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Multi-institutional approaches to graduate education continue to emerge as a way to better prepare students for collaborative work. In this article, we describe a graduate course designed to investigate application of conservation biology principles by local land use planners. "Where is Conservation Science in Local Planning?" was offered jointly by three institutions and integrated inquiry-guided, collaborative, and computer-mediated learning. Participants collaborated across universities to investigate this question and create products based on their work, including a presentation and two peer-reviewed manuscripts. We used a wiki for brainstorming and collaborative writing, a virtual classroom for work meetings, and video conferencing for building community and making complex decisions. Pre- and post-course questionnaires were used to evaluate the effectiveness of the course for improving skills in collaboration, use of collaborative technologies, and subject-area knowledge. Student development mirrored those areas where learning was required to consistently support class-wide activities. Students reported that they gained knowledge about collaboration, increased their mastery of communication skills and use of collaborative technologies, and gained knowledge about course subject matter. Students did not indicate significant changes in knowledge or activities related to leadership. Participants gained a fuller understanding of the benefits--collective creativity and enhanced accountability--and drawbacks--time required to build relationships and engage in deliberation--of collaborative research. Faculty participants suggest that future offerings continue to follow an inquiry-guided, collaborative learning approach using similar collaborative technologies, but include more explicit guidance about leadership and attempt to generate a smaller number of products. (Contains 5 tables.)

Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. In vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

The fabrication of microarrays containing PCR-amplified genomic DNA extracts from mice tumors on a Zetaprobe membrane using a modified thermal ink-jet printer is described. A simple and cost-effective procedure for the fabrication of microarrays containing biological samples using a modified bubble-jet printing system is presented. Because of their mass-produced design, ink-jet printers are a much cheaper alternative to conventional spotting techniques. The usefulness of the biochip microarray platform is illustrated by the detection of human fragile histidine triad (FHIT), a tumor suppressor gene. Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunodeficient mice. Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples. The extracted DNA samples were amplified by PCR (30 cycles) using the primers corresponding to nucleotides 2 to 18 of the FHIT sequence. The resulting solution was transferred to the individual reservoirs of a three-color cartridge from a conventional thermal ink-jet printer (HP 694C), and arrays were printed on to a Zetaprobe membrane. After spotting, these membranes were used in a hybridization assay, using fluorescent probes, and detected with a biochip.

Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8--10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover.

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps., (Copyright 2001 Academic Press.)

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.

Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+)-ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis.

Background: Structures have recently been solved at 8 A resolution for both Ca2+-ATPase from rabbit sarcoplasmic reticulum and H+-ATPase from Neurospora crassa. These cation pumps are two distantly related members of the family of P-type ATPases, which are thought to use similar mechanisms to generate ATP-dependent ion gradients across a variety of cellular membranes. We have undertaken a detailed comparison of the two structures in order to describe their similarities and differences as they bear on their mechanism of active transport., Results: Our first important finding was that the arrangement of 10 transmembrane helices was remarkably similar in the two molecules. This structural homology strongly supports the notion that these pumps use the same basic mechanism to transport their respective ions. Despite this similarity in the membrane-spanning region, the cytoplasmic regions of the two molecules were very different, both in their disposition relative to the membrane and in the juxtaposition of their various subdomains., Conclusions: On the basis of the crystallization conditions, we propose that these two crystal structures represent different intermediates in the transport cycle, distinguished by whether cations are bound to their transport sites. Furthermore, we propose that the corresponding conformational change (E2 to E1 ) has two components: the first is an inclination of the main cytoplasmic mass by 20 degrees relative to the membrane-spanning domain; the second is a rearrangement of the domains comprising the cytoplasmic part of the molecules. Accordingly, we present a rough model for this important conformational change, which relays the effects of cation binding within the membrane-spanning domain to the nucleotide-binding site, thus initiating the transport cycle.

There are many examples of macromolecules that form helical tubes or crystals, which are useful for structure determination by electron microscopy and image processing. Helical crystals can be thought of as two-dimensional crystals that have been rolled into a cylinder such that two lattice points are superimposed. In many real cases, helical crystals of a particular macromolecule derive from an identical two-dimensional lattice but have different lattice points superimposed, thus producing different helical symmetries which cannot be simply averaged in Fourier-space. When confronted with this situation, one can select images corresponding to one of the observed symmetries at the expense of reducing the number of images that can be used for data collection and averaging, or one can calculate separate density maps from each symmetry, then align and average them together in real-space. Here, we present a third alternative, which is based on averaging of the Fourier-Bessel coefficients, gn,l(r), and which allows the inclusion of data from all symmetry groups derived from a common two-dimensional lattice. The method is straightforward and simple in practice and is shown, through a specific example with real data, to give results comparable to real-space averaging., (Copyright 1999 Academic Press.)

Electron crystallography offers an increasingly viable alternative to X-ray crystallography for structure determination, especially for membrane proteins. The methodology has been developed and successfully applied to 2D crystals; however, well-ordered thin, 3D crystals are often produced during crystallization trials and generally discarded due to complexities in structure analysis. To cope with these complexities, we have developed a general method for determining unit cell geometry and for merging electron diffraction data from tilt series. We have applied this method to thin, monoclinic crystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 A resolution with tilt angles up to 30 degrees. The indexing of data from the tilt series has been verified by an analysis of Laue zones near the (h, k, 0) projection and the unit cell geometry is consistent with low-angle X-ray scattering from these crystals. Based on this unit cell geometry, we have systematically tilted crystals to record images of the (h, k, 0) projection. After averaging the corresponding phases to 8 A resolution, an (h, k, 0) projection map has been calculated by combining image phases with electron diffraction amplitudes. This map contains discrete densities that most likely correspond to Ca2+-ATPase dimers, unlike previous maps of untilted crystals in which molecules from successive layers are not aligned. Comparison with a projection structure from tubular crystals reveals differences that are likely due to the conformational change accompanying calcium binding to Ca2+-ATPase., (Copyright 1998 Academic Press)

Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail.

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.

We have used multilamellar crystals of the ATP-driven calcium pump from sarcoplasmic reticulum to address the structural effects of calcium binding to the enzyme. They are stacks of disk-shaped two-dimensional crystals. A density map projected along the lipid bilayer was obtained at 9-A resolution by frozen-hydrated electron microscopy. Although only in projection, much more details of the structure were revealed than previously available, especially in the transmembrane region. Quantitative comparison was made with the model obtained from the tubular crystals of this enzyme formed in the absence of calcium. Unexpectedly large differences in conformation were found, particularly in the cytoplasmic domain.

The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.

We report, for the first time, the use of surface-enhanced Raman (SERS)-active labels for primers used in polymerase chain reaction amplification of specific target DNA sequences. This method has the potential for combining the spectral selectivity and high sensitivity of the SERS technique with the inherent molecular specificity offered by DNA sequence hybridization. The effectiveness of the detection scheme is demonstrated using the gag gene sequence of the human immunodeficiency virus. The potential use of multiple probes for simultaneous detection of multiple biological targets is discussed.

Electron microscopy is gradually revealing more and more about the structure of the calcium pump from the sarcoplasmic reticulum, Ca(2+)-ATPase. The most recent result reveals the ATP-binding site, and two different avenues are being pursued towards achieving a higher resolution structure. Although no such structures are currently available for phospholamban, various spectroscopies and site-directed mutagenesis have been combined to produce a compelling structural model for its regulation of Ca(2+)-ATPase.

A new fluorescence-based fibre optic sensor is described which combines the sensitivity offered by laser-induced fluorescence with the selectivity offered by capillary electrophoresis (CE). A single optical fibre directly probes the terminus of a 5-8 cm separation capillary. The linear geometry associated with this sensor necessitates a 'single reservoir' design, thus presenting major challenges to overcome in comparison to the conventional two-reservoir configuration common to a typical laboratory setup. Some of the challenges confronted by the design features presented in this work include the reduction of gravity-driven hydrostatic flow, the ejection of electrolytic gases evolved at the detection-side electrode and the establishment a suitable compromise between detectability and separation performance. The success of such design features demonstrates the feasibility of a CE-based sensor which offers several amenities particularly useful for in situ sensing. Such attributes include selectivity, diminutive size, flexibility, reusability, high sensitivity, speed, and remote control. Detailed descriptions of sensor fabrication are included, including two variations on a general design concept. In addition, the single-fibre optical detection system is described. Separation characteristics of the new CE-based sensor are presented, highlighted by an observed separation efficiency of up to 8000 theoretical plates (for a 5 cm capillary). The separation of a three-component mixture of the laser dyes, Rhodamine 6G, fluorescein isothyocyanate and sodium fluorescein, is demonstrated.

In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.

The location of the ATP-binding site of a P-type ion pump, Ca(2+)-ATPase from rabbit sarcoplasmic reticulum, was examined by cryoelectron microscopy. A nonhydrolyzable analog of ATP, beta, gamma-bidentate chromium (III) complex of ATP (CrATP), was used to stabilize the enzyme in the Ca(2+)-occluded state. Tubular crystals were then induced by vanadate in the presence of EGTA, keeping CrATP bound to the enzyme. The three-dimensional structures of the crystals were determined at 14 A resolution by cryoelectron microscopy and helical image analysis. Statistical comparison of the structures with and without CrATP showed clear and significant differences at the groove proposed previously as the ATP-binding pocket.