Simulated fine-needle aspiration diagnosis of follicular thyroid nodules by hyperspectral Raman microscopy and chemometric analysis.

SIGNIFICANCE: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm. AIM: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms. APPROACH: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected. RESULTS: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%. CONCLUSIONS: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify "indeterminate" nodules and other cytologically challenging cases.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Evolutionary-driven C-MYC gene expression in mammalian fibroblasts.

The extent to which mammalian cells share similar transcriptomes remains unclear. Notwithstanding, such cross-species gene expression inquiries have been scarce for defined cell types and most lack the dissection of gene regulatory landscapes. Therefore, the work was aimed to determine C-MYC relative expression across mammalian fibroblasts (Ovis aries and Bos taurus) via cross-species RT-qPCR and comprehensively explore its regulatory landscape by in silico tools. The prediction of transcription factor binding sites in C-MYC and its 2.5 kb upstream sequence revealed substantial variation, thus indicating evolutionary-driven re-wiring of cis-regulatory elements. C-MYC and its downstream target TBX3 were up-regulated in Bos taurus fibroblasts. The relative expression of C-MYC regulators [RONIN (also known as THAP11), RXRß, and TCF3] and the C-MYC-associated transcript elongation factor CDK9 did not differ between species. Additional in silico analyses suggested Bos taurus-specific C-MYC exonization, alternative splicing, and binding sites for non-coding RNAs. C-MYC protein orthologs were highly conserved, while variation was in the transactivation domain and the leucine zipper motif. Altogether, mammalian fibroblasts display evolutionary-driven C-MYC relative expression that should be instructive for understanding cellular physiology, cellular reprogramming, and C-MYC-related diseases.

Raman-based cytopathology: an approach to improve diagnostic accuracy in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare form of thyroid malignancy that can be diagnostically challenging on fine needle aspiration (FNA) cytology. Ancillary tests such as elevated serum or immunohistochemical positive calcitonin have been helpful, yet they can occasionally provide false positive results. In search for an alternative method to improve diagnostic accuracy (DA), we applied hyperspectral Raman spectroscopy to characterize the biochemical composition of single cells from MTC and compared their spectral information to cells from other types of thyroid nodules. Hyperspectral Raman images of 117 MTC single cells from digested tissue were obtained with a line-scan hyperspectral Raman microscope and compared to 127 benign and 121 classic variant of papillary thyroid carcinoma (CVPTC) cells. When principal component analysis and linear discriminant analysis were used to classify the spectral data, MTC cells were differentiated from benign and CVPTC cells with 97% and 99% DA, respectively. In addition, MTC cells exhibited a prominent Raman peak at 1003 cm-1, whose intensity is 84% and 226% greater on average than that observed in benign and CVPTC cells, respectively. When specifically utilizing only this peak as a spectral marker, MTC cells were separated from benign and CVPTC cells with 87% and 95% DA, respectively. As this peak is linked to phenylalanine, which is known to be associated with calcitonin release in thyroid parafollicular cells, the increased intensity further suggests that this Raman peak could potentially be a new diagnostic marker for MTC. Furthermore, preliminary data from MTC cells (n=21) isolated from a simulated FNA procedure provided similar Raman signatures when compared to single cells from digestion. These results suggest that "Raman-based cytopathology" can be used as an adjunct technique to improve the diagnostic accuracy of FNA cytopathology at a single cell level.

Hyperspectral Raman microscopy can accurately differentiate single cells of different human thyroid nodules.

We report on the use of line-scan hyperspectral Raman microscopy in combination with multivariate statistical analyses for identifying and classifying single cells isolated from clinical samples of human thyroid nodules based on their intrinsic Raman spectral signatures. A total of 248 hyperspectral Raman images of single cells from benign thyroid (n = 127) and classic variant of papillary carcinoma (n = 121) nodules were collected. Spectral differences attributed to phenylalanine, tryptophan, proteins, lipids, and nucleic acids were identified for benign and papillary carcinoma cells. Using principal component analysis and linear discriminant analysis, cells were identified with 97% diagnostic accuracy. In addition, preliminary data of cells from follicular adenoma (n = 20), follicular carcinoma (n = 25), and follicular variant of papillary carcinoma (n = 18) nodules suggest the feasibility of further discrimination of subtypes. Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an objective approach for analyzing single cells from fine needle aspiration (FNA) biopsies to enable the minimally invasive diagnosis of "indeterminate" thyroid nodules and other challenging cases.

Evaluation of embryo collection and transfer days on pregnancy rate of Mangalarga Marchador mares during the breeding season / Evaluación de los días de recolección y transferencia de embriones sobre las tasas de preñez en yeguas Mangalarga Marchador durante la estación de monta / Avaliação do dia da colheita e da transferência de embriões equino da raça Mangalarga Marchador durante a estação de monta

Abstract Background: Proper timing for embryo collection and transfer in horses -which is critical for the success of this biotechnology- is still debated. Additionally, there is little information on this technology under tropical conditions. Objective: To determine the best day for collection and transfer of embryos in Mangalarga Marchador mares under Brazilian northeast's conditions. Methods: Donors (n= 30) and recipients (n= 76) in diestrus phase were selected based on both clinical and gynecology examinations. Estrus was induced on both donor and recipient mares by intramuscular injection of 5 mg Dinoprost, aiming to obtain an ovulation interval of -1 to +3 between recipient and donor. Ovulation was induced with buserelin acetate when the largest follicle reached at least 35 mm in diameter. At this time, mares were subjected to artificial insemination at 48-hour intervals until ovulation. The embryos were collected on days 7, 8, and 9 after ovulation. Results: The embryo collection on day 8 was more efficient (p<0.05) than on day 7, but it was not more effective (p>0.05) than day 9, which presented the same efficiency (p>0.05) as day 7. From a total of 76 embryos transferred to the recipients, that were between days 4 and 9 after ovulation, there was no influence (p>0.05) of the day of transfer on pregnancy rate. Conclusions: The embryo collection must be performed on day 8 after ovulation, and transfer can be performed on any day of that interval (4-9) without affecting the pregnancy rate.

Resumen Antecedentes: El momento mas apropiado para la recolección y transferencia de embriones en equinos -que es fundamental para el éxito de esta biotecnología- continua siendo sujeto de estudio. Además, es escasa la información sobre esta tecnología en condiciones tropicales. Objetivo: Determinar el momento mas adecuado para la recolecta y transferencia de embriones en yeguas Mangalarga Marchador, en las condiciones del nordeste Brasileño. Métodos: Donadoras (n= 30) y receptoras (n= 76) en la fase de diestro se seleccionaron con base en los exámenes clínicos y ginecológicos. El estro de las yeguas donadoras y receptoras fue inducido con 5 mg de Dinoprost, vía intramuscular, intentando obtener un intervalo de ovulación de -1 a +3 entre la receptora y la donadora. La ovulación fue inducida con acetato de buserelina cuando el folículo mayor alcanzó 35 mm de diámetro. En ese momento, las yeguas fueron sometidas a inseminación artificial en intervalos de 48 horas hasta la ovulación. Los embriones fueron recolectados en los días 7, 8 y 9 después de la ovulación. Resultados: La recolecta de embriones en el día 8 fue más eficiente (p<0,05) que en el día 7, pero no fue más efectivo (p>0,05) que en el día 9, el cuál presentó la misma eficiencia (p>0,05) que en el día 7. De un total de 76 embriones transferidos a las receptoras, que se encontraban entre el día 4 y 9 después de la ovulación, no se registró influencia (p>0,05) del día de la transferencia en la tasa de preñez. Conclusiones: La recolecta embrionaria debe ser realizada el día 8 después de la ovulación, y la transferencia puede ser realizada en cualquier día de este intervalo (4 a 9) sin que se afecte la tasa de preñez.

Resumo Antecedentes: A importância do momentoda colheita e da transferência do embrião equino para o sucesso dessa biotécnica em equino continua sem ser completamente entendida. Adicionalmente, existe pouca informação sobre essa tecnologia em condições tropicais. Objetivo: Determinar o melhor dia para colheita e para transferência de embriões em eguas manga larga marchador nas condições do nordeste brasileiro. Métodos: Doadoras (n = 30) e receptoras (n = 76) na fase de diestro foram selecionadas com base nos exames clínico e ginecológicos. O estro das éguas doadoras e receptoras foi induzido com 5 mg de Dinoprost administrado por via intramuscular, buscando obter um intervalo de ovulação de -1 a +3 entre a receptora e a doadora. A ovulação foi induzida com acetato de buserelina quando o foliculo maior alcançou o tamanho de 35 mm de diâmetro. Nesse momento, as éguas foram submetidas a inseminação artificial em intervalos de 48 horas até a ovulação. Os embriões foram colhidos nos dias 7, 8 e 9 depois da ovulação. Resultados: A colheita de embriões no dia 8 foi mais eficiente (p<0,05) do que no dia 7, porem não foi mais efetivo (p>0,05) do que o dia 9, o qual apresentou a mesma eficiência (p>0,05) que o dia 7. De um total de 76 embriões transferidos para as receptoras que se encontravam entre os dias 4 e 9 depois da ovulação, não se registrou influência (p>0,05) do dia da transferência sobre a taxa de prenhez. Conclusões: A colheita embrionária deve ser realizada no dia 8 depois da ovulação, e a transferência pode ser realizada em qualquer dia desse intervalo (4-9) sem que a taxa de prenhez seja afetada.

Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.

Disulfide biochemistry in 2-cys peroxiredoxin: requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin.

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8Å resolution of Tsa1(C47S) in the decameric form [(α(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys(P) α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6)M(-1)s(-1). Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.

Formigas em ambiente hospitalar e seu potencial como transmissoras de bactérias / Ants in a hospital environment and its importance as vector of bacteria

A comunidade de formigas da área externa ao hospital do Hospital Municipal de Morrinhos, GO, caracterizou-se pelos baixos índices de riqueza, diversidade, dominância e eqüidade de abundância das espécies. Pheidole sp.1, uma espécie poligínica, dominou esse ambiente apesar da coexistência com espécies potencialmente competitivas. A mesma espécie de formiga predominou no interior de praticamente todas as repartições do hospital e sua distribuição espaço-temporal aproximou-se da agregada (variância/média = 1.102, χ2 = 29.38, P < 0.01). Escherichia, Salmonella, Aeromonas, Enterococcus, Staphylococcus e Klebsiella foram os gêneros de bactérias associados a essa espécie de formiga em praticamente todas as repartições do hospital. O unicolonialismo de Pheidole sp.1 tende a potencializar o processo de contaminação e disseminação de agentes infecto-contagiosos. O manejo e controle da espécie devem ser acompanhados de técnicas que reduzam o processo de colonização por novas rainhas e a quantidade de locais de nidificação no interior do hospital.

The external ant community of Hospital Municipal de Morrinhos, in Morrinhos, Goiás State, was characterized by the low rates of richness, diversity, dominance and equity of species abundance. Pheidole sp.1, a polygynic species was numerically dominant in this environment, although it coexists with potentially competitive species. This ant species prevailed within all hospital departments and its space-time distribution was a little aggregated (variance/mean ratio = 1.102, χ2 = 29.38, P < 0.01). Escherichia, Salmonella, Aeromonas, Enterococcus, Staphylococcus and Klebsiella were the bacteria associated to this ant species in nearly all hospital annexes. The unicolonialism of Pheidole sp.1 tends to increase the contamination and dissemination process of infecto-contagious agents. The control and management of this ant species must be followed by practices that reduce the colonization process by other queens and the quantity of site nidification within the hospital.

Goal-directed fluid management based on pulse pressure variation monitoring during high-risk surgery: a pilot randomized controlled trial.

INTRODUCTION: Several studies have shown that maximizing stroke volume (or increasing it until a plateau is reached) by volume loading during high-risk surgery may improve post-operative outcome. This goal could be achieved simply by minimizing the variation in arterial pulse pressure (deltaPP) induced by mechanical ventilation. We tested this hypothesis in a prospective, randomized, single-centre study. The primary endpoint was the length of postoperative stay in hospital. METHODS: Thirty-three patients undergoing high-risk surgery were randomized either to a control group (group C, n = 16) or to an intervention group (group I, n = 17). In group I, deltaPP was continuously monitored during surgery by a multiparameter bedside monitor and minimized to 10% or less by volume loading. RESULTS: Both groups were comparable in terms of demographic data, American Society of Anesthesiology score, type, and duration of surgery. During surgery, group I received more fluid than group C (4,618 +/- 1,557 versus 1,694 +/- 705 ml (mean +/- SD), P < 0.0001), and deltaPP decreased from 22 +/- 75 to 9 +/- 1% (P < 0.05) in group I. The median duration of postoperative stay in hospital (7 versus 17 days, P < 0.01) was lower in group I than in group C. The number of postoperative complications per patient (1.4 +/- 2.1 versus 3.9 +/- 2.8, P < 0.05), as well as the median duration of mechanical ventilation (1 versus 5 days, P < 0.05) and stay in the intensive care unit (3 versus 9 days, P < 0.01) was also lower in group I. CONCLUSION: Monitoring and minimizing deltaPP by volume loading during high-risk surgery improves postoperative outcome and decreases the length of stay in hospital. TRIAL REGISTRATION: NCT00479011.

Desenvolvimento e validação de metodologia para quantificação de alcalóides totais como berberina em fitoterápico contendo Berberis vulgaris L. / Development and validation of a method for the quantification of total alkaloids as berberine in an herbal medicine containing Berberis vulgaris L.

A Robusterina® é um produto fitoterápico, com eficácia no tratamento de disfunções do ciclo menstrual. Em sua composição encontram-se Berberis vulgaris L., de ação sedativa e antiespasmódica; Gossypium herbaceum L., enemagoga, hemostática e ocitócica; Viburnum opulus L., antiespasmódico nas cólicas menstruais. De acordo com a Resolução RDC N° 48, de 16 de março de 2004, observa-se que o produto adequa-se na definição de Fitoterápico. A presença de alcalóides em Berberis vulgaris e a ausência de metodologias analíticas de quantificação para o produto, nos incentivaram a propor e validar um método apoiando-nos na Resolução RE N° 899, de 29 de Maio de 2003. Tal metodologia fundamenta-se na determinação espectrofotométrica de alcalóides utilizando-se Dragendorff como reagente precipitante, e o sulfato de berberina MerckÒ, como substância química de referência. A curva de calibração foi determinada com seis concentrações entre 40 e 200 mg/mL. A equação da reta é y = 0,0038x + 0,0092 com R² de 0,9996. Os parâmetros robustez, precisão, especificidade, limite de detecção e quantificação e exatidão foram avaliados estatisticamente com intervalo de confiança de 95 por cento (teste t de Student, ANOVA).

Robusterina® is a herbal medicine, with effectiveness in the treatment of menstrual cycle disfunctions. Its composition includes Berberis vulgaris L., with sedative and antispasmodic action; Gossypium herbaceum L., with emmenagogue, hemostatic and ocitocic action; Viburnum opulus L., with antispasmodic action for the treatment of menstrual colics. In accordance with Resolution RDC N° 48, of 16 of March of 2004 (ANVISA, Brazil) the product meets the definition of "Fitoterápico" (phytotherapeutic agent). The presence of alkaloids in Berberis vulgaris and the absence of analytical methodologies for quantification of the product, stimulated us to develop and validate a method in accordance with Resolution RE N° 899, of 29 of May of 2003. Such methodology is based on the determination of alkaloids using a spectrophotometric method, with Dragendorff as a precipitating reagent, and using berberine sulphate, as a standard. The calibration curve was determined with six concentrations ranging between 40 and 200 mg/mL. The equation of the calibration curve is y = 0.0038x + 0.0092 with R² of 0.9996. The parameters robustness, precision, specificity, limit of detection and quantification and accuracy have been evaluated using 95 percent confidence interval (test t of Student, ANOVA).

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.

Characterization of kinetics and products of the Baeyer-Villiger oxygenase MtmOIV, the key enzyme of the biosynthetic pathway toward the natural product anticancer drug mithramycin from Streptomyces argillaceus.

MtmOIV, the key oxygenase of the mithramycin biosynthetic pathway in Streptomyces argillaceus, was proven to act initially as Baeyer-Villiger monooxygenase, but may also catalyze various follow-up reaction steps. The reaction of the overexpressed pure His6-tagged enzyme with its substrate premithramycin B was studied. Various intermediates and products were isolated and physicochemically characterized, several of them being previously unknown compounds. This is the first example in which a bacterial enzyme was unequivocally proven to act as Baeyer-Villigerase with its natural substrate, that is, in its natural context.

Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom.

The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.

The transcription factor Snf1p is involved in a Tup1p-independent manner in the glucose regulation of the major methanol metabolism genes of Hansenula polymorpha

Hansenula polymorpha is a methylotrophic yeast widely employed in biotechnology as a ''protein factory''. Most promoters used for heterologous protein expression, like MOX (methanol oxidase) and DAS (di-hydroxy acetone synthase), are involved in the peroxisomal methanol metabolism (C1 metabolism) and are under strong glucose repression. Interestingly, the MOX promoter is subjected to glucose regulation also in Saccharomyces cerevisiae, a non-methylotrophic yeast in which this phenomenon is well studied. In this species, the transcription factor Tup1p plays an essential role in glucose repression of several genes. This effect is counteracted by the activator Snf1p when glucose is exhausted from medium. Therefore, to test whether this regulatory circuit has been conserved in H. polymorpha, HpTUP1 and HpSNF1 were partially cloned and disrupted. Deletion of HpTUP1 did not affect glucose repression of the major C1 metabolism genes (MOX, DAS). Thus, though conserved, HpTUP1 does not seem to take part in a general glucose repression in H. polymorpha. In contrast, the deletion of HpSNF1 led to significant decreases in the activation of these genes in the absence of glucose. Therefore, the effect of HpSNF1 in transcriptional activation may be through an HpTUP1- independent circuit.

Mithramycin SK, a novel antitumor drug with improved therapeutic index, mithramycin SA, and demycarosyl-mithramycin SK: three new products generated in the mithramycin producer Streptomyces argillaceus through combinatorial biosynthesis.

To gain initial structure-activity relationships regarding the highly functionalized pentyl side chain attached at C-3 of mithramycin (MTM), we focused on a post-polyketide synthase (post-PKS) tailoring step of the MTM biosynthesis by Streptomyces argillaceus ATCC 12956, which was proposed to be catalyzed by ketoreductase (KR) MtmW. In this last step of the MTM biosynthesis, a keto group of the pentyl side chain is reduced to a secondary alcohol, and we anticipated the generation of an MTM derivative with an additional keto group in the 3-side chain. Insertional inactivation of mtmW, a gene located ca. 8 kb downstream of the mithramycin-PKS genes, yielded an S. argillaceus mutant, which accumulated three new mithramycin analogues, namely mithramycin SA, demycarosyl-mithramycin SK, and mithramycin SK (MTM-SK). The structures of these three compounds confirmed indirectly the proposed role of MtmW in MTM biosynthesis. However, the new mithramycin derivatives bear unexpectedly shorter 3-side chains (ethyl or butyl) than MTM, presumably caused by nonenzymatic rearrangement or cleavage reactions of the initially formed pentyl side chain with a reactive beta-dicarbonyl functional group. The major product, MTM-SK, was tested in vitro against a variety of human cancer cell lines, as well as in an in vitro toxicity assay, and showed an improved therapeutic index, in comparison to the parent drug, MTM.

Estimating dispersal and gene flow in the neotropical freshwater turtle Hydromedusa maximiliani (Chelidae) by combining ecological and genetic methods

Hydromedusa maximiliani is a vulnerable neotropical freshwater turtle endemic to mountainous regions of the Atlantic rainforest in southeastern Brazil. Random amplified polymorphic DNA (RAPD) was used to estimate the gene flow and dispersal for individuals inhabiting rivers and streams within a drainage. Nine primers generated 27 scoreable bands, of which 9 (33 per cent) were polymorphic and produced 12 RAPD phenotypes. The gene flow estimates (Nm) among turtles inhabiting different rivers and streams were variable, ranging from 0.09 to 3.00 (mean: 0.60). For some loci, the rates of gene flow could offset population differentiation (Nm > 1), whereas for others random genetic drift could result in population divergence (Nm < 1). Since the genetic variation of this turtle seems to be structured according to the natural hierarchical system of rivers and streams within drainages, management programs involving translocations between different regions across the geographical range of H. maximiliani should be viewed with caution