Crystal structure of the lectin of Camptosema pedicellatum: implications of a conservative substitution at the hydrophobic subsite

Lectins have been used as models for studies of the molecular basis of protein-carbohydrate interaction and specificity by deciphering codes present in the glycan structures. The purpose of the present study was to purify and solve the complete primary and crystal structure of the lectin of Camptosema pedicellatum (CPL) complexed with 5-bromo-4-chloro-3-indolyl-α-d-mannose (X-Man) using tandem mass spectrometry. CPL was purified by single-step affinity chromatography. Mass spectrometry findings revealed that purified CPL features a combination of chains weighing 25,298 ± 2 (α-chain), 12,835 ± 2 (β-chain) and 12,481 ± 2 Da (γ-chain). The solved crystal structure of CPL features a conservative mutation in the hydrophobic subsite, a constituent of the carbohydrate recognition domain (CRD), indicating the relevance of hydrophobic interactions in the establishment of interactions with carbohydrates. The substitution and the analysis of the interactions with X-Man also revealed that the hydrophobic effect caused by a minor change in the hydrophobic subsite interferes in the formation of H-bonds due to the reorientation of the indolyl group in the CRD.     

Rab35 and glucocorticoids regulate APP and BACE1 trafficking to modulate Aβ production

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms, including overproduction of toxic amyloid-β (Aβ) peptides and intraneuronal accumulation of hyperphosphorylated Tau protein. The latter is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in Aβ overproduction remains an open question. Here, we find that hippocampal Rab35 levels are decreased not only by stress/GC but also by aging, another AD risk factor. Moreover, we show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking steps for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Finally, we demonstrate that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by high GC levels. These studies identify Rab35 as a key regulator of APP processing and suggest that its downregulation may contribute to stress-related and AD-related amyloidogenesis.Subject terms: Cellular neuroscience, Alzheimer''s disease     

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K _b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K _b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.     

苎麻光合生理生态特性研究

以大田栽培的苎麻植株为材料,用TPS-2便携式光合作用测定系统测定自然条件下生长的苎麻叶片的光合气体交换参数,以及光响应曲线和CO2响应曲线,并通过回归和相关法分析探讨净光合速率与主要生理、生态因子间的关系.结果表明:(1)苎麻叶片的净光合速率(Pn)日变化曲线呈现双峰型,2个光合峰高度接近,其净光合速率具有典型的午休"现象;蒸腾速率(Tr)日变化曲线呈现单峰型,其走势与气孔导度(Gs)日变化一致.(2)苎麻叶片光合作用的光饱和点(LSP)为1 568.5μmol?m-2?s-1,光补偿点(LCP)为54.18μmol?m-2?s-1,表观量子效率(AQY)为0.025 8 mol?mol-1;而其CO2补偿点(CCP)、饱和点(CSP)和羧化效率(CE)分别为49.25、1 746.9μmol?mol-1和0.045;因此,苎麻属于喜光性阳生植物,且对强光有一定的耐受能力.(3)苎麻叶片Pn日变化的主要限制因子是胞间CO2浓度(Ci),主要决定生理因子是气孔导度(Gs).     

A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field‐collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real‐time polymerase chain reaction (RT‐PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme‐linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP‐ELISA). However, because of the relatively high costs associated with equipment and reagents, RT‐PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA‐based nested PCR protocol, modified from a loop‐mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP‐ELISAs in field‐collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4‐fold higher sensitivity than the CSP‐ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium‐positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.     

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.     

Petaline nectaries in Swietenia macrophylla (Meliaceae): Distribution and structural aspects

There are few anatomical studies of the reproductive organs of Swietenia macrophylla, despite its economic importance. This study aims to describe the structural and ultrastructural organization of the petaline nectaries in mahogany flowers. Flower buds and flowers at anthesis were collected, fixed, and processed for studies under light and electron microscopy. Nectaries occur in the median region of the petal, on the abaxial surface. Nectar is produced at all stages, from the very young buds until anthesis. The nectary presents a uniseriate epidermis, without stomata; intercellular spaces among the epidermal cells are frequent and contiguous to the subcuticular space. The secretory tissue consists of two to five layers of cells, which are rich in organelles. The nectaries lack vasculature, and the secretory tissue is isolated from the petaline mesophyll by an endodermoid layer. In the staminate flowers, the number of nectaries is less than that observed in pistillate ones.     

Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.     

Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump

Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.     

Soybean Toxin (SBTX) Impairs Fungal Growth by Interfering with Molecular Transport,Carbohydrate/Amino Acid Metabolism and Drug/Stress Responses

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.