Integration of chitosan and plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus-W (PRSV-W) and Tomato chlorotic spot virus (TCSV) are common viruses infecting vegetables in south Florida. Application of plant growth-promoting rhizobacteria (PGPR) has emerged as a potential alternative of chemical pesticides to control these viruses. But, it is not sufficient to completely replace chemical control. This study aimed to investigate the synergistic effect of chitosan and PGPR to control PRSV-W and TCSV. The efficiency of PGPR to suppress PRSV-W and TCSV was significantly improved when they were accompanied with chitosan treatment. The highest reduction in disease severity of both PRSV-W and TCSV was achieved when chitosan treatment was accompanied with mixture of two PGPR (IN937a + SE34) or three PGPR strains (IN937a + SE34 + SE56). The results of this study proved that implementation of chitosan and PGPR could significantly restrict losses due to PRSV-W and TCSV in squash and tomato, in Florida and the United States.     

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.     

Nucleoside transport in mammalian cell membranes

Organomercurials form stable stoichiometric complexes with thiolated nucleosides. The complexes inhibited uptake of ribonucleosides and cytosine arabinoside (CAR) in various types of normal and transformed cells. The inhibition was competitive and reversible (Ki = 3--6 micrometer). The interaction between complexes and transport system displayed a 1:1 stoichiometry. Chemical factors which contributed to the inhibitory power were evaluated with a series of S-alkylated derivatives and S--Hg--R complexes of mercaptonucleosides. The inhibitory potency was not determined exclusively by the hydrophobic nature of either the S-alkylated or the S--Hg--R moieties. Chemical modification of cells with penetrating and nonpenetrating organomercurials lead to stimulation of nucleoside uptake and to an increase in its susceptibility to inhibition by S--Hg--R complexes or S-aklylated derivatives of mercaptopurine ribosides. The kinetic and chemical data obtained with nucleoside analogs and with chemical modifiers suggested complex features of nucleoside transport systems. Four distinct classes of sites were implied: (i) a substrate binding site susceptible directly to competitive inhibition by organomercurial-mercaptonucleoside complexes, (ii) an additional site susceptible either to S-arylalkylated or S-mercuriated derivatives of 6-mercaptopurine ribosides, (iii) SH-containing modifier sites which stimulate uridine uptake upon binding of organomercurials, and (iv) SH-containing modifier sites which inhibit the function upon binding of organomercurials. From the observation that only SH sites related to stimulation were susceptible to modification by macromolecular-SH modifier probes, some conclusions can be drawn regarding the disposition of the various sites in the cell membrane in general and among membrane components in particular.     

Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients

Summary A well-characterized 69.5×10³ dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100¢10³ dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.Supported in part by NIH grants CA 30019, CA 12582, CA 09010, CA 29605 awarded by DHSS     

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.     

Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.     

Promiscuity in multidrug recognition and transport: the bacterial MFS Mdr transporters

Multidrug (Mdr) transport is an obstacle to the successful treatment of cancer and infectious diseases, and it is mediated by Mdr transporters that recognize and export an unusually broad spectrum of chemically dissimilar toxic compounds. Therefore, in addition to its clinical significance, the Mdr transport phenomenon presents intriguing and challenging mechanistic queries. Recent studies of secondary Mdr transporters of the major facilitator superfamily (MFS) have revealed that they are promiscuous not only regarding their substrate recognition profile, but also with respect to matters of energy utilization, electrical and chemical flexibility in the Mdr recognition pocket, and surprisingly, also in their physiological functions.     

Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

Background

The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.

Methodology/Principal Findings

We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.

Conclusions

Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.     

Serum biomarkers differentiating Kawasaki disease from febrile infections: A pilot case-control study

Although some serum biomarkers are elevated in both Kawasaki disease (KD) and infections, these conditions have not been compared by individual or combined biomarkers. The aim of this study, undertaken between January 2016 and May 2018 in a large teaching hospital, was to compare the serum concentration of cytokines, metalloproteinases (MMP) and heat shock protein (HSP) between cases defined as children with Kawasaki disease (KD) and those with febrile infections (controls). Serum concentrations of tumour necrosis factor-alpha (TNF-alpha), interleukins (IL 1beta, 6, and 8), heat shock proteins (HSP 60 and 70) and matrix metalloproteinase (MMP 9) were measured on admission in 17 children under six years of age with a temperature >38.5 °C for ≥five days, and compared between the two groups. The median age was 25 months and the median duration of fever eight days. Seven children were diagnosed with KD and ten had a febrile infection. Only the serum concentrations of IL-6 and TNF-alpha were significantly higher in the former than in the latter group (P = 0.01 and 0.04 respectively). To differentiate between the two groups with the best sensitivity and specificity, the optimal cut-off value for IL-6 was 12.6 pg/mL, and for TNF-alpha 47.9 pg/mL. Their combined increase, however, outperformed their individual concentrations. The characteristic diagnostic “signature” of the combined elevation of IL-6 and TNF-alpha serum levels has the potential, in febrile children, to differentiate early KD from febrile infections, allowing the institution of appropriate therapy.     

Organocatalyzed Solvent Free and Efficient Synthesis of 2,4,5‐Trisubstituted Imidazoles as Potential Acetylcholinesterase Inhibitors for Alzheimer's Disease

The catalytic potential of pyridine‐2‐carboxlic acid has been evaluated for efficient, green and solvent free synthesis of 2,4,5‐trisubstituted imidazole derivatives 3a – 3m . The compounds 3a – 3m were synthesized by one pot condensation reaction of substituted aromatic aldehydes, benzil, and ammonium acetate in good to excellent yields (74–96 %). To explore the potential of these compounds against Alzheimer's disease, their inhibitory activities against acetylcholinesterase (AChE) were evaluated. In this series of compounds, compound 3m , bearing one ethoxy and a hydroxy group on the phenyl ring on 2,4,5‐trisubstituted imidazoles, proved to be a potent AChE inhibitor (102.56±0.14). Structure–activity relationship (SAR) of these compounds was developed. Molecular dockings were carried out for the compounds 3m , 3e , 3k , 3c , 3a , 3d , 3j , and 3f in order to further investigate the binding mechanism. The inhibitor molecule was molecularly docked with acetylcholinesterase to further study its binding mechanism. The amino group of the compound 3m forms an H‐bond with the oxygen atom of the residue (i. e., THR121) which has a bond length of 3.051 Å.