Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson’s disease and beta-amyloid and tau in Alzheimer’s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.     

Small-Colony Variant Selection as a Survival Strategy for Staphylococcus aureus in the Presence of Pseudomonas aeruginosa

Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of Pseudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hemB mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of patients with cystic fibrosis (CF). P. aeruginosa excretes an arsenal of small respiratory inhibitors, like pyocyanin (5), hydrogen cyanide (2), or quinoline N-oxides (9), that may act against the commensal microbiota as well as host cells. Previously it has been demonstrated that S. aureus is sensitive toward the toxic products generated by P. aeruginosa and that these exotoxins preferentially target the electron transport chain (17).Staphylococcal species can be divided into two groups: the sensitive group, comprising pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group was due to cydAB genes, which encode a pyocyanin- and cyanide-resistant cytochrome bd quinol oxidase (17). It has also been shown that the resistant or sensitive phenotype is determined by the CydB subunit, which is part of the cytochrome bd quinol oxidase of S. aureus. Despite its sensitivity to these exotoxins, S. aureus has frequently been coisolated with P. aeruginosa from the skin, eyes, and catheter infections and from the lungs of patients with CF. The aim of this study is to elucidate the escape mechanism of S. aureus by cocultivating S. aureus and P. aeruginosa. The findings indicate that a subpopulation of the staphylococcal community can survive in the presence of P. aeruginosa by the selection of small-colony variants (SCVs), which usually are defective in the electron transport chain. SCVs grow as tiny, nonpigmented colonies and are auxotrophic to hemin, menadione, or thymidine (14). Here we show that both a culture supernatant of P. aeruginosa and purified pyocyanin select for the SCV phenotype in S. aureus.     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Functional expression of horsegram (Dolichos biflorus) Bowman–Birk inhibitor and its self-association

Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, native to Southeast Asia and tropical Africa, contains multiple forms of Bowman–Birk inhibitors. The major Bowman–Birk inhibitor from horsegram (HGI-III) was cloned and functionally expressed in Escherichiacoli (rHGI), which moved as a dimer in solution similar to the natural inhibitor. The biochemical characterization of rHGI also points to its close resemblance with HGI-III not only in its structure but also in its inhibitory characteristics. To explore the electrostatic interactions involved in the dimerization, a site-directed mutagenesis approach was used. The role of reactive site residue K²⁴ and the C-terminal Asp in the structure and stability of the dimer was accomplished by mutating K²⁴ and D^75/76. The mutants produced in this study confirm that the self-association of HGI-III is indeed due to the electrostatic interaction between K²⁴ of one monomer and D^75/76 of the second monomer, in agreement with our previous data. The functional expression of a Bowman–Birk inhibitor minus a fusion tag serves as a platform to study the structural and functional effects of the special pattern of seven conserved disulphide bridges.     

Use of DNA Markers in Prediction of Hybrid Performance and Heterosis for a Three-Line Hybrid System in Rice

Two Cytoplasmic Male Sterile lines were crossed with fourteen restorer lines of rice widely grown in the western regions of Maharashtra, India, to produce 28 F₁ hybrids which were evaluated for eight agronomically important traits, contributing to yield potential, in replicated field trials. The hybrid performance was recorded along with heterosis and heterobeltiosis. All the rice lines under investigation were subjected to marker-based variability analysis. An attempt was made to correlate genetic distance based on specific markers for each trait individually, as well as average genetic distance based on all specific markers, with hybrid performance and heterosis, by regression analysis. Specific markers could cluster the parental lines in different groups and showed significant correlation with hybrid performance. The data also supports the proposition that epistasis is the basis of heterosis. The analysis, however, revealed a lack of significant predictive values for field application.     

25-hydroxycholesterol acts in the Golgi compartment to induce degradation of tyrosinase

Oxysterols play a significant role in cholesterol homeostasis. 25-Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol-binding protein and oxysterol-related proteins, the sterol regulatory element binding protein, and the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC(50) of 0.34 microM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse-chase studies of 25HC-treated cells demonstrated enhanced degradation of tyrosinase, the rate-limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis-Golgi SNARE protein complex, were also diminished in 25HC-treated melanocytes, however levels of the ER chaperone calnexin and the cis-Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4 alpha-allylcholestan-3 alpha-ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.     

Pathogen Induced Changes in the Protein Profile of Human Tears from Fusarium Keratitis Patients

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.