Breast cancer under age 40: a different approach.

Breast cancer (BC) under age 40 is a complex disease to manage due to the additionally fertility-related factors to be taken in consideration. More than 90% of young patients with BC are symptomatic. Women<40 years are more likely to develop BC with worse clinicopathological features and more aggressive subtype. This has been frequently associated with inferior outcomes. Recently, the prognostic significance of age<40 has been shown to differ according to the BC subtype, being associated with worst recurrence-free survival (RFS) and overall survival (OS) for luminal BC. The biology of BC<40 has also been explored through analysis of large genomic data set, and specific pathways overexpressed in these tumors have been identified which can lead to the development of targeted therapy in the future. A multidisciplinary tumor board should determine the optimal locoregional and systemic management strategies for every individual patient with BC before the start of any therapy including surgery. This applies to both early (early breast cancer (EBC)) and advanced (advanced breast cancer (ABC)) disease, before the start of any therapy. Mastectomy even in young patients confers no overall survival advantage when compared to breast-conserving treatment (BCT), followed by radiotherapy. Regarding axillary approach, indications are identical to other age groups. Young age is one of the most important risk factors for local recurrence after both breast-conserving surgery (BCS) and mastectomy, associated with a higher risk of distant metastasis and death. Radiation after BCS reduces local recurrence from 19.5 to 10.2% in BC patients 40 years and younger. The indications for and the choice of systemic treatment for invasive BC (both early and advanced disease) should not be based on age alone but driven by the biological characteristics of the individual tumor (including hormone receptor status, human epidermal growth factor receptor 2 (HER-2) status, grade, and proliferative activity), disease stage, and patient's comorbidities. Recommendations regarding the use of genomic profiles such as MammaPrint, Oncotype Dx, and Genomic grade index in young women are similar to the general BC population. Especially in the metastatic setting, patient preferences should always be taken into account, as the disease is incurable. The best strategy for these patients is the inclusion into well-designed, independent, prospective randomized clinical trials. Metastatic disease should always be biopsied whenever feasible for histological confirmation and reassessment of biology. Endocrine therapy is the preferred option for hormone receptor-positive disease (HR+ve), even in presence of visceral metastases, unless there is concern or proof of endocrine resistance or there is a need for rapid disease response and/or symptom control. Recommendations for chemotherapy (CT) should not differ from those for older patients with the same characteristics of the metastatic disease and its extent. Young age by itself should not be an indication to prescribe more intensive and combination CT regimens over the sequential use of monotherapy. Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) represent an important group of promising drugs in managing patients with breast cancer susceptibility gene (BRCA)-1- or BRCA-2-associated BC. Specific age-related side effects of systemic treatment (e.g., menopausal symptoms, change in body image, bone morbidity, cognitive function impairment, fertility damage, sexual dysfunction) and the social impact of diagnosis and treatment (job discrimination, taking care for children) should also be carefully addressed when planning systemic long-lasting therapy, such as endocrine therapy. Survivorship concerns for young women are different compared to older women, including issues of fertility, preservation, and pregnancy.

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbß3 or αVß3 and inhibits platelet aggregation and angiogenesis.

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbß3 and endothelial cell αVß3 integrins, but not for α5ß1 or α2ß1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 µM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbß3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbß3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbß3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Antimicrobial activity of Lippia sidoides Cham. (Verbenaceae) essential oil against Staphylococcus aureus and Escherichia coli / Atividade antimicrobiana do óleo essencial de Lippia sidoides Cham. (Verbenaceae) contra Staphylococcus aureus e Escherichia coli

The antibacterial effect of Lippia sidoides (rosemary pepper) essential oil was tested against the bacteria Staphylococcus aureus and Escherichia coli isolated from homemade Minas cheese produced in Brazil. The Minimum Inhibitory Concentration (MIC) determined in the Dilution Test was 13 µL oil mL-1 for both bacteria, which characterizes inhibitory action in broth for a 24-hour interaction period. The Minimum Bactericidal Concentration (MBC) determined in the Suspension Test, with one minute of contact, was 25 µL oil mL-1 for both tested bacteria, obtaining at this concentration a bactericidal effect of 99.9 percent on the viable bacterial cells from each sample. Results demonstrated the bacterial activity of Lippia sidoides essential oil against S. aureus and E. coli, suggesting its use as an antibacterial agent in foods.

O efeito antibacteriano do óleo essencial da Lippia sidoides (alecrim-pimenta) foi testado contra as bactérias Staphylococcus aureus e Escherichia coli isoladas de queijo minas artesanal, produzido no Brasil. A Concentração Inibitória Mínima (CIM) determinada por Teste de Diluição foi de 13 µL de óleo mL-1 para as duas bactérias, o que caracteriza ação inibitória em caldo durante vinte quatro horas de interação. A Concentração Bactericida Mínima (CBM) determinada através de Teste de Suspensão, com um minuto de contato, foi de 25 µL de óleo mL-1 para ambas as bactérias testadas, obtendo-se a esta concentração o efeito bactericida de 99,9 por cento sobre as células bacterianas viáveis em cada uma das amostras. Os resultados demonstraram atividade bactericida do óleo essencial de Lippia sidoides contra S. aureus e E. coli, sugerindo a possibilidade do uso como agente antibacteriano em alimentos.

An insight into the sialotranscriptome of the non-blood feeding Toxorhynchites amboinensis mosquito.

All adult mosquitoes take sugar meals, and most adult females also take blood meals to develop eggs. Salivary glands (SG) of males are thus much smaller and do not contain many of the antihemostatic and antiinflammatory compounds found in females. In the past 5 years, transcriptome analyses have identified nearly 70 different genes expressed in adult female SG. For most of these, no function can be assigned in either blood or sugar feeding. Exceptionally, Toxorhynchites mosquitoes are unusual in that they never feed on blood, and the SG of adults are identical in both sexes. Transcriptome analysis of the adult SG of this mosquito was performed to increase knowledge of the evolution of blood feeding--and to identify polypeptide families associated with sugar feeding--in mosquitoes.

Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids.

Dietary cis-9, trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect, in vitro incubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. Linoleic acid (LA) or alpha-linolenic acid (LNA) was incubated (1.67 g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay-concentrate ration. Adding fish oil (up to 4.17 g/l) tended to decrease the initial rate of LA (P=0.025) and LNA (P=0.137) disappearance, decreased (P<0.05) the transient accumulation of conjugated isomers of both fatty acids, and increased (P<0.05) the accumulation of trans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100 mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50 mg/l) to pure cultures inhibited the growth and isomerase activity of B. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised by B. fibrisolvens in pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects on B. fibrisolvens.

Comparative genomics of insect juvenile hormone biosynthesis.

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).

Analysis of networks based on styrene and divinylbenzene containing iron anchored using variable pressure scanning electron microscopy.

There is great demand for the development of composite materials containing small metal or metal oxides particles, owing to their variable properties and wide application. However, microscopic evaluation of these materials using high-vacuum scanning electron microscopy is difficult because the samples must undergo a series of preparation steps to reach a high image quality and to avoid becoming shrunk inside the microscope vacuum chamber. Thus, in this study, we used variable pressure scanning electron microscopy to evaluate the morphology and iron distribution on the surface of magnetic microspheres based on poly(styrene-co-divinylbenzene). These materials were obtained by suspension copolymerization of styrene and divinylbenzene in the presence of fine iron particles. Energy-dispersive X-rays were also used to analyse distribution of the iron particles. The results indicate that, under the conditions used, magnetic microspheres with a relatively narrow size distribution were formed. Moreover, the micrographs show that agglomerated iron particles appeared only on the microsphere surface.

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi.

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector-parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1-Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40-60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.

Toward a description of the sialome of the adult female mosquito Aedes aegypti.

To describe the set of mRNA and protein expressed in the salivary glands (sialome) of Aedes aegypti mosquitoes, we randomly sequenced a full-length cDNA library of this insect and performed Edman degradation of PVDF-transferred protein bands from salivary homogenates. We found 238 cDNA clusters which contained those coding for 10 of the 11 proteins found by aminoterminal degradation. All six previously described salivary proteins were found in this library. Full-length sequences of 32 novel cDNA sequences are reported, one of which is the product of a transposable element. Among the 31 novel protein sequences are 4 additional members of the D7 protein family; 4 novel members of the antigen 5 family (a protein family not reported in Aedes); a novel serpin; a novel member of the 30-kDa allergen of Ae. Aegypti; a secreted calreticulin; 2 proteins similar to mammalian angiopoietins; adenosine deaminase; purine hydrolase; lysozyme; a C-type lectin; 3 serine proteases, including one with high similarity to Bombyx prophenoloxidase activating enzyme; 2 proteins related to invertebrate immunity; and several sequences that have no significant matches to known proteins. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed.

The salivary adenosine/AMP content of Phlebotomus argentipes Annandale and Brunetti, the main vector of human kala-azar.

Adenosine and AMP in the salivary glands of the sand fly Phlebotomus argentipes were characterized by reversed-phase high-pressure liquid chromatography with diode-array detection and mass spectrometry. AMP and adenosine were measured in individual salivary gland pairs, yielding 76.8 +/- 8.6 and 380 +/- 25 pmoles per pair of salivary glands, respectively (mean +/- SE, n = 12). These values decrease to 45 +/- 7 and 181 +/- 21 pmoles following a blood meal, indicating that AMP and adenosine were secreted. Because adenosine and AMP have anti-platelet, vasodilatory, and immunomodulatory properties, it is proposed that these salivary nucleotides help the fly to blood feed and may affect Leishmania transmission.

The salivary adenosine deaminase activity of the mosquitoes Culex quinquefasciatus and Aedes aegypti.

A cDNA coding for a protein with significant similarity to adenosine deaminase (ADA) was found while randomly sequencing a cDNA library constructed from salivary gland extracts of adult female Culex quinquefasciatus. Prompted by this result, we found high ADA activities in two culicine mosquitoes, Culex quinquefasciatus and Aedes aegypti, but not in the anopheline Anopheles gambiae. Homogenates from Culex quinquefasciatus also have an AMP deaminase activity that is three times greater than the ADA activity, whereas in Aedes aegypti the AMP deaminase activity is less than 10% of the ADA activity. Evidence for secretion of ADA during blood feeding by Aedes aegypti includes the presence of ADA activity in warm solutions probed through a membrane by mosquitoes and in serotonin-induced saliva and a statistically significant reduction in the levels of the enzyme in Aedes aegypti following a blood meal. We could not demonstrate, however, that C. quinquefasciatus secrete ADA in their saliva. Male Aedes aegypti and C. quinquefasciatus, which do not feed on blood, have less than 3% of the levels of ADA found in females. We propose that ADA activity in A. aegypti may help blood feeding by removing adenosine, a molecule associated with both the initiation of pain perception and the induction of mast cell degranulation in vertebrates, and by producing inosine, a molecule that potently inhibits the production of inflammatory cytokines. The role of salivary ADA in Culex quinquefasciatus remains unclear.

The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins.

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.

Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase.

Free ADP-ribose has a reducing ribose moiety and it is hazardous due to its nonenzymic reactivity toward protein side chains. ADP-ribose hydrolases are putative protective agents to avoid the intracellular accumulation of ADP-ribose. In mammalian sources, two types of enzymes with ADP-ribose hydrolase activity are known: (i) highly specific ADP-ribose pyrophosphatases, which in a Mg(2+)-dependent fashion hydrolyse only ADP-ribose and the nonphysiological analogue IDP-ribose, and (ii) less specific nucleoside diphosphosugar or diphosphoalcohol (NDP-X) pyrophosphatases, which besides A(I)DP-ribose hydrolyse also some nonreducing NDP-X substrates. So far, of these two enzyme types only the less specific one has been reported in human sources: an ADP-sugar pyrophosphatase purified from erythrocytes or expressed from cDNA clones. Here we report that human placenta extracts contain two ADP-ribose hydrolases, which were characterised after a near 1000-fold purification. One is an ADP-sugar pyrophosphatase: it hydrolysed ADP-ribose, ADP-glucose and ADP-mannose, but not e.g. UDP-glucose, at similar rates. It resembles the erythrocyte and recombinant enzyme(s), but showed a 5-20-fold lower K(m) for ADP-ribose (7 microM). The other enzyme is a highly specific ADP-ribose pyrophosphatase (the first of this kind to be reported in humans): it hydrolysed only ADP-ribose and IDP-ribose at similar rates, with a very low, 0.4 microM K(m) for the former. This is a major candidate to control the accumulation of free ADP-ribose in humans. It remains to be seen whether it belongs to the 'nudix' protein family, which includes several ADP-ribose hydrolases and other 'housecleaning' enzymes (M.J. Bessman, D.N. Frick, S.F. O'Handley, J. Biol. Chem. 271 (1996) 25059-25062).

The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases.

Apyrases are enzymes that hydrolyze nucleotide di- and triphosphates to orthophosphate and mononucleotides. At least two families of enzymes, belonging to the 5'-nucleotidase and to the actin/heat shock 70/sugar kinase superfamily, have evolved independently to serve the apyrase reaction. Both families require either Ca(2+) or Mg(2+) for their action. A novel apyrase enzyme sequence, with no homology to any other known protein sequence, was found recently in the salivary glands of the hematophagous bed bug Cimex lectularius. This enzyme functions exclusively with Ca(2+). Here, we report the finding of a cDNA similar to that of the C. lectularius salivary apyrase isolated from a salivary gland cDNA library of Phlebotomus papatasi. Transfection of insect cells with the P. papatasi salivary gland apyrase cDNA resulted in the secretion of a Ca(2+)-dependent apyrase whose activity was indistinguishable from that in salivary homogenates of P. papatasi. Homologous sequences were found in humans, in another sand fly (Lutzomyia longipalpis), in the fruit fly Drosophila melanogaster, in the nematode Caenorhabditis elegans and in the protozoan Cryptosporidium parvum, indicating that this family of enzymes is widespread among animal species.

The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis.

In the process of sequencing a subtracted cDNA library from the salivary glands of the sand fly Lutzomyia longipalpis, we identified a cDNA with similarities to gene products of the adenosine deaminase family. Prompted by this cDNA finding, we detected adenosine deaminase activity at levels of 1 U/mg protein in salivary gland homogenates. The activity was significantly reduced following a blood meal indicating its apparent secretory fate. The native enzyme has a K(m) of approximately 10 microM, an isoelectric pH between 4.5 and 5.5, and an apparent molecular weight of 52 kDa by size exclusion chromatography. The possible role of this enzyme, which converts adenosine to inosine, in the feeding physiology of L. longipalpis is discussed.

Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus.

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut.

The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.