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.

Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression.

Abundant data support a key role for the transcription factor nuclear factor-kappaB (NF-kappaB) signaling pathway in controlling the initiation and progression of human cancer. NF-kappaB and associated regulatory proteins such as IkappaB kinase (IKK) are activated downstream of many oncoproteins and there is much evidence for the activation of NF-kappaB-dependent target genes in a variety of solid tumors and hematologic malignancies. This review focuses on the mechanisms by which the NF-kappaB pathway is activated in cancer and on the oncogenic functions controlled by activated NF-kappaB. Additionally, the effects of NF-kappaB activation in tumors relative to cancer therapy are also discussed.

Ca(2+)-dependent permeabilization of the inner mitochondrial membrane by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955-19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

Low frequency of ankyrin mutations in hereditary spherocytosis: identification of three novel mutations.

Hereditary spherocytosis (HS) is a common hemolytic anemia caused by defects in the erythrocyte membrane proteins. The screening of mutations in the ankyrin-1 (ANK1) gene of 28 Brazilian HS patients showed two new missense mutations (His276Arg and Ile1054Thr) and one novel promoter mutation (-153 G-->A). The His276Arg mutation affected the invariable TPLH sequence on repeat 9. The -153 mutation was linked in cis to the known -108 T-->C mutation. In contrast to other populations, we were able to detect mutations in the ankyrin-1 gene in only 10% of our patients. It is also interesting to point out that, from 15 informative subjects for the 3' Acn repeats, only one presented a loss of heterozigosity at the cDNA level. Taken together, these results suggest that mutations in the ankyrin-1 gene might not be as common in Brazil as described for other populations.

beta-Spectrin São PauloII, a novel frameshift mutation of the beta-spectrin gene associated with hereditary spherocytosis and instability of the mutant mRNA.

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. beta-Spectrin-related HS seems to be common. We report here a new mutation in the beta-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (beta-spectrin São PauloII), exon 20. The mRNA encoding beta-spectrin São PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the beta-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, beta-spectrin São PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes.

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.

Expression of spectrin alpha I/65 hereditary elliptocytosis in patients from Brazil.

We report the clinical and laboratory findings in three unrelated families from southeastern Brazil with Sp alpha I/65 hereditary elliptocytosis (HE), including one homozygote and a patient presenting an elongated beta-spectrin. In family 1, three patients presented the allele alpha-Lely in trans to the elliptocytogenic allele. In these three patients the blood smear showed pronounced elliptocytosis, poikilocytosis and a few small red cell fragments instead of the mild elliptocytosis observed in their father, who did not present the polymorphism. In family 2 we describe one homozygote, with consanguineous parents presenting with anaemia, splenomegaly, severe poikilocytosis and elliptocytosis, budding, microspherocytes and numerous fragments in the blood smear. In family 3 we found an elongated beta Sp in a patient with Sp alpha I/65. The cause of the HE was the Sp alpha I/65 since the elongated beta Sp was not found in his brother, who also presented with HE and Sp alpha I/65. Apparently the abnormal beta Sp did not aggravate the HE, because both individuals had the same clinical and laboratory findings. However, the propositus presented a few more elliptocytes and poikilocytes than his brother, probably because the elongated beta-spectrin may have disturbed the spectrin self-association. In fact, in the propositus an abnormal band was observed in the nondenaturing gels, just above the Sp dimer, probably as a result of the association of the abnormal beta Sp with the normal spectrin chains. In the family studied here, both brothers presented the allele alpha Lely, but as their mother was dead, it was not possible to determine the polymorphism transmission. However, the high number of poikilocytes observed in the blood smear of both cases suggests an association in trans with the Sp alpha I/65. Thus, taken together, the data in this report indicate that HE secondary to Sp alpha I/65 abnormality is frequent in Brazil, and in one case it was associated with an apparently novel abnormal large beta-spectrin.

Association of the alpha-spectrin R28H mutation with allele alphaLELY and with alphaI/alphaII domain haplotypes in three Brazilian families.

We have studied three Brazilian kindreds presenting spectrin alpha/74 hereditary elliptocytosis (HE) due to a G-->A substitution, responsible for the R28H mutation. The mutant allele was associated with alphaI domain haplotype 1 (XbaI-/MspI-/PvuII+) in all three families and with two different alphaII domain haplotypes (1/RIT, 4/RVR). This result may reflect that this mutation occurs in a "hot spot" and may have arisen more than once or that a crossing over event may have occurred between the two domains studied. We detected one new haplotype in the alphaI domain (haplotype 3 -XbaI(+)/MspI(-)/PvuII(+)). The mutant allele was associated with the lack of the alphaII domain Alu insertion in all three cases. Allele alphaLELY, detected by PCR and restriction enzyme digestion, was present in the heterozygous form in patient 1 (alphaHE/alphaLELY) and in the homozygous form in patients 2 and 3(alphaHE-LEL/alphaLELY). It was found to be associated with domain haplotypes I (RIT) and 4 (RVR) and with the presence and absence of the Alu insertion. This may have arisen through recombination events, since this polymorphism is located in the alphaIV-alphaV domain junction, which is far distant from the alphaII domain.

Presence of allele alphaLELY in an Amazonian Indian population.

Allele alphaLELY is a low-expression allele of the erythroid spectrin alpha-chain that is characterized by a C --> G mutation at position alpha1857 in exon 40 and a C --> T (nt -12) mutation in intron 45. This second mutation is probably responsible for the partial skipping of exon 46. This exon is essential for the nucleation of the alpha-chains by the beta-chains during erythropoeisis. Although allele alphaLELY remains asymptomatic in both heterozygotes and homozygotes, it enhances the expression of deleterious alpha-alleles that occur and, as such, has clinical importance. In this study, the frequency of allele alphaLELY was estimated in two ethnically different Brazilian populations: a random sample of blood donors from Campinas, a city located in São Paulo State, in the southeastern region of Brazil, and a sample of Parakanã Indians (Tupi tribe), a very isolated population with a high degree of inbreeding. The frequency of allele alphaLELY in the blood donor's sample (n = 54) was 24.1% whereas in the indigenous sample (n = 41), it was 15.9%. These frequencies were not significantly different at the 5% level (chi2 = 1.931). Similarly, when the frequencies of our samples were compared with those of the four ethnic groups studied by Maréchal et al. [Br J Haematol 90:553-556, 1995], no significant differences were found at the 5% level (chi2 = 6.686). These results suggest that allele alphaLELY is a very ancient allele since it occurs with a relatively uniform and high frequency in all human ethnic groups studied so far. These findings confirm the importance of allele alphaLELY in influencing the expression of deleterious alpha-spectrin alleles. To our knowledge, these are the first data concerning allele alphaLELY in native Americans.

Expression of spectrin alphaI/50 hereditary elliptocytosis and its association with the alphaLELY allele.

Hereditary elliptocytosis (HE) is a group of hemolytic anemias characterized by the presence of elliptical erythrocytes. The underlying alterations lie in the proteins of the membrane skeleton. Defects of the alphaI domain of spectrin have been defined based on a decrease in the normal 80-kD alphaI domain and a concomitant increase in one or more lower molecular weight peptides. We have studied three Brazilian kindreds with black ancestry, who presented mild common spalphaI/50 HE. Our aim was to determine the molecular alteration responsible for the spalphaI/50 HE observed in these three kindreds and to evaluate the presence and influence of allele alphaLELY in the expression of this type of HE. In order to establish the molecular defect, exons 5, 6 and 11 were amplified and submitted to a nonradioactive single strand conformation polymorphism protocol. An identical band shift in exon 6 was observed in all 3 patients and their affected relatives. Direct sequencing of the amplification products of exon 6 showed the same molecular defect in all patients: a T-->C substitution, responsible for the L260P mutation. Allele alphaLELY, detected by PCR and restriction enzyme digestion, was present in the heterozygous form in the three propositi and was associated in trans with the elliptocytogenic mutation. Blood smears of the patients with HE and alphaLELY in trans showed pronounced elliptocytosis, poikilocytosis and a few small red cell fragments, whereas the blood smears of their relatives, who had HE without allele alphaLELY, showed mild common HE with a predominance of ovalocytes and the absence of poikilocytes. We conclude that allele alphaLELY does not lead to the worsening of clinical conditions when associated in trans with mild HE, but can be easily distinguished by a blood smear analysis. The predominance of the L260P mutation in the kindreds studied could be related to the colonization of Brazil during the slave trade by Africans from the Benin-Togo area, where this mutation is particularly common.

beta-Spectrin S(ta) Bárbara: a novel frameshift mutation in hereditary spherocytosis associated with detectable levels of mRNA and a germ cell line mosaicism.

Hereditary spherocytosis (HS) is a common inherited anaemia characterized by the presence of spherocytic red cells and by a heterogeneous nature in terms of its clinical presentation, molecular basis and inheritance. Defects in several membrane protein genes have been involved in the pathogenesis of HS, including defects in the beta-spectrin gene. We detected a novel frameshift mutation in the beta-spectrin gene, a C deletion at codon 638, in a patient presenting with HS and spectrin deficiency. The mutant protein was not detected in the membrane or in other cellular compartments, but detectable levels of mutant mRNA were found in the patient. Interestingly, this mutation was not present in the patient's parents, suggesting a genetic mosaicism, especially as the patient has an affected brother with the same molecular defect. We analysed DNA from different tissues of the parents and the mutation was absent from all tissues analysed. This mutation seems to be confined to the germ cell lineage of the patient's mother and must present a mosaic pattern in these cells as the patient also has unaffected siblings.

Beta-spectrin Campinas: a novel shortened beta-chain variant associated with skipping of exon 30 and hereditary elliptocytosis.

Beta-Spectrin Campinas is a novel spectrin variant associated with a shortened beta-chain in a kindred with hereditary elliptocytosis (HE). The propositus and her mother exhibited increased amounts of spectrin dimers and an increase in the alphaI 74 kD fragment from the alpha-chain after partial tryptic digestion of spectrin. The shortened beta-chain appeared as an additional band of approximately 200 kD on SDS-PAGE. In order to delineate the molecular defect of this abnormality at the gene level, reticulocyte mRNA was transcribed into cDNA and the last four exons of the beta-spectrin gene were amplified. Agarose gel of the amplification product of the propositus revealed the expected band of 487 bp as well as a shortened band of approximately 300 bp (size determined on gel). This shortened cDNA amplification product was cloned and nucleotide sequencing revealed the absence of the entire exon 30. In order to determine the underlying mutation responsible for this abnormal splicing, a genomic DNA fragment containing exons 30 and 31 was amplified and nucleotide sequencing revealed a G-->A substitution at the 5' donor splice site consensus sequence of intron 30 (nt + 1 IVS30). The skip splicing observed in this study results in a frameshift, creating a new stop codon and causing a deletion of 129 amino acids at the very COOH-terminus of the protein, thus impairing spectrin dimers self-association. We classified this HE as spherocytic HE because the propositus presented a few spherocytes in addition to many elliptocytes in the blood smear, whereas her mother, who was splenectomized, showed many schizocytes, poikilocytes and spherocytes.

Mutation analysis of the HFE gene in Brazilian populations.

We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 227 individuals from Brazil comprising 71 Caucasians, 91 racially mixed Caucasian African-derived Amerindians (both populations from Southeast Brazil), 85 African-derived subjects (from Northeast Brazil) and 75 Parakanã Indians. Allelic frequency of the mutation C. 845G(A (C282Y) was 1.4% in the Caucasian population, 1.1% in the African-derived population, 1.1% in the racially mixed normal controls and 0% in the Parakanã Indians. In the African-derived population, the C282Y mutation was present on chromosomes bearing the haplotype 6/1h according to Beutler and West (1997). Allelic frequency of the mutation C. 187C(G (H63D) was 16.3% in the Caucasian population, 7.5% in the African-derived population, 9.8% in the racially mixed controls and 0% in the Amerindians. The presence of these mutations in the African-derived population reflects the fact that these subjects may have undergone a non-identified racial admixture in their past history. The absence of both defects in the Amerindians suggests that these mutations have emerged after the migration of Polynesians to America, or that they may not have reached the Polynesian population until after the migration to America had occurred.

Erythrocyte ankyrin promoter mutations associated with recessive hereditary spherocytosis cause significant abnormalities in ankyrin expression.

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, -108T to C and -108T to C in cis with -153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human (A)gamma-globin gene expressed gamma-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant -108 promoter demonstrated variegated gamma-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked -108/-153 mutations. Three transgenic lines had undetectable levels of (A)gamma-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of (A)gamma-globin mRNA than wild type. Two of four expressing lines showed variegated gamma-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations.

ß-Spectrin Säo PauloII, a novel frameshift mutation of the ß-spectrin gene associated with hereditary spherocytosis and instability of the mutant mRNA

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. ß-Spectrin-related HS seems to be common. We report here a new mutation in the ß-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (ß-spectrin Säo PauloII), exon 20. The mRNA encoding ß-spectrin Säo PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the ß-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, ß-spectrin Säo PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes