Rapid identification of monospecific monoclonal antibodies using a human proteome microarray

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.     

Aspergillus Gracilis Bainier var. Sartoryi (Biourge) Batista,Lima e Vital n. var. — Sua importáncia filogenética

Summary We deal with a new variety ofAspergillus which we callAspergillus gracilis Bainiervar. sartoryi (Biourge) Batista, Lima and Vital.This variety appears to be very important under the phylogenetic point of view, apparently being an intermediate form betweenAspergillus andPenicillium.Its morphology resemblesA. gracilis but the conidia characteristics are distinct; we took the epithetsartoryi fromA. sartoryi Biourge which was not described by the respective author.We suggest, too, the transfer of the seriesA. restrictus, where we put our new variety, from the groupA. glaucus, to become independant, as a connective series in the phylogenetic development betweenAspergillus andPenicillium, since the type of the series and the other elements that it embraces does not produce sclerotia or cleistothecia and the conidial apparatus offers curious similarity toPenicillium. Besides, some fungi of the series grow well in certain culture media, in complete discordance with the members of the groupA. glaucus.

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Publiçacão no 4

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Chefe do Instituto de Micologia;

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Diretor do Instituto de Antibióticos e Prof. de Microbiologia da Escola de Química;

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Assistente micologista do Instituto de Micologia — (Todos da Universidade do Recife).

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Os autores declinam o seu agradecimento ao Dr.Heraldo da Silva maia, Assistente micologista eD. Marilene Maranhão Moreira, Auxiliar-Técnico, do Instituto de Micologia, pela colaboração que lhes prestaram, durante a realização do presente trabalho.     

In vivo and laccase-catalysed decolourization of xenobiotic azo dyes by a basidiomycetous fungus: characterization of its ligninolytic system

Bioremediation is considered a promising eco-efficient alternative for industrial wastewater treatment. Particular attention is currently being given to biological degradation of synthetic dyes and more specifically to colour removal by fungi. This work looks at the extracellular enzymatic system of strain Euc-1. Its ability to decolourize 14 xenobiotic azo dyes was evaluated and compared with the well-known species Phanerochaete chrysosporium. Strain Euc-1 is a mesophilic white-rot basidiomycete, the main secreted ligninolytic enzyme being laccase (0.38 U ml^–1). Although low manganese-dependent peroxidase activity (0.05 U ml^–1) was also detected, neither lignin peroxidase nor aryl alcohol oxidase could be found in batch culture. Optimum pH values of 4.0 and 5.0 were obtained in the laccase-catalysed oxidation of guaiacol and syringaldazine, respectively. Laccase activity increased with the temperature rise up to 50–60 °C and remarkable thermal stability was observed at 50 °C with a half-life of 12 h and no deactivation within the first 2 h. Solid-plate decolourization studies showed that basidiomycete Euc-1 decolourized 11 azo dyes whereas P. chrysosporium only two. Moreover, it is shown that purified laccase from basidiomycete Euc-1 efficiently decolourizes the azo dye acid red 88.     

Traffic-related air pollution and respiratory symptoms among asthmatic children,resident in Mexico City: the EVA cohort study

Background

Taffic-related air pollution has been related to adverse respiratory outcomes; however, there is still uncertainty concerning the type of vehicle emission causing most deleterious effects.

Methods

A panel study was conducted among 147 asthmatic and 50 healthy children, who were followed up for an average of 22 weeks. Incidence density of coughing, wheezing and breathing difficulty was assessed by referring to daily records of symptoms and child''s medication. The association between exposure to pollutants and occurrence of symptoms was evaluated using mixed-effect models with binary response and poisson regression.

Results

Wheezing was found to relate significantly to air pollutants: an increase of 17.4 μg/m³ (IQR) of PM_2.5 (24-h average) was associated with an 8.8% increase (95% CI: 2.4% to 15.5%); an increase of 34 ppb (IQR) of NO₂ (1-h maximum) was associated with an 9.1% increase (95% CI: 2.3% to16.4%) and an increase of 48 ppb (IQR) in O₃ levels (1 hr maximum) to an increase of 10% (95% CI: 3.2% to 17.3%). Diesel-fueled motor vehicles were significantly associated with wheezing and bronchodilator use (IRR = 1.29; 95% CI: 1.03 to 1.62, and IRR = 1.32; 95% CI: 0.99 to 1.77, respectively, for an increase of 130 vehicles hourly, above the 24-hour average).

Conclusion

Respiratory symptoms in asthmatic children were significantly associated with exposure to traffic exhaust, especially from natural gas and diesel-fueled vehicles.     

A closer look at intraspecific variation of Cerataphis brasiliensis (Hempel) (Hemiptera: Hormaphididae)

The aim of our study was to characterize Cerataphis brasiliensis Hempel, an aphid of native palm tree of tropical savannas (cerrado), and to record intraspecific variation. We found two parthenogenetic forms of apterous viviparae females of C. brasiliensis. We differentiate the two forms through measurable and qualitative characters: (i) length of cephalic horns, (ii) number of antennal segments, (iii) presence or absence of wax glands, (iv) location of siphunculi.     

The Arabidopsis kinase-associated protein phosphatase regulates adaptation to Na+ stress

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.     

Developmental changes in plant resistance to water flow in Pisum sativum (L.)

Soil and plant resistance to water flow under field conditions in pea (Pisum sativum L.) plants were measured at six ages. Transpiration flux, leaf and soil water potentials were used to calculate the total resistance to water flow using the Ohm's law analogy. Plant resistance was estimated from the slope of the water potential difference () vs. transpiration (Q) relationship. Plant growth, root density and soil water content distribution were measured. Leaf area and root length both increased until the end of seed filling and decreased during seed maturation. Total resistance decreased with the transpiration flux in a non-linear relationship. Plant resistance estimated as the slope of the vs. Q regression line increased until pod filling and then decreased. The increased resistance to water flow during pod filling was associated with a 10% increase in cell wall thickness.     

Isolation and characterization of <Emphasis Type="Italic">shs1</Emphasis>, a sugar-hypersensitive and ABA-insensitive mutant with multiple stress responses

To identify salt tolerance determinants, we screened for double mutants from a T-DNA tagged sos3-1 mutant population in the Arabidopsis Col-0 gl1 background. The shs1-1 (sodium hypersensitive) sos3-1 mutant was isolated as more sensitive to NaCl than sos3-1 plants. TAIL-PCR revealed that the introduced T-DNA was located 62 bp upstream of the initiation codon of an adenylate translocator-like protein gene on chromosome IV. SHS1 mRNA did not accumulate in shs1-1 sos3-1 plants although it accumulated in shoots of both sos3-1 and the wild type plants, indicating that this gene is inactive in the mutant. Genetic co-linkage analysis revealed that the mutation causing the phenotype segregated as a recessive, single gene mutation. This mutant showed altered sensitive responses to salt as well as to cold stress. It also demonstrated sugar sensitive and ABA insensitive phenotypes including enhanced germination, reduced growth, altered leaf morphology, and necrosis on leaves at an early growth stage. Sensitivity of sos3-1 shs1-1 root growth to LiCl, KCl, and mannitol was not significantly different from growth of sos3-1 roots. Further, expression of 35S::SHS1 in sos3-1 shs1-1 plants complemented NaCl and sugar sensitivity and partially restored the leaf morphology. G. Inan and F. Goto contributed equally in this work.     

Developmental regulation of water uptake in wheat

The discovery of aquaporins has provided a new basis for studying and interpreting water relations in plants. However, slow progress has been made in elucidating the functional facets of the aquaporin-mediated water pathway in whole plant systems. While increasing experimental evidence suggests that these proteins are directly involved in mediating water homeostasis at varying environmental conditions, only a few attempts have been made to understand their contribution to overall water transport at different developmental stages. By using a chemical inhibitor (HgCl(2)) of aquaporins function, here we present in planta evidence for both diurnal and developmental regulation of aquaporin activity in wheat. We demonstrate that the greatest sensitivity of water flux to pharmacological blockage occurs at the stage of ear emergence and does not coincide with the phenological stage at which the greatest plant water uptake occurs (milky ripeness). The relationship transpiration flux (Q) vs. soil-leaf water potential difference (DeltaPsi(soil-leaves)) revealed a gradual decrease of plant resistance to water flux from tillering to milky ripeness, both in HgCl(2)-treated and untreated control plants. However, the mercury-inhibition of water flux began to gradually increase at ear emergence, suggesting that a larger portion of water moves through aquaporins from this developmental stage on. Although the intercept of the DeltaPsi(soil-leaves)/Q regression line, i.e. the DeltaPsi required to initiate the water flux through the soil-plant-air continuum, was generally not affected by mercury treatment, a significant mercury effect on the intercept was observed at the stage of ear formation. These findings may have important implications for predicting which strategy plants utilize to optimize water use during their life cycle.     

Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.