Detection of protamine 2 in bovine spermatozoa and testicles.

BACKGROUND: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. OBJECTIVES: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. MATERIALS AND METHODS: For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. RESULTS: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. CONCLUSION: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Concentração sérica de progesterona, pH da vagina e morfometria corpórea de cervas gestantes (Cervus unicolor) em cativeiro / Serum concentration of progesterone, pH of the vagina and body morphometryof pregnant deer (Cervus unicolor)in captivity

The aim was to study pregnant cervus in captivity, to obtain body morphometric data, serum progesterone concentrations, and pH of the vagina. Females of cervus (Cervus unicolor), (n=8) between 2 and 3 years old were used in November during breeding season. After sedation, in one collection, the following was measured: body weight 54.50 ± 18.70kg, body length 1.21 ± 0.16m, height at withers 0.75 ± 0.08m, thoracic perimeter 0.84 ± 0.12m, and body mass index 97.32 ± 10.50kg / m2. After sedation, blood samples were collected to measure progesterone concentration by radioimmunoassay and the use of an indicator tape to obtain the pH of the vagina was performed eight times at four day intervals in the eight females, in a total of 64 samples for each exam. Data were analyzed, and significance was at 5%. There were no differences (P> 0.05) between measurements for serum progesterone concentrations and pH of the vagina. The correlations between body weight, progesterone, pH, and body mass index were not significant (P> 0.05). There was no significant oscillation of progesterone concentration and pH of the vagina in the pregnancy period studied.

Concentração sérica de progesterona, pH da vagina e morfometria corpórea de cervas gestantes (Cervus unicolor) em cativeiro / Serum concentration of progesterone, pH of the vagina and body morphometryof pregnant deer (Cervus unicolor)in captivity

The aim was to study pregnant cervus in captivity, to obtain body morphometric data, serum progesterone concentrations, and pH of the vagina. Females of cervus (Cervus unicolor), (n=8) between 2 and 3 years old were used in November during breeding season. After sedation, in one collection, the following was measured: body weight 54.50 ± 18.70kg, body length 1.21 ± 0.16m, height at withers 0.75 ± 0.08m, thoracic perimeter 0.84 ± 0.12m, and body mass index 97.32 ± 10.50kg / m2. After sedation, blood samples were collected to measure progesterone concentration by radioimmunoassay and the use of an indicator tape to obtain the pH of the vagina was performed eight times at four day intervals in the eight females, in a total of 64 samples for each exam. Data were analyzed, and significance was at 5%. There were no differences (P> 0.05) between measurements for serum progesterone concentrations and pH of the vagina. The correlations between body weight, progesterone, pH, and body mass index were not significant (P> 0.05). There was no significant oscillation of progesterone concentration and pH of the vagina in the pregnancy period studied.(AU)

Resistance of mice to Leptospira infection and correlation with chemokine response.

Leptospirosis is globally widespread neglected disease, affecting most mammalian species. Clinical signs can be confused with other diseases which make the diagnosis and treatment difficult. Chemokines and cytokines are known for their role in the inflammatory and immune response to infections. The profile determination of chemokines' expressions in the course of infection may elucidate the defense mechanisms of the host and support the search for effective treatment strategies. We investigated the mechanisms of innate immunity through the comparison of chemokines induced during infection with L. interrogans in mice with different levels of susceptibility. We used lung and spleen tissues samples of mice from C3H/HeJ, C3H/HePas and Balb/c, respectively sensitive, intermediate susceptibility and resistant to the pathogen. The inoculation of L. interrogans in C3H/HeJ mice led a comparatively smaller change in chemokines expression in both spleen and lung tissues. In samples from spleens and lungs of C3H/HePas and Balb/c the higher increases occurred on CXCL9, CXCL16, CXCL5, CCL8 and CCL5 in Balb/c. Given the same genetic background, the differences in the responses of C3H/HePas compared to C3H/HeJ mice strongly suggest the role of chemokines for the survival of parental strain. Therefore, the greatest increase in CXC chemokines appears to be efficient to induce migration of cells to the secondary lymphoid organs and affected tissues, which is important to control infection. Overall, CXC chemokines are important for the activation and attraction of T cell and may influence the course and control of the infection in resistant Balb/c mice.

Morfometria corpórea, características do sêmen, proteínas seminais e testosterona em cervos Cervus unicolor, em cativeiro / Body morphometry, semen characteristics, seminal proteins and testosterone in sambar deer (Cervus unicolor) in captivity

Objetivou-se estudar a morfometria corpórea, as características do sêmen, o perfil proteico do plasma seminal em SDS-PAGE e a concentração sérica de testosterona em cervos-sambar (Cervus unicolor), criados em cativeiro, na estação reprodutiva da primavera. Quatro machos com idades entre 12 e 36 meses foram avaliados em quatro momentos, com intervalos de sete dias, com peso corpóreo (60,5 a 89,0kg), índice de massa corporal (93,07kg/m2 a 126,56kg/m2), volume do ejaculado (0,50±0,35mL a 0,75±0,28mL), motilidade espermática (87,75±4,78% a 90,00±7,07%), defeitos totais (17,25±5,81% a 47,72±17,55%), testosterona sérica (6,43±4,33ng/dL a 166,00±64,48ng/dL) e proteínas do plasma seminal com bandas entre 7,6 e 142kDa. As características dos ejaculados não diferiram (P>0,05) entre as três primeiras colheitas. Houve diferença (P<0,05) para os defeitos espermáticos com elevação na quarta colheita. No plasma seminal de cada cervo, foram identificadas de 16 a 27 bandas de proteínas entre 7,6 e 142kDa. Conclui-se que a qualidade espermática foi satisfatória na primavera. O estresse das contenções sucessivas causou queda da qualidade espermática. A idade influi na concentração sérica de testosterona, a qual foi maior nos cervos aos 36 meses...

The aim of this work was to study the body morphometry, semen characteristics, seminal plasma protein profile in SDS-PAGE and serum testosterone concentration in Sambar Deer (Cervus unicolor), in captivity in the breeding season (spring). Four males aged between 12 and 36 months were assessed in four moments with intervals of seven days with body weight (60.5 to 89.0kg), body mass index (93.07 to 126.56kg/m2), ejaculate volume (0.50±0.35mL to 0.75±0.28mL), sperm motility (87.75±4.78% to 90.00±7.07% ), total defects (17.25±5.81% to 47.72±17.55%), serum testosterone (6.43±4.33 ng/dL to 166.00±64.48ng/dL) and seminal plasma proteins with bands between 7.6 and 142 kDa. The characteristics of ejaculates did not differ (P>0.05) among ejaculates (1st, 2nd and 3rd). There were differences (P<0.05) for sperm defects elevation on the fourth ejaculate. In seminal plasma 16 to 27 protein bands between 7.6 and 142 kDa were identified. In conclusion, sperm quality was satisfactory in the spring and the stress of successive contentions decreased sperm quality. Also, there is influence of age upon serum testosterone concentration which was higher in deer at 36 months...

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1á (MIP-1á) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 106 cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1á were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1á observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis.

Patellar instability following experimental medial patellar desmotomy in horses.

A controlled study was designed in order to evaluate the effects of medial patellar desmotomy (MPD), combined with exercise restriction, on clinically and radiographically normal femoropatellar joints, and to identify the patellar instability by radiographic examination. MPD was performed on the right hind limb and the horses were rested for 120 days. Both hind limbs were radiographed before surgery and at 15, 30, 60, 90, and 120 days after MPD, obtaining lateromedial, flexed lateromedial, caudocranial and cranioproximal-craniodistal (skyline) views. The femorotibial and femoropatellar angles were assessed on the lateromedial views, and the distance between the medial and lateral cortex of the femur, and the distance between the medial cortex of the femur and the lateral border of the patella were evaluated in the caudocranial views. An increase in the angle between the proximal articular surface of the patella and the cranial distal surface of the femur along with enthesophyte formation and lateral deviation of the patella were found on the radiographs of the right stifles after MPD. Therefore, MPD leads to patellar instability, which can be demonstrated by the radiographic changes. The 120 days rest did not prevent the lesions caused by postsurgical patellar instability.

A Comparative Histomorphometric Study of the Stomach of Rat (Rattus norvegicus), Bat (Eidolon helvum) and Pangolin (Manis tricuspis) in Relation to Diet / Estudio Comparativo Histomorfométrico del Estómago de Rata (Rattus norvegicus), Murciélago (Eidolon helvum) y Pangolin (Manis tricuspis) en Relación con la Dieta

This study verified the comparative histomorphometric adaptations in the stomach of rat, bat and pangolin in relation to diet. Ten rats, ten bats and ten pangolins of both sexes were used for this investigation. The animals were sacrificed after slight anesthesia under chloroform inhalation. The stomach were excised, fixed in 10 percent formol saline and processed for light microscopic study. Stained slides were also subjected to morphometric analysis at a magnification of 400x. The results revealed that the cellular diameter/ density of parietal and zymogenic cells are significantly different in the three mammals (p<0.05) with the exception of the diameter of the zymogenic cells in pangolin which was not statistically significant (p>0.05) when compared with that of rat. Also, histological analysis revealed slight differences in the pattern of organization and distribution of connective tissue fibers. All these observations were reflections of the different pattern the stomachs of the three mammals have adopted to cope with their respective diets.

En este estudio se verificaron las adaptaciones histomorfométricas comparativas en el estómago de ratas, murciélagos y pangolines en relación a la dieta. Se utilizaron para esta investigación 10 ejemplares de cada especie, de ambos sexos. Los animales fueron sacrificados después de anestesia bajo inhalación de cloroformo. Los estómagos fueron extirpados, fijados en formol al 10 por ciento de solución salina y procesados para su estudio microscópico de luz. Los cortes teñidos fueron también objeto de análisis morfométrico con un aumento de X 400. Los resultados revelaron que el diámetro/densidad celular de parietal y las células cimógenas son significativamente diferentes en los tres mamíferos (p <0,05), con la excepción del diámetro de la células cimógenas de pangolines que no era estadísticamente significativa (p> 0,05) en comparación con la de rata. Por otra parte, el análisis histológico reveló ligeras diferencias en las características de organización y distribución de las fibras del tejido conjuntivo. Todas estas observaciones son un reflejo del patrón de los diferentes estómagos de los tres mamíferos, que han adoptado para hacer frente a sus respectivas dietas.

Microarchitectural adaptations in the stomach of African tree pangolin (Manis tricuspis) / Adaptaciones microarquitecturales en el estómago del pangolin africano (Manis tricuspis)

The microarchitecture of the pangolin's stomach favouring the high chitinous diet has been less waived into, despite extensive morphological investigations. Histological analysis of the microanatomy will provide powerful tools for interpretation to yield reliable insights. We investigated this by fixing the tissues in 10 percent formol saline for histological analysis. Serial sections at 5 micron m thickness were subjected to general staining methods for light microscopic study (Haematoxylin and eosin, Van Gieson's and Verhoeff's). The results revealed basic structural arrangements in their coats, with a modification of the epithelial lining of cardia and fundus into stratified squamous keratinized epithelium. These modifications were also reflected in the distribution of collagen and elastic fibers in the various layers (coats) of the stomach. The present study has shown that there was an adaptation of the stomach of African tree pangolin to its diet as reflected in the microarchitectural configuration.

La micro arquitectura del estómago de los pangolines que favorece la alta dieta de chitinous sido poco tomada en cuenta, a pesar de las amplias investigaciones morfológicas. El análisis histológico de la microanatomía proporcionará herramientas de gran importancia para la interpretación, junto con dar una información confiable. Se investigó mediante la fijación de los tejidos en solución salina de formol al 10 por ciento para análisis histológico. Las serie de secciones fueron sometidos a métodos de tinción estándar para el estudio con microscopía de luz (hematoxilina y eosina, Van Gieson y Verhoeff s). Los resultados revelaron adaptaciones estructurales básicas en sus capas, con una modificación del revestimiento epitelial del cardias y fundus en epitelio escamoso estratificado (queratinizado). Estas modificaciones también se reflejan en la distribución de colágeno y fibras elásticas en las diversas capas del estómago. El presente estudio ha demostrado que es una adaptación del estómago a la dieta como se refleja en la configuración de la microarquitectura.

Effect of quinine administration on Purkinje cells in the cerebellar cortex of adult Wistar rats.

OBJECTIVE: The effect of quinine commonly used for the treatment of Chloroquine resistant malaria and cerebral malaria on the population and transverse diameters of Purkinje cells in the cerebellar cortex was investigated. METHODS: Twenty-seven adult male wistar rats weighing between 150 g and 190 g were separated into three groups, each containing nine rats. The rats in group I were injected intramuscularly with equivalent volume of physiological saline, while group II rats were injected intramuscularly with an initial 20 mg/kg body weight dose of quinine followed by a 10 mg/kg body weight dose given 8 hourly for 7 days. The group III rats received the same treatment as group II, but were subjected to a withdrawal period of one week. The cerebellum of each rat was removed and fixed in 10% formol saline for routine histological procedures. RESULTS: The Purkinje cell population reduced significantly (P < 0.05) from the mean value of 363 +/- 5.2 cells/mm2 in group I to a mean value of 239 +/- 9.5 cells/mm2 in group II and 220 +/- 6.6 cells/mm2 in group III rats. The transverse diameters of the Purkinje cells also reduced significantly (p < 0.05) from the mean value of 1.20 +/- 0.02 microm in the group I to a mean value of 1.09 +/- 0.1 microm in group II and 0.75 +/- 0.03 microm in group III. CONCLUSION: The observed decrease in population and diameters of Purkinje cells in the treatment groups may impair cerebellar functions since they are the principal neurons of the cerebellum.

A comparative study of the ileum in rat (Rattus norvegicus), Bat (Eidolon helvum) and Pangolin (Manis tricuspis) as investigated using histological method / Estudio comparativo del íleon en rata (Rattus norvegicus), murciélago (Eidolon helvum) y pangolín (Manis tricuspis) utilizando como investigación el método histológico

The histological and morphometric differences in some parts of the gastrointestinal tracts of rat, bat and pangolin have been well established. This investigation aims at comparatively elucidating any adaptational changes the ileum of the three mammals must have adopted to meet its dietary requirements and also cope with their morphological differences. The investigation was carried out using ten rats, ten bats and ten pangolins of both sexes. The animals were slightly anaesthetized under chloroform inhalation. The intestines were harvested and the ileum excised and fixed in 10 percent formol saline. The tissues were processed for light microscopic study. The following stains were employed: Haematoxylin and eosin (H & E), Van Gieson and Verhoeff's haematoxylin elastic tissue stain. Stained slides were also analysed morphometrically. The results revealed microstructural modifications which characterized the mucosa as well as the pattern of distribution of the conjuntive tissue fibers in the ileum of the three mammals. These modifications are means of successfully coping with their respective diets and morphological differences.

Las diferencias histológicas y morfométricas en algunas partes del tubo digestivo de ratas, murciélagos y pangolines han sido bien establecidos. Esta investigación tiene por objeto aclarar comparativamente cualquier cambio de adaptación del íleon de los tres mamíferos que hayan adoptado para satisfacer sus necesidades dietéticas y también hacer frente a sus diferencias morfológicas. La investigación se llevó a cabo usando 10 ratas, 10 murciélagos y 10 pangolines, de ambos sexos. Los animales fueron ligeramente anestesiados bajo inhalación de cloroformo. Los intestinos fueron expuestos, el íleon extirpado y fijado en formol salino al 10 por ciento. Los tejidos fueron procesados para su estudio por microscopía de luz. Los siguientes tinciones fueron empleadas: Hematoxilina y eosina (H & E), Van Gieson y tinción de hematoxilina para tejidos elásticos de Verhoeff. Los cortes teñidos también se analizaron morfométricamente. Los resultados revelaron modificaciones microestructurales características de la mucosa, así como el patrón de distribución de las fibras de tejido conjuntivo en el íleon de los tres mamíferos. Estas modificaciones son el medio para entender sus diferencias en la dieta y morfología respectivamente.

Propolis: Effect of different concentrations, extracts and intake period on seric biochemical variables.

Propolis is a resinous substance produced by honeybees that possesses many biological activities, such as antitumor, antioxidant, antimicrobial, anti-inflammatory, and immunomodulatory, among others. The purpose of the present study was to investigate the biochemical profile of propolis-treated rats to observe whether propolis might lead to side effects after administration. Three different treatments were analyzed: (1) rats were treated with different concentrations of propolis (1, 3 and 6 mg/kg/day) during 30 days; (2) rats were treated with 1 mg/kg/day of ethanolic or water extracts of propolis (EEP, WEP) during 30 days; (3) rats were treated with 1 mg/kg/day of ethanolic extract of propolis during 90 and 150 days. Our results demonstrated no alterations in the seric levels of cholesterol, HDL-cholesterol, total lipids, triglycerides and in the specific activity of aminotransferases (AST) and lactic dehydrogenase (LDH) of propolis-treated groups when compared to controls. On the basis of our findings, since propolis does not induce any significant change in seric parameters, it is claimed that long-term administration of propolis might not have any cardiac injury.

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.

Genome features of Leptospira interrogans serovar Copenhageni.

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Genome features of Leptospira interrogans serovar Copenhageni

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Fixaçäo externa de fratura metacarpiana com pinos transcorticais e gesso sintético em bezerro Simental / External fixation of the metacarpal fracture with transcortical pins and fiberglass cast in Simmental calf

A six-month-old 245 kg male Simmental calf was referred to the Veterinary Hospital in Cuiabá, MT, with closed comminuted diaphyseal fracture in metacarpus. It was given preference to external fixation as means of fracture immobilization, and transcortical pins and fiberglass cast were used. This technique showed effective immobilization of the fracture, less expensive and feasible to be done in the field

An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.