Imaging and tracking of single hyaluronan molecules diffusing in solution

Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D = 3.0 ± 0.2 μm²/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension

Monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) in rats is preceded by an inflammatory response, progressive endothelial cell membrane disruption, reduction in the expression of caveolin-1, and reciprocal activation of STAT3 (PY-STAT3). Superoxide and NF-kappaB have been implicated in PAH. To evaluate the role of caveolin-1, PY-STAT3 activation, and superoxide in PAH, MCT-injected rats were treated daily with pyrrolidine dithiocarbamate (PDTC; starting on days 1, 3, and 14 x 2 wk), an inhibitor of inflammation and NF-kappaB activation. Hemodynamic data, the expression of inhibitory (I)-kappaBalpha, caveolin-1, and Tie2 (a membrane protein), activation of PY-STAT3 and NF-kappaB, and superoxide chemiluminescence were examined. Rats developed progressive PAH at 2 wk post-MCT. There was progressive reduction in the expression of caveolin-1, Tie2, and activation of PY-STAT3 in the lungs. Reduction in I-kappaBalpha expression was present at 2 and 4 wk post-MCT. Superoxide chemiluminescence and NF-kappaB activation were observed only at 2 wk post-MCT and both decreased by 4 wk post-MCT despite progressive PAH. PDTC (starting on days 1 and 3) rescued caveolin-1 and Tie2, reversed MCT-induced PY-STAT3 activation, and attenuated PAH. In addition, PDTC restored I-kappaBalpha expression and reduced superoxide chemiluminescence at 2 wk but did not inhibit NF-kappaB activation despite attenuation of PAH. PDTC had no effect on established PAH. Increased superoxide chemiluminescence and NF-kappaB activation appear to be a transient phenomenon in the MCT model. Thus the disruption of endothelial cell membrane integrity resulting in caveolin-1 loss and reciprocal activation of PY-STAT3 plays a key role in the MCT-induced PAH.     

In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution

Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.     

9G4+ Antibodies Isolated from HIV-Infected Patients Neutralize HIV-1 and Have Distinct Autoreactivity Profiles

Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these “9G4+” antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.