Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-beta-synthase domains are required for its osmoregulatory function

The plant pathogen Pseudomonas syringae may cope with osmotic stress on plants, in part, by importing osmoprotective compounds. In this study, we found that P. syringae pv. tomato strain DC3000 was distinct from most bacterial species in deriving greater osmoprotection from exogenous choline than from glycine betaine. This superior osmoprotection was correlated with a higher capacity for uptake of choline than for uptake of glycine betaine. Of four putative osmoregulatory ABC transporters in DC3000, one, designated OpuC, functioned as the primary or sole transporter for glycine betaine and as one of multiple transporters for choline under high osmolarity. Surprisingly, the homolog of the well-characterized ProU transporter from Escherichia coli and Salmonella enterica serovar Typhimurium did not function in osmoprotection. The P. syringae pv. tomato OpuC transporter was more closely related to the Bacillus subtilis and Listeria monocytogenes OpuC transporters than to known osmoprotectant transporters in gram-negative bacteria based on sequence similarity and genetic arrangement. The P. syringae pv. tomato OpuC transporter had a high affinity for glycine betaine, a low affinity for choline, and a broad substrate specificity that included acetylcholine, carnitine, and proline betaine. Tandem cystathionine-beta-synthase (CBS) domains in the ATP-binding component of OpuC were required for transporter function. The presence of these CBS domains was correlated with osmoregulatory function among the putative transporters examined in DC3000 and was found to be predictive of functional osmoregulatory transporters in other pseudomonads. These results provide the first functional evaluation of an osmoprotectant transporter in a Pseudomonas species and demonstrate the usefulness of the CBS domains as predictors of osmoregulatory activity.     

Development and pilot evaluation of novel genetic educational materials designed for an underserved patient population

Genetic counseling for BRCA1 and BRCA2 mutations involves teaching about hereditary cancer, genetics and risk, subjects that are difficult to grasp and are routinely misunderstood. Supported by a grant from the Avon Foundation, the UCSF Cancer Risk Program started the first genetic testing and counseling service for a population of traditionally underserved women of varied ethnic and social backgrounds at the San Francisco General Hospital (SFGH). Informed by educational theory and clinical experience, we devised and piloted two simplified explanations of heredity and genetic risk, with the aim of uncovering how to best communicate genetics and risk to this underserved population. A "conventional" version comprised pictures of genes, pedigrees, and quantitative representations of risk. A "colloquial" pictorial version used an analogy of the "information book" of genes, family stories and vignettes, and visual representations of risk, without using scientific words such as genes or chromosomes. A verbal narrative accompanied each picture. We presented these modules to four focus groups of five to eight women recruited from the SFGH Family Practice Clinic. Overall, women preferred a picture-based approach and commented that additional text would have been distracting. The majority of women preferred the colloquial version because it was easier to understand and better conveyed a sense of comfort and hope. We conclude that simplicity, analogies, and familiarity support comprehension while vignettes, family stories, and photos of real people provide comfort and hope. These elements may promote understanding of complex scientific topics in healthcare, particularly when communicating with patients who come from disadvantaged backgrounds.     

Stable isotope pilot study of exchangeable copper kinetics in human blood plasma

To develop further our understanding of initial dietary copper metabolism, a method has been developed to separate plasma copper that is bound to albumin, from that bound to ceruloplasmin. This method has been tested using plasma samples from a pilot study involving six human volunteers who consumed 3mg oral doses of the stable isotope (65)Cu and gave blood samples at timed intervals up to 7 days. The results suggest that this method can be used to monitor dynamic fluctuations in newly absorbed copper over a short time frame.     

Multimodality registration without a dedicated multimodality scanner

Multimodality scanners that allow the acquisition of both functional and structural image sets on a single system have recently become available for animal research use. Although the resultant registered functional/structural image sets can greatly enhance the interpretability of the functional data, the cost of multimodality systems can be prohibitive, and they are often limited to two modalities, which generally do not include magnetic resonance imaging. Using a thin plastic wrap to immobilize and fix a mouse or other small animal atop a removable bed, we are able to calculate registrations between all combinations of four different small animal imaging scanners (positron emission tomography, single-photon emission computed tomography, magnetic resonance, and computed tomography [CT]) at our disposal, effectively equivalent to a quadruple-modality scanner. A comparison of serially acquired CT images, with intervening acquisitions on other scanners, demonstrates the ability of the proposed procedures to maintain the rigidity of an anesthetized mouse during transport between scanners. Movement of the bony structures of the mouse was estimated to be 0.62 mm. Soft tissue movement was predominantly the result of the filling (or emptying) of the urinary bladder and thus largely constrained to this region. Phantom studies estimate the registration errors for all registration types to be less than 0.5 mm. Functional images using tracers targeted to known structures verify the accuracy of the functional to structural registrations. The procedures are easy to perform and produce robust and accurate results that rival those of dedicated multimodality scanners, but with more flexible registration combinations and while avoiding the expense and redundancy of multimodality systems.     

Ecology and bioprospecting

Bioprospecting is the exploration of biodiversity for new resources of social and commercial value. It is carried out by a wide range of established industries such as pharmaceuticals, manufacturing and agriculture as well as a wide range of comparatively new ones such as aquaculture, bioremediation, biomining, biomimetic engineering and nanotechnology. The benefits of bioprospecting have emerged from such a wide range of organisms and environments worldwide that it is not possible to predict what species or habitats will be critical to society, or industry, in the future. The benefits include an unexpected variety of products that include chemicals, genes, metabolic pathways, structures, materials and behaviours. These may provide physical blueprints or inspiration for new designs. Criticism aimed at bioprospecting has been addressed, in part, by international treaties and legal agreements aimed at stopping biopiracy and many activities are now funded by agencies that require capacity-building and economic benefits in host countries. Thus, much contemporary bioprospecting has multiple goals, including the conservation of biodiversity, the sustainable management of natural resources and economic development. Ecologists are involved in three vital ways: first, applying ecological principles to the discovery of new resources. In this context, natural history becomes a vast economic database. Second, carrying out field studies, most of them demographic, to help regulate the harvest of wild species. Third, emphasizing the profound importance of millions of mostly microscopic species to the global economy.     

Imaging of alkaline phosphatase activity in bone tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Strong TCR conservation and altered T cell cross-reactivity characterize a B*57-restricted immune response in HIV-1 infection

HLA-B*57 is associated with slower disease progression to AIDS, and CD8+ T cell responses to B*57-restricted epitopes are thought to contribute to this protective effect. In this study, we evaluate the B*57-restricted p24 KAFSPEVIPMF (KF11) immune response which is immunodominant during chronic infection. Previously, we observed that the KF11 clade variants KGFNPEVIPMF [A2G,S4N] and KAFNPEIIMPF [S4N,V7I], sharing a position 4 mutation, are differentially recognized by KF11-specific T cells. By combining structural and cellular studies, we now demonstrate that the KF11 and [A2G,S4N] epitopes induce distinct functional responses in [A2G,S4N] and KF11-specific T cells, respectively, despite minimal structural differences between the individual B*57-peptide complexes. Recently, we also elucidated the highly distinct structure of KF11 in complex with B*5703, and have now characterized the CD8+ T cell repertoire recognizing this epitope. We now report striking features of TCR conservation both in terms of TCR Valpha and Vbeta chain usage, and throughout the hypervariable region. Collectively, our findings highlight unusual features of the B*5701/B*5703-KF11-specific immune responses which could influence disease progression and that might be important to consider when designing future vaccine regimens.