbamidomethylated HCA were, respectively, 9196.6 Da and 9988.2 Da, indicating that the primary structure of the protein is crosslinked by 7 disulfide bonds. This unusual structural feature among lectins, along with its N-terminal sequence and amino-acid composition, clearly shows that HCA belongs to a protein family distinct from the isolectins Hypnin A1 and A2 isolated from the related Japanese alga Hypnea japonica. On the other hand, HCA displayed a high degree of similarity to the agglutinin from the Brazilian species Hypnea musciformis. Our data indicate the occurrence of structural diversity among lectins of closely related species living in distant ecosystems, i.e., the Pacific coast of Japan and the Atlantic coast of Brazil, and support the hypothesis that the lectin content (lectinome) might serve as a biomarker for taxonomical purposes.

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose-response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydrodynamic injection mediates a transient inversion of intrahepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydrodynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes.

Antagonists Mo and Cu in a heterometallic cluster present on a novel protein (orange protein) isolated from Desulfovibrio gigas.

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS(4)(2-) moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed.