Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Camelid nanobodies raised against an integral membrane enzyme,nitric oxide reductase

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N₂O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.     

Observation of brown-throated three-toed sloths: mating behaviour and the simultaneous nurturing of two young

Bradypus variegatus is a member of the Order Pilosa, Family Bradypodidae, and is distributed in many subtropical and tropical countries in South and Central America. However, studies on this species in the wild are relatively limited and many aspects of its reproductive behaviour are unknown or unclear. The current report presents new observations of the reproductive behaviour of B. variegatus in its natural environment. These include details of both a male–female copulation and the simultaneous nurturing of two young sloths.     

Hunting restraint by Creoles at the Community Baboon Sanctuary, Belize: a preliminary survey

This study surveyed 33 male hunters between the ages of 17 and 54 at the Community Baboon Sanctuary (CBS), Belize, to evaluate attitudes and behaviors in relation to hunting black howler monkeys (Alouatta pigra). The study defined hunting restraint as a learned predisposition not to hunt 1 or more species of nonhuman animal. Consistent with Belizean folklore, Creoles at the CBS exhibited hunting restraint with respect to black howlers, preferring to kill bushmeat other than monkeys. The most cited reasons for the observed hunting restraint were utilitarian. Historical and cultural factors also appeared significant. The study results are interpreted in terms of economic theory and suggest that disinhibition of hunting restraint might depend on changes in opportunity costs of hunting these primates. Nonetheless, a change in hunting attitudes and behaviors by Belizean Creoles seems unlikely in the near future because the local and national government and the benefits of ecotourism economically and legally protect howlers. The Creoles' culturally transmitted hunting restraint also culturally protects A. pigra, and the species is not a preferred source of food.     

Norfloxacin Zn(II)-based complexes: acid base ionization constant determination, DNA and albumin binding properties and the biological effect against Trypanosoma cruzi

Zn(II) complexes with norfloxacin (NOR) in the absence or in the presence of 1,10-phenanthroline (phen) were obtained and characterized. In both complexes, the ligand NOR was coordinated through a keto and a carboxyl oxygen. Tetrahedral and octahedral geometries were proposed for [ZnCl₂(NOR)]·H₂O (1) and [ZnCl₂(NOR)(phen)]·2H₂O (2), respectively. Since the biological activity of the chemicals depends on the pH value, pH titrations of the Zn(II) complexes were performed. UV spectroscopic studies of the interaction of the complexes with calf-thymus DNA (CT DNA) have suggested that they can bind to CT DNA with moderate affinity in an intercalative mode. The interactions between the Zn(II) complexes and bovine serum albumin (BSA) were investigated by steady-state and time-resolved fluorescence spectroscopy at pH 7.4. The experimental data showed static quenching of BSA fluorescence, indicating that both complexes bind to BSA. A modified Stern–Volmer plot for the quenching by complex 2 demonstrated preferential binding near one of the two tryptophan residues of BSA. The binding constants obtained (K _b ) showed that BSA had a two orders of magnitude higher affinity for complex 2 than for 1. The results also showed that the affinity of both complexes for BSA was much higher than for DNA. This preferential interaction with protein sites could be important to their biological mechanisms of action. The analysis in vitro of the Zn(II) complexes and corresponding ligand were assayed against Trypanosoma cruzi, the causative agent of Chagas disease and the data showed that complex 2 was the most active against bloodstream trypomastigotes.     

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7 Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.     

Burkholderia sp. SCMS54 reduces cadmium toxicity and promotes growth in tomato

Cadmium (Cd) can enter soil through the use of fertilisers, calcareous, pesticides and industrial and/or domestic effluents. Cd can leach into groundwater and be taken up by plants, potentially leading to reductions in plant growth and yield. In soil, plant roots interact with heavy metal (HM)‐tolerant microorganisms that may promote plant growth. Soil microorganisms may also be able to solubilise or mobilise soil metals, thereby acting as bioremediators. A better understanding of the interaction among plants, metals, microorganisms and soil will lead to improved plant tolerance. Two multi‐tolerant bacteria from the Burkholderia genus were isolated from Cd‐contaminated and Cd‐uncontaminated soil of a coffee plantation. In addition to its high tolerance to Cd, the strain SCMS54 produces indole‐acetic acid (IAA), solubilises inorganic phosphate and produces siderophores, demonstrating its potential to contribute to beneficial plant–microorganism interactions. When interacting with tomato plants exposed to Cd, the bacterium led to decreases in plant peroxide and chlorosis levels, promoted relative plant growth and decreased the root absorption of Cd, resulting in increased plant tolerance to this highly toxic HM. The results indicated that the inoculation of tomato plants with Burkholderia sp. SCMS54 promotes better growth in plants cultivated in the presence of Cd. This phenomenon appears to be attributed to a mechanism that decreases Cd concentrations in the roots via a beneficial interaction between the bacteria and the plant roots.