Estímulo no crescimento e na hidrólise de ATP em raízes de alface tratadas com humatos de vermicomposto: i - efeito da concentração.

O vermicomposto contém uma concentração elevada de substâncias húmicas e já é bem conhecido o efeito do seu uso sobre as propriedades do solo. No entanto,a ação direta das substâncias húmicas sobre o metabolismo das plantas é menos conhecida. O objetivo deste trabalho foi avaliar o uso de humatos extraídos de vermicomposto de esterco de curral com KOH 0,1 mol L-1 sobre o desenvolvimento e metabolismo de ATP em plântulas de alface. Após a germinação, plântulas de alface foram tratadas com os humatos em concentrações que variaram de 0 a 100 mg L-1 de C, durante quinze dias. Foram avaliados o crescimento da raiz e a atividade das bombas de H+ isoladas da fração microssomal do sistema radicular. Foi observado aumento na matéria fresca e seca do sistema radicular, bem como no número de sítios de mitose, raízes emergidas do eixo principal, na área e no comprimento radiculares, com o uso do humato na concentração de 25 mg L-1 de C. Também foi observado, nessa concentração, aumento significativo na hidrólise de ATP pelas bombas de H+, responsáveis pela geração de energia necessária à absorção de íons e pelo crescimento celular

Structure of a bacterial type III secretion system in contact with a host membrane in situ

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform– ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection

Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples

Structural Biology by NMR: Structure, Dynamics, and Interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data

Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Refined methodology for implantation of a head fixation device and chronic recording chambers in non-human primates

The present study was aimed at developing a new strategy to design and anchor custom-fitted implants, consisting of a head fixation device and a chronic recording chamber, on the skull of adult macaque monkeys. This was done without the use of dental resin or orthopedic cement, as these modes of fixation exert a detrimental effect on the bone. The implants were made of titanium or tekapeek and anchored to the skull with titanium screws. Two adult macaque monkeys were initially implanted with the head fixation device several months previous to electrophysiological investigation, to allow optimal osseous-integration, including growth of the bone above the implant's footplate. In a second step, the chronic recording chamber was implanted above the brain region of interest. The present study proposes two original approaches for both implants. First, based on a CT scan of the monkey, a plastic replicate of the skull was obtained in the form of a 3D print, used to accurately shape and position the two implants. This would ensure a perfect match with the skull surface. Second, the part of the implants in contact with the bone was coated with hydroxyapatite, presenting chemical similarity to natural bone, thus promoting excellent osseous-integration. The longevity of the implants used here was 4 years for the head fixation device and 1.5 years for the chronic chamber. There were no adverse events and daily care was easy. This is clear evidence that the present implanting strategy was successful and provokes less discomfort to the animals