The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

The histogenesis of lymphoid organs in Tilapia mossambica and antibody responses in adults and fry

The thymic anlagen appears in Tilapia mossambica at 2 days post hatching and becomes lymphoid at 5 days. Lymphoid cells were first seen in the pronephros at 14 days and in the spleen at approximately five weeks of age. Differentiation into red and white pulp regions was seen by 10 weeks of age. Light and electron microscopic studies of adult lymphoid organ revealed increases in size and lymphoid cell numbers. Adult thymus develops a clearer corticomedullary differentiation of thymic corpuscles in the medulla and in the splenic red and white pulp became more distinct. Melanomacrophage centres were seen in spleen and pronephros. Adult fish gave primary and secondary antibody responses following challenge with sheep red bloods cells (SRBC), Escherichia coli (E. coli) and human gamma globulin (HGG). Plaque forming cell and immunocytoadherence assays revealed that head kidney and spleen were major sites for antibody production and development of antigen reactive cells. Proliferative activity in these organs was revealed using autoradiography and scintillation counting. Increased levels of pyroninophilia were also seen following antigenic challenge. Pilot studies on adults revealed that they were capable of rejecting first and second set allografts and leucocytes from spleen and head kidney proliferated in mixed leucocyte cultures. Antibody responses to SRBC, E. coli and HGG develop at about 10-12 weeks of age. Fry given either a single injection of SRBC at 10 weeks or two injections of the same antigen at 10 weeks and 12 days later, failed to respond to a further challenge with SRBC 56 days after the first injection (A time when animals would normally respond positively to this antigen). Injection of E. coli at the same times resulted in a prolonged antibody response.

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

Heterogeneously grown tunable group-IV laser on silicon

Tunable tensile-strained germanium (epsilon-Ge) thin films on GaAs and heterogeneously integrated on silicon (Si) have been demonstrated using graded III-V buffer architectures grown by molecular beam epitaxy (MBE). epsilon-Ge epilayers with tunable strain from 0% to 1.95% on GaAs and 0% to 1.11% on Si were realized utilizing MBE. The detailed structural, morphological, band alignment and optical properties of these highly tensile-strained Ge materials were characterized to establish a pathway for wavelength-tunable laser emission from 1.55 μm to 2.1 μm. High-resolution X-ray analysis confirmed pseudomorphic epsilon-Ge epitaxy in which the amount of strain varied linearly as a function of indium alloy composition in the InxGa1-xAs buffer. Cross-sectional transmission electron microscopic analysis demonstrated a sharp heterointerface between the epsilon-Ge and the InxGa1-xAs layer and confirmed the strain state of the epsilon-Ge epilayer. Lowtemperature micro-photoluminescence measurements confirmed both direct and indirect bandgap radiative recombination between the Γ and L valleys of Ge to the light-hole valence band, with L-lh bandgaps of 0.68 eV and 0.65 eV demonstrated for the 0.82% and 1.11% epsilon-Ge on Si, respectively. The highly epsilon-Ge exhibited a direct bandgap, and wavelength-tunable emission was observed for all samples on both GaAs and Si. Successful heterogeneous integration of tunable epsilon-Ge quantum wells on Si paves the way for the implementation of monolithic heterogeneous devices on Si.

(Table 1) Types of giant magnetofossils in ODP holes

Magnetotactic bacteria biomineralize magnetic minerals with precisely controlled size, morphology, and stoichiometry. These cosmopolitan bacteria are widely observed in aquatic environments. If preserved after burial, the inorganic remains of magnetotactic bacteria act as magnetofossils that record ancient geomagnetic field variations. They also have potential to provide paleoenvironmental information. In contrast to conventional magnetofossils, giant magnetofossils (most likely produced by eukaryotic organisms) have only been reported once before from Paleocene-Eocene Thermal Maximum (PETM; 55.8 Ma) sediments on the New Jersey coastal plain. Here, using transmission electron microscopic observations, we present evidence for abundant giant magnetofossils, including previously reported elongated prisms and spindles, and new giant bullet-shaped magnetite crystals, in the Southern Ocean near Antarctica, not only during the PETM, but also shortly before and after the PETM. Moreover, we have discovered giant bullet-shaped magnetite crystals from the equatorial Indian Ocean during the Mid-Eocene Climatic Optimum (~40 Ma). Our results indicate a more widespread geographic, environmental, and temporal distribution of giant magnetofossils in the geological record with a link to "hyperthermal" events. Enhanced global weathering during hyperthermals, and expanded suboxic diagenetic environments, probably provided more bioavailable iron that enabled biomineralization of giant magnetofossils. Our micromagnetic modelling indicates the presence of magnetic multi-domain (i.e., not ideal for navigation) and single domain (i.e., ideal for navigation) structures in the giant magnetite particles depending on their size, morphology and spatial arrangement. Different giant magnetite crystal morphologies appear to have had different biological functions, including magnetotaxis and other non-navigational purposes. Our observations suggest that hyperthermals provided ideal conditions for giant magnetofossils, and that these organisms were globally distributed. Much more work is needed to understand the interplay between magnetofossil morphology, climate, nutrient availability, and environmental variability.

Quartz grain microtextures of sediments from ODP Hole 188-1166A (Table 1)

This paper presents the results of the scanning electron microscopic (SEM) analysis of quartz grains from a selection of samples at Site 1166. Ocean Drilling Program Leg 188 drilled Site 1166 on the Prydz Bay continental shelf, Antarctica, to document onset and fluctuations of East-Antarctic glaciation. This site recovered Upper Pliocene-Holocene glacial sediments directly above Cretaceous through Lower Oligocene sediments recording the transition from preglacial to early glacial conditions. SEM analysis of quartz grains at Site 1166 was used to characterize the glacial and preglacial sediments by their diagnostic textures. Angular edges, edge abrasion as well as arcuate to straight steps, are the most frequent features in glacial deposits. The highest frequency of grains with round edges is present in Middle-Late Eocene fluvio-deltaic sands. However, angular outlines, fractured plates with subparallel linear fractures and edge abrasion indicating glacier influence are also present. Preglacial carbonaceous mudstone and laminated gray claystone show distinctive high relief quartz grains and some chemical weathering on grain surfaces. The results of the microtextural analysis of quartz grains are used to verify some critical periods of ice sheet evolution, such as the transition from the East Antarctic preglacial to glacial conditions on the continental shelf from Middle/Late Eocene to Late Eocene/Early Oligocene time.

Organisation chimioanatomique des afférences pallidales chez le primate

L’élucidation de la position qu’occupent les projections sérotoninergique (5-HT), cholinergique (ACh) et dopaminergique (DA) du tronc cérébral dans l’organisation anatomofonctionelle du globus pallidus externe (GPe) et interne (GPi) au sein des ganglions de la base chez le primate est primordiale à la compréhension de ce système neuronal hautement complexe impliqué dans le contrôle du comportement moteur. Les travaux de recherche consolidés dans la présente thèse rapportent les résultats principalement obtenus chez le singe écureuil (Saimiri sciureus) à l’aide de marquages immunohistochimiques et de quantifications stéréologiques servant à évaluer la distribution régionale et les caractéristiques ultrastructurales des varicosités axonales 5-HT, ACh et DA observées dans le pallidum. Nos données ont permis l’éloboration d’un nouveau modèle du neurone pallidal en tenant compte de la hiérarchie et des caractéristiques neurochimiques de ses entrées synaptiques. Ainsi, l’analyse quantitative en microscopie optique révèle que le GPe et le GPi reçoivent des innervations 5-HT, ACh et DA de densités variables et distribuées de façon hétérogène. Plus particulièrement, le GPe est innervé par 600 000 varicosités 5-HT/mm3 de tissu, 500 000 varicosités ACh/mm3 et 170 000 varicosités DA/mm3. En revanche, le GPi reçoit 600 000 varicosités 5-HT/mm3, 250 000 varicosités ACh/mm3 et 190 000 varicosités DA/mm3. De plus, la 5-HT, l’ACh et la DA ciblent préférentiellement les secteurs correspondant aux territoires fonctionnels associatifs et limbiques du pallidum, suggérant un rôle de ces projections dans la planification du comportement moteur ainsi que dans la régulation de l’attention et de l’humeur. Nos analyses en microscopie électronique révèlent que très peu de ces varicosités axonales établissent un contact synaptique, puisque plus de 70% des varicosités 5-HT, ACh et DA sont complètement dépouvues de jonction synaptique. Ainsi, bien que la 5-HT, l’ACh et la DA seraient en mesure de moduler directement les neurones pallidaux grâce à la transmission synaptique, leur plus grande influence s’opérerait par la transmission volumique, permettant d’influencer à la fois les neurones pallidaux et leurs afférences, principalement du striatum et noyau subthalamique. L’ensemble de ces résultats indique que les projections 5-HT, ACh et DA du tronc cérébral agissent de concert avec les afférences plus robustes en provenance du striatum et du noyau subthalamique. Ces nouvelles données neuroanatomiques positionnent le tronc cérébral en tant qu’acteur important dans l’organisation anatomique et fonctionnelle du pallidum chez le primate et doivent être prises en considération dans l’élaboration de nouvelles approches thérapeutiques visant à contrer les processus neurodégénératifs qui affectent les ganglions de la base, tel que la maladie de Parkinson.

The Performance and Service Life Prediction of High Performance Concrete in Sulfate and Acidic Environments

Concrete substructures are often subjected to environmental deterioration, such as sulfate and acid attack, which leads to severe damage and causes structure degradation or even failure. In order to improve the durability of concrete, the High Performance Concrete (HPC) has become widely used by partially replacing cement with pozzolanic materials. However, HPC degradation mechanisms in sulfate and acidic environments are not completely understood. It is therefore important to evaluate the performance of the HPC in such conditions and predict concrete service life by establishing degradation models. This study began with a review of available environmental data in the State of Florida. A total of seven bridges have been inspected. Concrete cores were taken from these bridge piles and were subjected for microstructural analysis using Scanning Electron Microscope (SEM). Ettringite is found to be the products of sulfate attack in sulfate and acidic condition. In order to quantitatively analyze concrete deterioration level, an image processing program is designed using Matlab to obtain quantitative data. Crack percentage (Acrack/Asurface) is used to evaluate concrete deterioration. Thereafter, correlation analysis was performed to find the correlation between five related variables and concrete deterioration. Environmental sulfate concentration and bridge age were found to be positively correlated, while environmental pH level was found to be negatively correlated. Besides environmental conditions, concrete property factor was also included in the equation. It was derived from laboratory testing data. Experimental tests were carried out implementing accelerated expansion test under controlled environment. Specimens of eight different mix designs were prepared. The effect of pozzolanic replacement rate was taken into consideration in the empirical equation. And the empirical equation was validated with existing bridges. Results show that the proposed equations compared well with field test results with a maximum deviation of ± 20%. Two examples showing how to use the proposed equations are provided to guide the practical implementation. In conclusion, the proposed approach of relating microcracks to deterioration is a better method than existing diffusion and sorption models since sulfate attack cause cracking in concrete. Imaging technique provided in this study can also be used to quantitatively analyze concrete samples.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

Characterization of the external female genitalia of six species of Triatominae (Hemiptera: Reduviidade) by scanning electron microscopy

By macroscopic and microscopic dorsal side observation, it was noted that the IX and X segments of two species each of Panstrongylus and Triatoma terminate in an elongated way, whereas they terminate abruptly in the two species of Rhodnius. Scanning observation of the dorsal, ventral, lateral and posterior sides of the female genitalia of Panstrongylus herreri, Panstrongylus megistus, Rhodnius colombiensis, Rhodnius prolixus, Triatoma infestans and Triatoma vitticeps revealed that these six species are generally and specifically distinguished based on these elements. We describe several components that distinguish P. herreri from P. megistus: four on the dorsal side: the VII, VIII, IX and X segments, on the ventral view, three: VII sternite, VIII gonocoxite and VIII gonapophyse, on the lateral view one character, VIII gonocoxite and on the posterior view three characters: VIII and IX gonocoxite and XI gonopophyse. Comparing R. colombiensis and R. prolixus, there were three distinct characters on the dorsal side: the VII, VIII and X segments, on the ventral view three characters: the IX and X segments and VIII gonocoxite and on the posterior view four characters: the VIII, IX, X segments and VIII gonapophyse that distinguish the two species. T. infestans and T. vitticeps have four different characters on the dorsal side: the VII, VIII, IX and X segments, on the ventral view four characters: the VII and X segments, VIII gonocoxite and VIII gonapophyse, on the lateral view two characters, IX and X segments and on the posterior view four characters: the IX and X segments, VIII gonocoxite and VIII gonapophyse that distinguish the two species. Examination of the external female genitalia of six triatomine species by scanning suggests that these components are useful for taxonomical studies.

Characterization of the external female genitalia of six species of Triatominae (Hemiptera: Reduviidade) by scanning electron microscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of vascular cavity coatings and microscopic tube-shaped structures from Upper Cretaceous dinosaur bone: evidence of a bacterial origin for dinosaur blood vessels""

Recent claims of blood vessels extracted from dinosaur fossils challenge classical views of soft-tissue preservation. Alternatively, these structures may represent postdepositional,diagenetic biofilms that grew on vascular cavity surfaces within the fossil. Similar red, hollow, tube-shaped structures were recovered from well-preserved and poorly-preserved (abraded, desiccated, exposed) Upper Cretaceous dinosaur fossils in this study. Integration of light microscopy, scanning electron microscopy, and energy dispersive x-ray spectroscopy was used to compare these vessel structures to the fossils from which they are derived. Vessel structures are typically 100-400 μm long, 0.5-1.5 μm thick, 10-40 μm in diameter and take on a wide range of straight, curved, andbranching morphologies. Interior surfaces vary from smooth to globular and typically contain spheres, rods, and fibrous structures (< 2 μm in diameter) incorporated into the surface. Exterior surfaces exhibit 2-μm-tall converging ridges, spaced 1-3 μm apart, that are sub-parallel to the long axis of the vessel structure. Fossil vascular cavities are typically coated with a smooth or grainy orange layer that shows a wide range of textures including smooth, globular, rough, ropy, and combinations thereof. Coatings tend to overlay secondary mineral crystals and framboids, confirming they are not primary structures of the fossil. For some cavity coatings, the surface that had been in contact with the bone exhibits a ridged texture, similar to that of vessel structures, having formed as a mold of the intravascular bone surface. Thus, vessel structures are interpreted as intact cavity coatings isolated after the fossil is demineralized. The presence of framboids and structures consistent in size and shape with bacteria cells, the abundance of iron in cavity coatings, and the growth of biofilms directly from the fossil that resemble respective cavity coatings support the hypothesis that vessel structures result from ironconsuming bacteria that form biofilms on the intravascular bone surfaces of fossil dinosaur bone. This also accounts for microstructures resembling osteocytes as some fossil lacunae are filled with the same iron oxide that comprises vessel structures andcoatings. Results of this study show that systematic, high-resolution SEM analyses of vertebrate fossils can provide improved insight on microtaphonomic processes, including the role of bacteria in diagenesis. These results conflict with earlier claims of dinosaurblood vessels and osteocytes.

Characterization of oil shale residue and rejects from irati formation by electron paramagnetic resonance

In this study, sedimentary organic matter of oil shale rejects, calschist, shale fine and the so called retorted shale from Irati formation was characterized. EPR was used to analyse the samples regarding loss of signal in g = 2.003 associated to the organic free radical with the calcined samples and washing with hydrogen peroxide. The radical signal was detected in all samples, however, for the calschist and shale fine samples another signal was identified at g = 2.000 which disappeared when the sample was heated at 400 ºC. Hydrogen peroxide washing was also performed and it was noted that after washing the signal appeared around g = 2.000 for all samples, including retorted shale, which might be due to the quartz E1 defect.

Design and characterization of bridged loop-gap resonators for use in electron paramagnetic resonance measurements

This paper revisits the design of L and S band bridged loop-gap resonators (BLGRs) for electron paramagnetic resonance applications. A novel configuration is described and extensively characterized for resonance frequency and quality factor as a function of the geometrical parameters of the device. The obtained experimental results indicate higher values of the quality factor (Q) than previously reported in the literature, and the experimental analysis data should provide useful guidelines for BLGR design.