Revisiting the birth of NaCl crystals using molecular dynamics simulation.

Molecular dynamics simulations were used to investigate the initial stage of phase separation mechanisms for an oversaturated electrolytic solution. We developed a low computational cost methodology to determine the simulation frames where the first ionic clusters are formed. By discretizing the simulation box, we obtain a density profile in the moments preceding and succeeding the nuclei's formation. The growth of the clusters identified with our methodology was analyzed until the end of the simulation. Calculation of the Steinhardt parameter showed symmetry of the solid, giving indications that the classical nucleation theory explains the mechanism of the solid formation. The methodology developed was useful for identifying phase separation mechanisms in the nucleation process. At lower concentrations, there was no formation of stable clusters. At intermediate concentrations, the analyses indicate a transition of phases in one stage, from a oversaturate electrolytic solution to a crystalline solid. At high concentration, a transition of phases in two stages, initially, is the formation of a dense liquid, and only after that, crystalline solid formed inside the dense liquid. The change in phase separation mechanism due to increasing oversaturation underscores the importance of precise determination of the driving force for phase separation and concentration limits for each mechanism.

Phytochemical characterization, antioxidant potential and antimicrobial activity of Averrhoa carambola L. (Oxalidaceae) against multiresistant pathogens / Caracterização fitoquímica, potencial antioxidante e atividade antimicrobiana de Averrhoa carambola L. (Oxalidaceae) frente a patógenos multirresistentes

Abstract The objective of this work was to perform the phytochemical characterization, to determine total phenols, antioxidant (AAO%) and antimicrobial potential of the ethanolic extracts of carambola. The phytochemical study was carried out through a qualitative analysis of the chemical constituents and quantitative determination of the phenol content By the Folin-Ciocalteu test. Qualitative and quantitative antioxidant tests were performed using the DPPH method (2,2 diphenyl-1-picryl-hydrazila) and iron reduction (FRAP). The minimum inhibitory concentration (MIC) was determined by microdilution in 96-well plates. The presence of pyrogallic tannins, steroids and saponins has been identified. The highest total phenol content, quantified in the samples, was found in the stem bark (0.0866 mgEAG/g) and in the fruit (0.0734 mgEAG/g). In the antioxidant evaluation, the extracts of the green fruit bagasse (AAO% 71.9%,) and stem bark at 50 μg/mL (AAO% 94%) with CE50 23.7 μg/mL. Leaf extracts, stem bark, ripe fruit bagasse and green fruit bagasse presented MICs of 100 μg/mL against multiresistant pathogenic bacteria and fungi.

Resumo O objetivo desse trabalho foi realizar a caracterização fitoquímica, determinar fenóis totais, potencial antioxidante (AAO%) e antimicrobiano dos extratos etanólicos de carambola O estudo fitoquímico foi realizado por meio de análise qualitativa dos constituintes químicos e determinação quantitativa do teor de fenóis totais pelo teste de Folin-Ciocalteu. Os testes antioxidantes qualitativos e quantitativos foram realizados pelo método do DPPH (2,2 difenil-1- picril-hidrazila) e redução do ferro (FRAP). A concentração inibitória mínima (CIM) foi determinada por microdiluição em placas de 96 poços. Foi identificada a presença de taninos pirogálicos, esteroides e saponinas. O maior teor de fenóis totais, quantificado nas amostras, foi encontrado na casca do caule (0,0866 mg EAG/g) e no fruto (0,0734 mg EAG/g). Na avaliação antioxidante destacaram-se a 500 µg/mL os extratos do bagaço do fruto verde (AAO% 71,9%,), e casca do caule a 50 µg/mL (AAO% 94%) com CE50 23,7 µg/mL. Os extratos das folhas, casca do caule, bagaço do fruto maduro e bagaço do fruto verde apresentaram CIM de 100 µg/mL contra bactérias e fungos patogênicos multirresistentes.

Phytochemical characterization, antioxidant potential and antimicrobial activity of Averrhoa carambola L. (Oxalidaceae) against multiresistant pathogens.

The objective of this work was to perform the phytochemical characterization, to determine total phenols, antioxidant (AAO%) and antimicrobial potential of the ethanolic extracts of carambola. The phytochemical study was carried out through a qualitative analysis of the chemical constituents and quantitative determination of the phenol content By the Folin-Ciocalteu test. Qualitative and quantitative antioxidant tests were performed using the DPPH method (2,2 diphenyl-1-picryl-hydrazila) and iron reduction (FRAP). The minimum inhibitory concentration (MIC) was determined by microdilution in 96-well plates. The presence of pyrogallic tannins, steroids and saponins has been identified. The highest total phenol content, quantified in the samples, was found in the stem bark (0.0866 mgEAG/g) and in the fruit (0.0734 mgEAG/g). In the antioxidant evaluation, the extracts of the green fruit bagasse (AAO% 71.9%,) and stem bark at 50 µg/mL (AAO% 94%) with CE50 23.7 µg/mL. Leaf extracts, stem bark, ripe fruit bagasse and green fruit bagasse presented MICs of 100 µg/mL against multiresistant pathogenic bacteria and fungi.

Cross-Sectional Imaging of Third Molar-Related Abnormalities.

Third molars may be associated with a wide range of pathologic conditions, including mechanical, inflammatory, infectious, cystic, neoplastic, and iatrogenic. Diagnosis of third molar-related conditions can be challenging for radiologists who lack experience in dental imaging. Appropriate imaging evaluation can help practicing radiologists arrive at correct diagnoses, thus improving patient care. This review discusses the imaging findings of various conditions related to third molars, highlighting relevant anatomy and cross-sectional imaging techniques. In addition, key imaging findings of complications of third molar extraction are presented.

Soil attributes in coal mining areas under recovery with bracatinga (Mimosa scabrella).

The coal reserves in the south of Brazil were intensely exploited at the time of great demand for such fuel. This resulted in changes in the environment, mainly in the chemical, physical and biological characteristics of the soil. Due to the potential to control erosive processes, increase soil quality and restore biological diversity, revegetation is a promising alternative to recover those impacted areas. In that respect, bracatinga is a pioneering tree species that easily grow in different environments and has being planted as vegetation cover in areas under recovery. Therefore, the objective of this work was to characterize the chemical features and to evaluate the soil microbiological attributes in areas degraded by coal mining and under recovery using bracatinga as cover plant. In the bracatinga canopy projection area, soil samples were collected in the environmental restoration areas that have been, at the time of collecting, under a regime of 2, 4, 6 and 12 years of restoration. In addition an area with natural occurrence of bracatinga was used as control. Microbial biomass nitrogen, microbial biomass carbon and microbial biomass respiration increase in average 281, 230 and 157% respectively, when the 12-year-old areas were compared to the 2-year-old-areas. Likewise, a decrease in qCO2 in the order of 60% was observed for that same comparison. The 12-year-old areas reached the same values of qCO2 found in the reference area. The data suggest an improvement in the microbiological attributes of the soil with the increase in recovery time for the studied areas. SIGNIFICANCE AND IMPACT OF THE STUDY: In coal mining areas under recovery with typically acid soils, the use of the current recovery strategies (revegetation mainly) has been efficient to increase the quality of soils, especially in the environmental restoration areas. Soil microbiological attributes such as microbial biomass nitrogen, microbial biomass carbon, microbial basal respiration and metabolic quotient (qCO2 ) are dynamic and highly sensitive. These parameters have the potential to be adopted together with conventional attributes, such as floristic composition indices and species diversity indices, to evaluate the degree of any particular environmental recovery process being conducted at previously explored mining areas.

Bradyrhizobium and Pseudomonas strains obtained from coal-mining areas nodulate and promote the growth of Calopogonium muconoides plants used in the reclamation of degraded areas.

AIMS: The objective of this work was to isolate and characterize indigenous rhizobia from coal-mining areas able to efficiently nodulate and fix nitrogen in association with Calopogonium mucunoides (calopo). METHODS AND RESULTS: Isolation, authentication and morphological, biochemical and molecular characterization of the autochthonous rhizobia were performed and their symbiotic efficiency (SE) evaluated. Efficient rhizobial isolates suitable for the inoculation of calopo in coal-mining regions were obtained. A total of 30 isolates were obtained after nodulation authentication, of which five presented high SE with plant-growth promoting traits such as indole-3-acetic acid production, phosphate solubilization and biofilm formation. These isolates were identified as belonging to Bradyrhizobium, Pseudomonas and Rhizobium. CONCLUSIONS: Bradyrhizobium sp. A2-10 and Pseudomonas sp. A6-05 were able to promote calopo plant growth using soil obtained from coal-mining degraded areas, thus indicating their potential as inoculants aiming at land reclamation. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of Pseudomonas nodule formation in calopo. Furthermore, the results demonstrated that autochthonous rhizobia obtained from degraded soils presented high SE in calopo and possess a wide range of plant-growth promoting traits. Ultimately, they may all contribute to an increased leguminous plant growth under stress conditions. The selected rhizobia strains may be used as inoculants and present a valuable role in the development of strategies aiming to recover coal-mining degraded areas. Bacterial inoculants would greatly reduce the use of often harmful nitrogen fertilizers vastly employed in revegetation programmes of degraded areas.

Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Phytochemical characterization, antioxidant potential and antimicrobial activity of Averrhoa carambola L. (Oxalidaceae) against multiresistant pathogens

Abstract The objective of this work was to perform the phytochemical characterization, to determine total phenols, antioxidant (AAO%) and antimicrobial potential of the ethanolic extracts of carambola. The phytochemical study was carried out through a qualitative analysis of the chemical constituents and quantitative determination of the phenol content By the Folin-Ciocalteu test. Qualitative and quantitative antioxidant tests were performed using the DPPH method (2,2 diphenyl-1-picryl-hydrazila) and iron reduction (FRAP). The minimum inhibitory concentration (MIC) was determined by microdilution in 96-well plates. The presence of pyrogallic tannins, steroids and saponins has been identified. The highest total phenol content, quantified in the samples, was found in the stem bark (0.0866 mgEAG/g) and in the fruit (0.0734 mgEAG/g). In the antioxidant evaluation, the extracts of the green fruit bagasse (AAO% 71.9%,) and stem bark at 50 g/mL (AAO% 94%) with CE50 23.7 g/mL. Leaf extracts, stem bark, ripe fruit bagasse and green fruit bagasse presented MICs of 100 g/mL against multiresistant pathogenic bacteria and fungi.

Resumo O objetivo desse trabalho foi realizar a caracterização fitoquímica, determinar fenóis totais, potencial antioxidante (AAO%) e antimicrobiano dos extratos etanólicos de carambola O estudo fitoquímico foi realizado por meio de análise qualitativa dos constituintes químicos e determinação quantitativa do teor de fenóis totais pelo teste de Folin-Ciocalteu. Os testes antioxidantes qualitativos e quantitativos foram realizados pelo método do DPPH (2,2 difenil-1- picril-hidrazila) e redução do ferro (FRAP). A concentração inibitória mínima (CIM) foi determinada por microdiluição em placas de 96 poços. Foi identificada a presença de taninos pirogálicos, esteroides e saponinas. O maior teor de fenóis totais, quantificado nas amostras, foi encontrado na casca do caule (0,0866 mg EAG/g) e no fruto (0,0734 mg EAG/g). Na avaliação antioxidante destacaram-se a 500 µg/mL os extratos do bagaço do fruto verde (AAO% 71,9%,), e casca do caule a 50 µg/mL (AAO% 94%) com CE50 23,7 µg/mL. Os extratos das folhas, casca do caule, bagaço do fruto maduro e bagaço do fruto verde apresentaram CIM de 100 µg/mL contra bactérias e fungos patogênicos multirresistentes.

Isolation of the pituitary gonadotrophic α-subunit hormone of the giant amazonian fish: pirarucu (Arapaima gigas).

The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

Novel series of (177)Lu-labeled bombesin derivatives with amino acidic spacers for selective targeting of human PC-3 prostate tumor cells.

AIM: Bombesin (BBN) has demonstrated the ability to bind with high affinity and specificity to GRP receptor, overexpressed on human prostate cancer. A large number of BBN derivatives have been synthesized for this purpose but most of them exhibit high abdominal accumulation, which may represent a problem in their clinical use due to serious side effects to patients. In this study we describe the results of radiolabeling with lutetium-177, stability and in vivo studies of novel phenyl-glycine-extended bombesin derivatives. The spacers were inserted to improve bombesin in vivo properties and to reduce its target to non-tumor sites. METHODS: Preliminary studies were done to establish the ideal conditions for labeling bombesin derivatives. Chromatography systems were applied to determine free lutetium and the stability of the preparations was evaluated either after storing at 2-8 ºC or incubation in human serum at 37 ºC. In vivo experiments included biodistribution, pharmacokinetics and SPECT images and were performed in Balb-c and Nude mice bearing PC-3 xenografts. RESULTS: The derivatives were labeled with high yield and kept stable at 2-8 ºC and are metabolized by human serum enzymes. In vivo studies showed fast blood clearance of labeled peptides and rapid excretion, performed mainly by renal pathway. In addition, biodistribution and imaging studies showed low abdominal accumulation and significant and specific tumor uptake of (177)Lu-labeled derivatives. CONCLUSIONS: The derivative with longer spacer holds a higher potential as radiopharmaceutical for prostate tumor diagnosis and the derivatives with shorter spacers are potential radiopharmaceuticals for prostate tumor treatment.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

Lymphocytic prolactin does not contribute to systemic lupus erythematosus hyperprolactinemia.

OBJECTIVES: Lymphocytic prolactin (PRL) gene expression is detected in the majority of the immune cells and it is not known if this source contributes to hyperprolactinemia in systemic lupus erythematosus (SLE). We have therefore evaluated lymphocytic PRL secretion and gene expression in SLE and healthy controls. METHODS: Thirty SLE patients (ACR criteria) and 10 controls were selected for the study. Serum levels of PRL and macroprolactin were detected by immunofluorometric assay and gel filtration chromatography, respectively. The lymphocytic biological activity was determined by Nb2 cells bioassays. Lymphocytic PRL gene expression was evaluated by RT-PCR assay. RESULTS: The median serum PRL levels of the 30 SLE patients was higher than the control group (9.65 (1.9-38.9) vs. 6.40 (2.4-10.3) ng/mL, p=0.03). A significant difference was detected between median serum PRL levels of active SLE, inactive SLE and controls (10.85 (5-38.9) vs. 7.65 (1.9-15.5) vs. 6.40 (2.4-10.3) ng/mL), p=0.01). The higher frequency of mild hyperprolactinemia was detected among active SLE in comparison with inactive SLE and controls (7 (38.9%) vs. 1 (8.3%) vs. 0 (0%)), with statistical significance (p=0.02). Nb2 cells assay revealed uniformly low levels of lymphocytic PRL in active, inactive and control groups without statistical significance among them (24.2 (8-63) vs. 27 (13.6-82) vs. 29.5 (8-72) ng/mL), p=0.84). Furthermore, median lymphocytic PRL gene expression evaluated by RT-PCR assay was comparable in both active and inactive SLE groups (p=0.12). CONCLUSIONS: This is the first study to exclude a lymphocytic source of PRL, pointing out a pituitary etiology for hyperprolactinemia in SLE. However, other sources from the immune system cannot be ruled out.

Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells.

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.

Morbidity of adjuvant high-dose-rate brachytherapy for low to intermediate risk endometrial adenocarcinoma completely resected.

The aim of this retrospective analysis was to evaluate bowel and urinary acute and late morbidity in patients with low to intermediate risk endometrial carcinoma, submitted to total abdominal hysterectomy and bilateral salpingo-oophorectomy, without lymphadenectomy, and postoperative high-dose-rate brachytherapy (HDR-B) as the sole treatment. From March 1996 to June 1998, 70 patients were treated on an outpatient basis, to a total dose of 30-50 Gy, given in two fractions per week. A total of 4-5 fractions of 6-10 GY was delivered. Three patients (4.2%) developed severe bowel complications, with one patient experiencing severe rectal bleeding. Local control was observed in 68 (97.1%) patients. Five-year actuarial disease and complication-free survival were 94.3% and 96.8%, respectively. In conclusion, it seems that a modest dose fraction schedule of HDR-B yields very high local control rates and low morbidity rates.

[Influence of different methods of chemical disinfection on the physical properties of type IV and V gypsum dies]. / Influência de diferentes métodos de desinfecção química nas propriedades físicas de troquéis de gesso tipo IV e V.

Several instruments and materials frequently used in prosthodontics--such as stone casts, dental impressions, interocclusal records--are classified, by the dental literature, as vehicles of transmission of infectious diseases to those who handle them. The present study aims at comparing dimensional alteration, superficial texture and compression resistance of stone dies submitted to different disinfection methods: 30-minute immersion in 1% sodium hypochlorite or in 2.2% alkaline glutaraldehyde (with or without previous ultrasonic washing) and addition of 2.2% alkaline glutaraldehyde or 5% sodium hypochlorite to the gypsum during its preparation. It was possible to conclude that: (1) chemical disinfection did not cause significant dimensional alteration in stone dies; (2) superficial texture was altered according to the disinfection method utilized; (3) immersion in disinfectant solution during 30 min, as well as the addition of disinfectant to the gypsum during its preparation, reduced the compression resistance of dies.

High-level synthesis of human prolactin in Chinese-Hamster ovary cells.

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower. However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.

Synthesis and characterization of recombinant, authentic human prolactin secreted into the periplasmic space of Escherichia coli.

Recombinant, fully bioactive, authentic human prolactin (aut-hPRL) has been synthesized in transformed Escherichia coli HB2151 bacteria in a soluble, non-glycosylated form, which is secreted into the bacterial periplasm. Use was made of a bacterial expression vector, containing tac promoter-controlled sequences for the translation enhancer from bacteriophage T7 gene 10, and for a cellulase leader peptide from Cellulomonas fimi joined to sequences coding for aut-hPRL. This vector was derived from a previously described vector containing sequences of an hPRL variant, tag-hPRL (containing a 12-amino-acid peptide tag at the N-terminal end), using site-specific mutagenesis to delete the tag sequence. SDS/PAGE, partial N-terminal amino acid sequence analysis, Western blot analysis and Nb2 lymphoma cell in vitro bioassay indicated correct processing of the hormone. Periplasmic secretion of aut-hPRL, as measured by immunoassay, was relatively low (approx. 0.08 microgram/ml per A600 unit), in contrast to that of tag-hPRL which was approximately 8-fold higher, apparently a consequence of the tag sequence. This is the first report describing periplasmic secretion of biologically active, authentic hPRL.