Recent advances into understanding some aspects of the structure and function of mammalian and avian lungs

Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include (a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical, compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultrafine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions-including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors-is reflected in its "compromise design."

Structural plasticity in the language system related to increased second language proficiency

While functional changes linked to second language learning have been subject to extensive investigation, the issue of learning-dependent structural plasticity in the fields of bilingualism and language comprehension has so far received less notice. In the present study we used voxel-based morphometry to monitor structural changes occurring within five months of second language learning. Native English-speaking exchange students learning German in Switzerland were examined once at the beginning of their stay and once about five months later, when their German language skills had significantly increased. We show that structural changes in the left inferior frontal gyrus are correlated with the increase in second language proficiency as measured by a paper-and-pencil language test. Contrary to the increase in proficiency and grey matter, the absolute values of grey matter density and second language proficiency did not correlate (neither on first nor on second measurement). This indicates that the individual amount of learning is reflected in brain structure changes, regardless of absolute proficiency.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Immunologic privilege in the central nervous system and the blood-brain barrier

The brain is in many ways an immunologically and pharmacologically privileged site. The blood-brain barrier (BBB) of the cerebrovascular endothelium and its participation in the complex structure of the neurovascular unit (NVU) restrict access of immune cells and immune mediators to the central nervous system (CNS). In pathologic conditions, very well-organized immunologic responses can develop within the CNS, raising important questions about the real nature and the intrinsic and extrinsic regulation of this immune privilege. We assess the interactions of immune cells and immune mediators with the BBB and NVU in neurologic disease, cerebrovascular disease, and intracerebral tumors. The goals of this review are to outline key scientific advances and the status of the science central to both the neuroinflammation and CNS barriers fields, and highlight the opportunities and priorities in advancing brain barriers research in the context of the larger immunology and neuroscience disciplines. This review article was developed from reports presented at the 2011 Annual Blood-Brain Barrier Consortium Meeting.

Examining the gateway to the limbic system with diffusion tensor imaging: the perforant pathway in dementia

Current treatments for Alzheimer's disease (AD) are only able to slow the progression of mental deterioration, making early and reliable diagnosis an essential part of any promising therapeutic strategy. In the initial stages of AD, the first neuropathological alterations occur in the perforant pathway (PP), a large neuronal fiber tract located at the entrance to the limbic system. However, to date, there is no sensitive diagnostic tool for performing in vivo assessments of this structure. In the present bimodal magnetic resonance imaging (MRI) study, we examined 10 elderly controls, 10 subjects suffering from mild cognitive impairment (MCI), and 10 AD patients in order to evaluate the sensitivity of diffusion tensor imaging (DTI), a new MRI technique, for detecting changes in the PP. Furthermore, the diagnostic explanatory power of DTI data of the PP should be compared to high-resolution MRI volumetry and intervoxel coherences (COH) of the hippocampus and the entorhinal cortex, two limbic regions also involved in the pathophysiology of early AD. DTI revealed a marked decrease in COH values in the PP region of MCI (right side: 26%, left side: 29%, as compared to controls) and AD patients (right side: 37%, left side: 43%, as compared to controls). Reductions in COH values of the PP region were significantly correlated with cognitive impairment. DTI data of the PP zone were the only parameter differing significantly between control subjects and MCI patients, while the volumetric measures and the COH values of the hippocampus and the entorhinal cortex did not. DTI of medial temporal brain regions is a promising non-invasive tool for the in vivo diagnosis of the early/preclinical stages of AD.