John Goers

Methionine oxidation inhibits fibrillation of human α-synuclein in vitro

We examined the effect of methionine oxidation of human recombinant α‐synuclein on its structural properties and propensity to fibrillate. Both oxidized and non‐oxidized α‐synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered. Both proteins adopted identical partially folded conformations under conditions of acidic pH. The fibrillation of α‐synuclein at neutral pH was completely inhibited by methionine oxidation. This inhibitory effect was eliminated at low pH. The addition of oxidized α‐synuclein to the unoxidized form led to a substantial inhibition of α‐synuclein fibrillation.

Polycation-induced oligomerization and accelerated fibrillation of human α-synuclein in vitro

The aggregation and fibrillation of α‐synuclein has been implicated as a causative factor in Parkinsons disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that α‐synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze α‐synuclein oligomerization. The formation of α‐synuclein–polycation complexes was not accompanied by significant structural changes in α‐synuclein. However, α‐synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating α‐synuclein aggregation.

An enzyme-linked immunoassay for lipoprotein lipase

Polyclonal antibodies against bovine milk lipoprotein lipase (LPL) were used to generate an enzyme-linked immunosorbent assay (ELISA) for rat LPL. The antibodies to LPL were affinity purified on bovine LPL columns and were shown to be specific for LPL by immunoprecipitation and enzyme inhibition. The solid-phase ELISA was sensitive from 1.0 to 20 ng/ml of LPL and paralleled enzyme activity. Denatured rat LPL showed the same LPL mass as undenatured samples, allowing LPL mass to be quantitated effectively in a variety of rat tissue extracts.

Effects of Exercise Training and Feeding on Lipoprotein Lipase Gene Expression in Adipose Tissue, Heart, and Skeletal Muscle of the Rat

Lipoprotein lipase (LPL) is found in adipose tissue and muscle, and is important for the uptake of triglyceride-rich lipoproteins from plasma. This study examined the regulation of LPL in adipose tissue and muscle by exercise training in combination with the fed or fasted state. After training male rats on a treadmill for 6 weeks, LPL activity, mass, and mRNA levels were measured in adipose tissue, heart, soleus, and extensor digitorum longus (EDL) muscles and compared with levels in sedentary rats. Tissue LPL was measured as the heparin-released (HR) and cellular-extracted (EXT) fractions 16 hours following the last bout of exercise, during which time some animals were fasted and others were allowed free access to food. Training led to an increase in HR LPL activity and LPL protein mass in soleus and EDL, but had no effort on adipose tissue and heart LPL. The increase in soleus LPL with exercise was in the HR fraction only, whereas the increase in EDL LPL with training was in both the HR and EXT fractions. All these changes in LPL activity were accompanied by similar changes in LPL immunoreactive mass. However, there were no changes in LPL mRNA levels with training. Feeding induced a large increase in adipose tissue LPL activity and mass in both the HR and EXT fractions: however, there was no change in mRNA levels. In heart, feeding yielded a decrease in HR but no consistent change in EXT activity or mass, and a consistent decrease in mRNA levels. As compared with control rats, trained rats demonstrated different responses to feeding in all tissues, especially in soleus and EDL. Whereas feeding had no effect on LPL in soleus and EDL of control rats, feeding induced a decrease in HR and EXT LPL in the soleus of trained rats. In addition, feeding yielded a significant decrease in EXT LPL of the EDL of trained rats. Thus, these data demonstrate that adipose tissue and heart LPL are highly regulated by feeding and are not responsive to long-term exercise training. On the other hand, skeletal muscle LPL is increased in trained rats, but this increase is blunted considerably by feeding following the last bout of exercise. These changes in LPL activity and mass are mostly unaccompanied by changes in LPL mRNA levels, demonstrating that much physiologic regulation occurs posttranscriptionally.

Regulation of lipoprotein lipase immunoreactive mass in isolated human adipocytes.

Previous studies of human adipose tissue lipoprotein lipase (LPL) have focused on enzyme catalytic activity, and have not measured the LPL protein directly. To study the regulation of the LPL protein, an antibody against purified bovine LPL was used. To demonstrate the specificity of the antiserum, adipose homogenates were Western blotted, and adipocytes were radiolabeled and the cell homogenates immunoprecipitated, yielding a single specific band at 53 kD. Breakdown products of LPL were demonstrated at 35 and 20 kD by Western blotting. An ELISA for human adipose LPL was established, in which LPL was sandwiched between affinity-purified antibody and biotinylated affinity-purified antibody. The standard curves for bovine LPL and human adipose LPL were parallel, and LPL activity correlated strongly with LPL immunoreactive mass. Thus, the bovine LPL standard curve was used to estimate LPL immunoreactive mass from human adipose tissue. The regulation of LPL activity and immunoreactive mass were compared in cultured adipocytes in the presence an absence of insulinlike growth factor-I/somatomedin C (IGF-I), insulin, and fetal bovine serum. IGF-I and a high insulin concentration (70 nM) stimulated only the heparin-releasable (HR) component of LPL activity and immunoreactive mass, and neither IGF-I nor insulin affected LPL specific activity. In contrast, 10% fetal bovine serum stimulated HR activity, HR mass, and cellular extractable (EXT) immunoreactive mass, with no effect on EXT activity. This resulted in a decrease in EXT specific activity in response to serum. The effects of the locally produced nucleosides adenosine and inosine were studied in a similar manner. As with serum, adenosine stimulated HR activity, HR mass, and EXT immunoreactive mass, resulting in a decrease in EXT specific activity. Inosine stimulated an increase in HR activity and HR mass, but had no effect on EXT, and thus did not change LPL specific activity. Thus, a sensitive ELISA for adipose tissue LPL has been developed using a specific, well-characterized antibody. Regulation of human LPL immunoreactive mass was demonstrated in vitro by IGF-I, serum, high concentrations of insulin, adenosine, and inosine. This method will permit further investigations into the regulation of the LPL protein.

Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal

ABSTRACT The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal. IMPORTANCE Bacterial biofilms are ubiquitous in nature, but the mechanisms governing their assembly and spatial organization are not fully understood. Bacterial communication through quorum sensing has been shown to influence biofilm growth through the regulation of biofilm genes. Our study revealed a new role for quorum sensing in biofilms through rapid chemotactic responses to quorum signals. Specifically, we studied how chemorepulsion of Helicobacter pylori from the universal quorum signal autoinducer-2 (AI-2) shapes the spatial organization of its biofilms. We demonstrate that the chemorepulsive response of H. pylori to AI-2 is necessary to promote its dispersal from biofilms grown on both abiotic and biotic surfaces and is sufficient to promote dispersal in a chemotaxis-dependent manner. This work has broad implications for understanding the mechanisms by which endogenously produced microbial compounds shape the assembly and spatial organization of microbial communities in their environments. Bacterial biofilms are ubiquitous in nature, but the mechanisms governing their assembly and spatial organization are not fully understood. Bacterial communication through quorum sensing has been shown to influence biofilm growth through the regulation of biofilm genes. Our study revealed a new role for quorum sensing in biofilms through rapid chemotactic responses to quorum signals. Specifically, we studied how chemorepulsion of Helicobacter pylori from the universal quorum signal autoinducer-2 (AI-2) shapes the spatial organization of its biofilms. We demonstrate that the chemorepulsive response of H. pylori to AI-2 is necessary to promote its dispersal from biofilms grown on both abiotic and biotic surfaces and is sufficient to promote dispersal in a chemotaxis-dependent manner. This work has broad implications for understanding the mechanisms by which endogenously produced microbial compounds shape the assembly and spatial organization of microbial communities in their environments.

Differential sedimentation study on the dissociation and conformational change in hemoglobin

Abstract X-ray studies suggest that hemoglobin undergoes a subunit shift when oxygen is bound. The crystallographic measurements indicate that the oxygenated hemoglobin molecule occupies a slightly smaller volume; thus, an increase in sedimentation coefficient would be predicted upon oxygenation. As a complicating factor, the oxygenated hemoglobin begins to dissociate into dimers and even monomers as the protein concentration is lowered, while the deoxygenated molecule retains its tetrameric structure. Using differential sedimentation techniques, these expected changes have been measured. First, the deoxygenated hemoglobin is shown not to be appreciably dissociated in the concentration range between 0.3 to 50 mg/ml., and the sedimentation data are closely approximated by the expression 1 S = 1 S 20, w 0 (1 + kc) . Second, the sedimentation coefficient difference between deoxygenated and oxygenated hemoglobin changes by 0.5 s over this concentration range, behaving as predicted from the known dissociation constants. Finally, at the highest concentrations, the oxygenated hemoglobin sediments more rapidly than the deoxygenated form, as would be predicted from the subunit rearrangement determined from X-ray crystallographic studies.

Studies on C3ahu binding to human eosinophils: characterization of binding.

C3a purified to chemical homogeneity from human serum binds preferentially to human eosinophils greater than neutrophils. Little or no binding is found with human platelets. Maximum binding to eosinophils at 37 degrees C occurs within 15 min. Dilution of 125I-C3a by either cold C3a or washing away unbound 125I-C3a and reincubating at 37 degrees C reveals a T1/2 of approximately 30 min. C3adesArg neither binds to eosinophils nor inhibits the binding of 125I-C3a. The binding of C3a to human eosinophils may reflect a physiologic role of C3a in eosinophil motility or function.