Configurational effects in antibody–antigen interactions studied by microcalorimetry

In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high-sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is ?350 ± 60 cal K^?1 mol^?1 while for E8 it is ?165 ± 40 cal K^?1 mol^?1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25°C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (?77 ± 15 vs. ?34 ± 11 cal K^?1 mol^?1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen–deuterium exchange data, detected using 2D NMR, on the two antibody–antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3. These results were interpreted as suggesting that a larger surface area was buried on cytochrome c upon binding to E3 than to E8, and that larger changes in configurational entropy occur upon binding of E3 than E8. These findings are confirmed by the present study using isothermal titration calorimetry. © 1995 Wiley-Liss, Inc.     

Genomic sequencing of different sequevars of Ralstonia solanacearum belonging to the Moko ecotype

Banana vascular wilt or Moko is a disease caused by Ralstonia solanacearum. This study aimed to sequence, assemble, annotate, and compare the genomes of R. solanacearum Moko ecotypes of different sequevar strains from Brazil. Average nucleotide identity analyses demonstrated a high correlation (> 96%) between the genome sequences of strains CCRMRs277 (sequevar IIA-24), CCRMRs287 (IIB-4), CCRMRs304 (IIA-24), and CCRMRsB7 (IIB-25), which were grouped into phylotypes IIA and IIB. The number of coding sequences present in chromosomes and megaplasmids varied from 3,070 to 3,521 and 1,669 to 1,750, respectively. Pangenome analysis identified 3,378 clusters in the chromosomes, of which 2,604 were shared by all four analyzed genomes and 2,580 were single copies. In megaplasmids, 1,834 clusters were identified, of which 1,005 were shared by all four genomes and 992 were identified as single copies. Strains CCRMRsB7 and CCRMRs287 differed from the others by having unique clusters in both their chromosomes and megaplasmids, and CCRMRsB7 possessed the largest genome among all Moko ecotype strains sequenced to date. Therefore, the genomic information obtained in this study provides a theoretical basis for the identification, characterization, and phylogenetic analysis of R. solanacearum Moko ecotypes.     

Pathogenic Variability of Isolates of Pseudocercospora griseola, the Cause of Common Bean Angular Leaf Spot, and its Implications for Resistance Breeding

The pathogenic variability was evaluated of 48 Pseudocercospora griseola isolates collected in the State of Minas Gerais, Brazil. Isolates were inoculated to a set of 12 international differential cultivars in a greenhouse. Ten pathotypes (55-15, 63-7, 63-15, 63-23, 63-25, 63-27, 63-31, 63-47, 63-55 and 63-63) were identified, showing the great pathogenic variability of this fungus in Minas Gerais State. Pathotypes 55-15, 63-15, 63-25 and 63-27 had not previously been reported in the State. Of the 48 isolates, all except pathotype 55-1547 induced a compatible reaction with all cultivars from the Andean group. Isolates were highly pathogenic in both Andean and Mesoamerican cultivars, thus being classified as Mesoamerican pathotypes. Pathotype 63-63 was the most widespread, and overcame the resistance genes present in all differential cultivars.     

MDM2, P53, P21WAF1 and pAKT protein levels in genesis and behaviour of adenoid cystic carcinoma

Background: MDM2, P53, P21^WAF1 and pAKT are proteins associated with the balance between cell death and survival. There are many hypotheses regarding the role of these proteins in salivary gland tumours. However, many molecular events that activate or inactivate regulatory genes remain unknown. The aim of this study was to evaluate and to correlate MDM2, P53, P21^WAF1 and pAKT protein expressions in adenoid cystic carcinomas (ACC). Methods: Twenty-two cases of ACC were evaluated by immunohistochemistry and one cell line derived from ACC was analyzed by Western Blotting and immunofluorescence techniques. Results: Strong MDM2 and pAKT, variable P53 and null P21 expressions were found in the cases analyzed, but no statistical correlation was established when comparing MDM2 and pAKT expressions in the 3 different ACC subtypes. The ACC cell line showed intense nuclear and cytoplasmatic MDM2 and pAKT expressions and null P53 and P21 expressions. Conclusions: Results indicate that MDM2 and pAKT are related to the tumorigenesis of ACC, but they might not be directly connected to tumour progression. We also demonstrate that the pAKT pathway is active in ACC and it seems to be activating the MDM2 shuttle from the cytoplasm to the nucleus, where it phosphorylates P53 and carries it to the cytoplasm for degradation.     

Intestinal helminths in wild Peruvian red uakari monkeys (Cacajao calvus ucayalii) in the northeastern Peruvian Amazon

Parasites are important in the management of the health of primate populations. We examined 36 fecal samples from Peruvian red uakari monkeys (Cacajao calvus ucayalii) collected from wild animals in the northeastern Peruvian Amazon. Samples were positive for helminth infection. Nematodes egg: Strongyloididae, Trypanoxyuris sp., Spirurid, and a cestode egg were identified.     

Physicochemical and biological properties of the coffee (Coffea arabica) rhizosphere suppress the root-knot nematode Meloidogyne exigua

ABSTRACT

We investigated the properties of rhizospheric soils infested with root-knot nematode (RKN) Meloidogyne exigua in 17 coffee (Coffea arabica) farms from the Southern region of Minas Gerais, Brazil. Physicochemical (pH, clay and organic matter) and biological properties (RKN parasites and microbiota volatile toxicity on M. exigua) were correlated with the number of second-stage juveniles (J₂) and the egg hatching of M. exigua extracted from those rhizospheres. In the five most suppressive farms, the number of J₂ was less than 50/100?g of soil and the egg hatching was significantly low. The bacterium Pasteuria penetrans was found in four of the most suppressive farms with an average of 30% of J₂ infected with endospores. By using in vitro experiments the microbiota volatiles emitted from the most suppressive soils killed more than 83% of the J₂. Additionally, volatiles produced by Fusarium oxysporum, Cladosporium sp. and Syncephalastrum sp. isolated from M. exigua eggs, significantly killed the J₂. Identification of nematicidal compounds from the soils by GC-MS supported the strong involvement of the microbiota volatile toward RKN suppressiveness. Clay percentage and pH were similar in farms with the most suppressive soils (42.5% and 6.6%, respectively). Finally, the most suppressive soils came from farms with the highest coffee bean yields. Collectively, these results suggest the strong involvement of parasitic microorganisms, clay percentage and the pH suppressing RKN in soils from the major coffee production region in Brazil, and that volatiles emitted from total microbiota and exclusively from egg-isolated fungi are toxic to M. exigua.     

Effects of phosphonium-based ionic liquids on the lipase activity evaluated by experimental results and molecular docking

In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P₄₄₄₄]⁺, [P_444(14)]⁺, [P_666(14)]⁺) and anions (Cl⁻, Br⁻, [Deca]⁻, [Phosp]⁻, [NTf₂]⁻) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]⁻ and [NTf₂]⁻ increase the BCL activity, while the remaining [P_666(14)]-based ILs with the Cl⁻, Br⁻, and [Deca]⁻ anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P_666(14)][NTf₂] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L⁻¹). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]⁻ has a higher binding affinity compared to Cl⁻ and Br⁻, and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]⁻ and [NTf₂]⁻ have high binding energies (−6.2 and −5.6 kcal·mol⁻¹, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.