Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).

A P-type ATPase from the aquatic fungus Blastocladiella emersonii similar to animal Na,K-ATPases.

We have cloned a P-type ATPase gene from the aquatic fungus Blastocladiella emersonii (BePAT1) using a probe obtained with degenerate oligonucleotides, corresponding to two amino acid sequences highly conserved among all P-type ATPase isoforms, and the polymerase chain reaction technique. Nucleotide sequence analysis revealed a 3.4 kb open reading frame encoding a putative peptide of 1080 amino acid residues with a calculated molecular mass of 119 kDa, which presents all diagnostic features of P-type transporting ATPases. Comparison to other members of the family and phylogenetic analyses have shown that the BePAT1 protein belongs to the subfamily of Na,K- and H,K-ATPases, indicating that the divergence between the alpha-subunit of the Na,K-ATPase and other members of the P-type ATPase family has occurred before the divergence between the animal and fungal lineages in evolution.

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter.

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.

Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell.

The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.

Genetic analysis of a temporally transcribed chemotaxis gene cluster in Caulobacter crescentus.

Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division.

General nonchemotactic mutants of Caulobacter crescentus.

We have examined 35 mutants that have defects in general chemotaxis. Genetic analysis of these mutants resulted in the identification of at least eight che genes located at six different positions on the Caulobacter crescentus chromosome. The cheR, cheB and cheT genes appeared to be located in a three-gene cluster. Mutations in these three genes resulted in the inability of the flagellum to reverse the direction of rotation. Defects in the cheR gene resulted in a loss of the ability to methylate the methyl-accepting chemotaxis proteins. In vitro experiments showed that the lack of in vivo methylation in cheR mutants was due to the absence of methyltransferase activity. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. The specific defects responsible for the lack of a chemotactic response have not been determined for the other identified che genes.

A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42 degrees C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Sp1 and two metal-responsive elements.

Cloning, structural analysis and expression of the gene encoding Hsp32 from Dictyostelium discoideum.

We have isolated and characterized genomic clones encoding a novel small heat shock (HS) protein (Hsp32) from Dictyostelium discoideum that is not homologous to the alpha-crystallin family Hsps. Besides its induction by HS, this gene is also regulated during the life cycle of the organism. At physiologic temperatures hsp32 is expressed at high levels in growing cells and at low levels in cells starved to initiate their developmental programme. However, in both cases the gene can be induced by HS. A DNA fragment containing the upstream region of hsp32 was shown to confer HS induction to a cat reporter gene, indicating a transcriptional regulation for this gene. A single transcription start site, located at position -152 relative to the initiator Met, 17 nucleotides downstream from a putative TATA box, was determined both in vegetative cells and cells starved for 6 h. This site was unchanged when either vegetative or starved cells were submitted to HS at 30 degrees C for 30 min. Despite HS induction, a perfect HSE element was not found in the 5' regulatory region of the gene. The hsp32 coding region is interrupted by a single intron located near its 5' end, which is properly spliced even under HS conditions.

Cloning of a cDNA encoding a novel heat-shock protein from Dictyostelium discoideum.

We have cloned, from Dictyostelium discoideum, a cDNA encoding a new heat-shock (HS) protein (Hsp) with a predicted molecular mass of 31,447 Da. Outside of its low molecular mass, this Hsp does not share any similarity with the small Hsp currently identified or with alpha-crystallins. Northern blot analysis indicates that this HS-inducible gene is also developmentally regulated

Investigation of 5-nitrofuran derivatives: synthesis, antibacterial activity, and quantitative structure-activity relationships.

Three sets of antibacterial nitrofuran derivatives [set I, 5-R-substituted (Z)-2-(5-nitrofuran-2-ylmethylene)-3(2H)-benzofuranones (R = OCH(3), H, CH(3), C(2)H(5), nC(3)H(7), Cl, Br, CN, and NO(2)) and their 2-hydroxyphenyl and 2-acetoxyphenyl analogues; set II, 5-R-substituted (E)-1-(2-hydroxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3), C(2)H(5), Cl, and NO(2)); and set III, 5-R-substituted (E)-1-(2-acetoxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3); C(2)H(5), Cl, and NO(2))] were prepared and tested against a Gram-positive (Staphylococcus aureus, strain ATCC-25923) and a Gram-negative bacterium (Caulobacter crescentus, strain NA 1000). QSAR equations derived for the IC(50) values against both bacteria show negative contributions of two terms: an electronic one, expressed either by sigma, the Hammett substituent constant, or by E, the cyclic voltametric reduction potential. Another term described by an indicator variable, I(abs), is assigned the value of 0 for set I compounds and the value of 1 for sets II and III. No important contribution of the hydrophobic factor was found. For the three sets, the QSAR regressions suggest that the same structural features describe the activities for both bacteria and that, although reduction is a necessary step, it should not be the determining one. These results agree with those found for the QSAR of 5-nitroimidazole analogues.

Heat shock alters poly(A) tail length of Dictyostelium discoideum hsp32 RNA.

Hsp32 is a heat shock gene in D. discoideum. We have previously observed that heat stress-induced change produces a broad band on Northern blots, suggesting that more than one population of mRNA is present under those conditions. This was not the result of a defect in the splicing of the hsp32 mRNA, nor did it result from the use of a different transcription start site under heat shock conditions. Here, we show that the broad banding pattern reflects the appearance of a transcript with a poly(A) tail that is approximately 100 nt longer than that seen in unstressed cells. Experiments indicated that this tail was not a property of newly synthesized mRNA but rather a response to heat stress. This response appeared to be specific to the hsp32 transcript and did not result in the retention of the RNA in the nucleus. These results document a relatively unusual heat shock response and also indicate that the nature of the response differs among RNAs and has selective consequences.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Efeito embriotóxico, teratogênico e abortivo de plantas medicinais / Embryotoxic, teratogenic and abortive effects of medicinal plants

O uso milenar de plantas medicinais mostrou ao longo dos anos, que determinadas plantas apresentam substâncias potencialmente perigosas. Do ponto de vista científico, algumas pesquisas mostraram que muitas dessas plantas possuem substâncias agressivas e por essa razão devem ser utilizadas com cuidado, respeitando seus riscos toxicológicos. Os efeitos mais preocupantes do uso indiscriminado de plantas medicinais são embriotóxico, teratogênico e abortivo, uma vez, que os constituintes da planta podem atravessar a placenta, chegar ao feto e gerar um desses efeitos. Este estudo objetiva fornecer uma listagem das principais plantas medicinais que tenham efeitos embriotóxicos, teratogênicos e abortivos comprovados, conhecendo as partes da planta utilizadas e seus respectivos nomes científicos, com a finalidade de alertar gestantes quanto aos riscos de seu uso. Realizou-se buscas nas bases eletrônicas de dados SciELO, PubMed, MEDLINE, LILACS, CAPES e Google acadêmico. Nos resultados encontrados, plantas como Arnica (Arnica montana), Artemísia (Artemisia vulgaris), Arruda (Ruta chalepensis/ Ruta graveolens), Barbatimão (Stryphnodendron polyphyllum), Boldo (Vernonia condensata) dentre outras, podem vir a gerar um desses efeitos. A partir deste estudo comprova-se que para a maioria das plantas medicinais não há dados a respeito da segurança de seu uso durante a gravidez.

The ancient use of medicinal plants has shown over the years that certain plants have potentially dangerous substances. From a scientific point of view, some studies have shown that many of these plants contain aggressive substances and therefore should be used with caution, respecting their toxicological risks. The most important effects of the indiscriminate use of medicinal plants are embryotoxic, teratogenic and abortifacient since the plant constituents can cross the placenta, reaching the fetus and leading to one of these effects. This study aimed to provide a list of the major medicinal plants that have proven embryotoxic, teratogenic and abortifacient effects, including the used plant parts and their respective scientific names, in order to warn pregnant women about the risks of its use. Searches were carried out in the electronic databases SciELO, PubMed, MEDLINE, LILACS, CAPES and Google Scholar. Results indicated that plants such as mountain arnica (Arnica montana), mugwort (Artemisia vulgaris), fringed rue (Ruta chalepensis / Ruta graveolens), "Barbatimão" (Stryphnodendron polyphyllum) and "Boldo" (Vernonia condensata) are likely to generate such an effect. This study shows that for most medicinal plants there are not data regarding the safety of their use during pregnancy.

PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases.

During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N-terminal extension may only play a role in C subunit degradation.