Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^

Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.

Characterization of a high-voltage-activated IA current with a role in spike timing and locomotor pattern generation

Transient A-type K+ channels (IA) in neurons have been implicated in the delay of the spike onset and the decrease in the firing frequency. Here we have characterized biophysically and pharmacologically an IA current in lamprey locomotor network neurons that is activated by suprathreshold depolarization and is specifically blocked by catechol at 100 μM. The biophysical properties of this current are similar to the mammalian Kv3.4 channel. The role of the IA current both in single neuron firing and in locomotor pattern generation was analyzed. The IA current facilitates Na+ channel recovery from inactivation and thus sustains repetitive firing. The role of the IA current in motor pattern generation was examined by applying catechol during fictive locomotion induced by N-methyl-d-aspartate. Blockade of this current increased the locomotor burst frequency and decreased the firing of motoneurons. Although an alternating motor pattern could still be generated, the cycle duration was less regular, with ventral roots bursts failing on some cycles. Our results thus provide insights into the contribution of a high-voltage-activated IA current to the regulation of firing properties and motor coordination in the lamprey spinal cord.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

Molecular and Functional Characterization of Histone Derived Antimicrobial Peptides from Marine Organisms

Antimicrobial peptides (AMPs) are gene encoded, small sized, generally cationic, amphiphathic peptides characterized by antimicrobial activity against bacteria, fungi, viruses and other pathogens. They are a major component of the innate immune defense system of almost all living organisms, ranging from bacteria to humans and represent the first line of defense against the invading microbial pathogens (Boman, 1995; Zasloff, 2002). Antimicrobial peptides represent a heterogeneous group displaying multiple modes of action that are determined by the sequence and concentration of peptides. Their remarkable specificity for prokaryotes with low toxicity for eukaryotic cells has favored their investigation and exploitation as new antibiotics

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. The microfluidic technology described in this thesis has the potential to significantly advance our understanding of the structure and function of membrane proteins, thereby aiding the elucidation of human biology, the development of pharmaceuticals with fewer side effects for a wide range of diseases. References (1) Quick, M.; Javitch, J. A. P Natl Acad Sci USA 2007, 104, 3603. (2) Trubetskoy, V. S.; Burke, T. J. Am Lab 2005, 37, 19. (3) Pecina, P.; Houstkova, H.; Hansikova, H.; Zeman, J.; Houstek, J. Physiol Res 2004, 53, S213. (4) Arinaminpathy, Y.; Khurana, E.; Engelman, D. M.; Gerstein, M. B. Drug Discovery Today 2009, 14, 1130. (5) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. Nat Rev Drug Discov 2006, 5, 993. (6) Dauter, Z.; Lamzin, V. S.; Wilson, K. S. Current Opinion in Structural Biology 1997, 7, 681. (7) Hansen, C.; Quake, S. R. Current Opinion in Structural Biology 2003, 13, 538. (8) Govada, L.; Carpenter, L.; da Fonseca, P. C. A.; Helliwell, J. R.; Rizkallah, P.; Flashman, E.; Chayen, N. E.; Redwood, C.; Squire, J. M. J Mol Biol 2008, 378, 387. (9) Hansen, C. L.; Skordalakes, E.; Berger, J. M.; Quake, S. R. P Natl Acad Sci USA 2002, 99, 16531. (10) Leng, J.; Salmon, J.-B. Lab Chip 2009, 9, 24. (11) Zheng, B.; Gerdts, C. J.; Ismagilov, R. F. Current Opinion in Structural Biology 2005, 15, 548. (12) Lorber, B.; Delucas, L. J.; Bishop, J. B. J Cryst Growth 1991, 110, 103. (13) Talreja, S.; Perry, S. L.; Guha, S.; Bhamidi, V.; Zukoski, C. F.; Kenis, P. J. A. The Journal of Physical Chemistry B 2010, 114, 4432. (14) Chayen, N. E. Current Opinion in Structural Biology 2004, 14, 577. (15) He, G. W.; Bhamidi, V.; Tan, R. B. H.; Kenis, P. J. A.; Zukoski, C. F. Cryst Growth Des 2006, 6, 1175. (16) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew Chem Int Edit 2004, 43, 2508. (17) Li, L.; Mustafi, D.; Fu, Q.; Tereshko, V.; Chen, D. L. L.; Tice, J. D.; Ismagilov, R. F. P Natl Acad Sci USA 2006, 103, 19243. (18) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew Chem Int Edit 2006, 45, 7336. (19) van der Woerd, M.; Ferree, D.; Pusey, M. Journal of Structural Biology 2003, 142, 180. (20) Ng, J. D.; Gavira, J. A.; Garcia-Ruiz, J. M. Journal of Structural Biology 2003, 142, 218. (21) Talreja, S.; Kenis, P. J. A.; Zukoski, C. F. Langmuir 2007, 23, 4516. (22) Hansen, C. L.; Quake, S. R.; Berger, J. M. US, 2007. (23) Newman, J.; Fazio, V. J.; Lawson, B.; Peat, T. S. Cryst Growth Des 2010, 10, 2785. (24) Newman, J.; Xu, J.; Willis, M. C. Acta Crystallographica Section D 2007, 63, 826. (25) Collingsworth, P. D.; Bray, T. L.; Christopher, G. K. J Cryst Growth 2000, 219, 283. (26) Durbin, S. D.; Feher, G. Annu Rev Phys Chem 1996, 47, 171. (27) Talreja, S.; Kim, D. Y.; Mirarefi, A. Y.; Zukoski, C. F.; Kenis, P. J. A. J Appl Crystallogr 2005, 38, 988. (28) Yoshizaki, I.; Nakamura, H.; Sato, T.; Igarashi, N.; Komatsu, H.; Yoda, S. J Cryst Growth 2002, 237, 295. (29) Anderson, M. J.; Hansen, C. L.; Quake, S. R. P Natl Acad Sci USA 2006, 103, 16746. (30) Hansen, C. L.; Sommer, M. O. A.; Quake, S. R. P Natl Acad Sci USA 2004, 101, 14431. (31) Lounaci, M.; Rigolet, P.; Abraham, C.; Le Berre, M.; Chen, Y. Microelectron Eng 2007, 84, 1758. (32) Zheng, B.; Roach, L. S.; Ismagilov, R. F. J Am Chem Soc 2003, 125, 11170. (33) Zhou, X.; Lau, L.; Lam, W. W. L.; Au, S. W. N.; Zheng, B. Anal. Chem. 2007. (34) Cherezov, V.; Caffrey, M. J Appl Crystallogr 2003, 36, 1372. (35) Qutub, Y.; Reviakine, I.; Maxwell, C.; Navarro, J.; Landau, E. M.; Vekilov, P. G. J Mol Biol 2004, 343, 1243. (36) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I.; Landau, E. M.; Rosenbusch, J. P. Journal of Structural Biology 1998, 121, 82. (37) Gavira, J. A.; Toh, D.; Lopez-Jaramillo, J.; Garcia-Ruiz, J. M.; Ng, J. D. Acta Crystallogr D 2002, 58, 1147. (38) Stevens, R. C. Current Opinion in Structural Biology 2000, 10, 558. (39) Baker, M. Nat Methods 2010, 7, 429. (40) McPherson, A. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 5. (41) Gabrielsen, M.; Gardiner, A. T.; Fromme, P.; Cogdell, R. J. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 127. (42) Page, R. In Methods in Molecular Biology: Structural Proteomics - High Throughput Methods; Kobe, B., Guss, M., Huber, T., Eds.; Humana Press: Totowa, NJ, 2008; Vol. 426, p 345. (43) Caffrey, M. Ann Rev Biophys 2009, 38, 29. (44) Doerr, A. Nat Methods 2006, 3, 244. (45) Brostromer, E.; Nan, J.; Li, L.-F.; Su, X.-D. Biochemical and Biophysical Research Communications 2009, 386, 634. (46) Li, G.; Chen, Q.; Li, J.; Hu, X.; Zhao, J. Anal Chem 2010, 82, 4362. (47) Jia, Y.; Liu, X.-Y. The Journal of Physical Chemistry B 2006, 110, 6949. (48) RCSB Protein Data Bank. http://www.rcsb.org/ (July 11, 2010). (49) Membrane Proteins of Known 3D Structure. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html (July 11, 2010). (50) Michel, H. Trends Biochem Sci 1983, 8, 56. (51) Rosenbusch, J. P. Journal of Structural Biology 1990, 104, 134. (52) Garavito, R. M.; Picot, D. Methods 1990, 1, 57. (53) Kulkarni, C. V. 2010; Vol. 12, p 237. (54) Landau, E. M.; Rosenbusch, J. P. P Natl Acad Sci USA 1996, 93, 14532. (55) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (56) Cherezov, V.; Liu, W.; Derrick, J. P.; Luan, B.; Aksimentiev, A.; Katritch, V.; Caffrey, M. Proteins: Structure, Function, and Bioinformatics 2008, 71, 24. (57) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C. Science 2007, 318, 1258. (58) Cherezov, V.; Yamashita, E.; Liu, W.; Zhalnina, M.; Cramer, W. A.; Caffrey, M. J Mol Biol 2006, 364, 716. (59) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211. (60) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.; Stevens, R. C.; Kobilka, B. K. Science 2007, 318, 1266. (61) Wacker, D.; Fenalti, G.; Brown, M. A.; Katritch, V.; Abagyan, R.; Cherezov, V.; Stevens, R. C. J Am Chem Soc 2010, 132, 11443. (62) Höfer, N.; Aragão, D.; Caffrey, M. Biophys J 2010, 99, L23. (63) Li, L.; Ismagilov, R. F. Ann Rev Biophys 2010. (64) Pal, R.; Yang, M.; Lin, R.; Johnson, B. N.; Srivastava, N.; Razzacki, S. Z.; Chomistek, K. J.; Heldsinger, D. C.; Haque, R. M.; Ugaz, V. M.; Thwar, P. K.; Chen, Z.; Alfano, K.; Yim, M. B.; Krishnan, M.; Fuller, A. O.; Larson, R. G.; Burke, D. T.; Burns, M. A. Lab Chip 2005, 5, 1024. (65) Jayashree, R. S.; Gancs, L.; Choban, E. R.; Primak, A.; Natarajan, D.; Markoski, L. J.; Kenis, P. J. A. J Am Chem Soc 2005, 127, 16758. (66) Wootton, R. C. R.; deMello, A. J. Chem Commun 2004, 266. (67) McPherson, A. J Appl Crystallogr 2000, 33, 397.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Characterization Of Microsatellite Markers Developed From Prosopis Rubriflora And Prosopis Ruscifolia (leguminosae - Mimosoideae), Legume Species That Are Used As Models For Genetic Diversity Studies In Chaquenian Areas Under Anthropization In South America.

Prosopis rubriflora and Prosopis ruscifolia are important species in the Chaquenian regions of Brazil. Because of the restriction and frequency of their physiognomy, they are excellent models for conservation genetics studies. The use of microsatellite markers (Simple Sequence Repeats, SSRs) has become increasingly important in recent years and has proven to be a powerful tool for both ecological and molecular studies. In this study, we present the development and characterization of 10 new markers for P. rubriflora and 13 new markers for P. ruscifolia. The genotyping was performed using 40 P. rubriflora samples and 48 P. ruscifolia samples from the Chaquenian remnants in Brazil. The polymorphism information content (PIC) of the P. rubriflora markers ranged from 0.073 to 0.791, and no null alleles or deviation from Hardy-Weinberg equilibrium (HW) were detected. The PIC values for the P. ruscifolia markers ranged from 0.289 to 0.883, but a departure from HW and null alleles were detected for certain loci; however, this departure may have resulted from anthropic activities, such as the presence of livestock, which is very common in the remnant areas. In this study, we describe novel SSR polymorphic markers that may be helpful in future genetic studies of P. rubriflora and P. ruscifolia.

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction.

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes.

Development And Characterization Of Microsatellite Loci For Tabebuia Cassinoides (bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.

Characterization Of Morphology And Composition Of Inorganic Fillers In Dental Alginates.

Energy dispersive X-ray spectroscopy microanalysis (EDX), scanning electron microscopy (SEM), and Archimedes' Principle were used to determine the characteristics of inorganic filler particles in five dental alginates, including Cavex ColorChange (C), Hydrogum 5 (H5), Hydrogum (H), Orthoprint (O), and Jeltrate Plus (JP). The different alginate powders (0.5 mg) were fixed on plastic stubs (n = 5) and sputter coated with carbon for EDX analysis, then coated with gold, and observed using SEM. Volume fractions were determined by weighing a sample of each material in water before and after calcining at 450(°)C for 3 h. The alginate materials were mainly composed of silicon (Si) by weight (C-81.59%, H-79.89%, O-78.87%, H5-77.95%, JP-66.88%, wt). The filler fractions in volume (vt) were as follows: H5-84.85%, JP-74.76%, H-70.03%, O-68.31%, and C-56.10%. The tested materials demonstrated important differences in the inorganic elemental composition, filler fraction, and particle morphology.

Development And Characterization Of 32 Microsatellite Loci In Genipa Americana (rubiaceae).

• Microsatellite primers were developed for the tree species Genipa americana (Rubiaceae) for further population genetic studies. • We identified 144 clones containing 65 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 32 microsatellite loci and validated in 40 individuals of two natural G. americana populations. Seventeen loci were polymorphic, revealing from three to seven alleles per locus. The observed and expected heterozygosities ranged from 0.24 to 1.00 and from 0.22 to 0.78, respectively. • The 17 primers identified as polymorphic loci are suitable to study the genetic diversity and structure, mating system, and gene flow in G. americana.

The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Phtx-ii A Basic Myotoxic Phospholipase A2 From Porthidium Hyoprora Snake Venom, Pharmacological Characterization And Amino Acid Sequence By Mass Spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.