Severe MgADP Inhibition of Bacillus subtilis F1-ATPase Is Not Due to the Absence of Nucleotide Binding to the Noncatalytic Nucleotide Binding Sites

F₁-ATPase from Bacillus subtilis (BF₁) is severely suppressed by the MgADP inhibition. Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF₁ indicated that the noncatalytic sites could bind ATP normally. Furthermore, the mutant BF₁ that cannot bind ATP to the noncatalytic sites showed much lower ATPase activity. It was concluded that the cause of strong MgADP inhibition of BF₁ is not the weak nucleotide binding to the noncatalytic sites but the other steps required for the activation.     

Peyer’s Patches and Mesenteric Lymph Nodes Cooperatively Promote Enteropathy in a Mouse Model of Food Allergy

Background and Objective

To improve the efficacy and safety of tolerance induction for food allergies, identifying the tissues responsible for inducing intestinal inflammation and subsequent oral tolerance is important. We used OVA23-3 mice, which express an ovalbumin-specific T-cell receptor, to elucidate the roles of local and systemic immune tissues in intestinal inflammation.

Methods and Results

OVA23-3 mice developed marked enteropathy after consuming a diet containing egg white (EW diet) for 10 days but overcame the enteropathy (despite continued moderate inflammation) after receiving EW diet for a total of 28 days. Injecting mice with anti-IL-4 antibody or cyclosporine A confirmed the involvement of Th2 cells in the development of the enteropathy. To assess the individual contributions of Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and the spleen to the generation of effector CD4⁺ T-cells, we analyzed the IL-4 production, proliferation in response to ovalbumin, and CD4⁺ T-cell numbers of these tissues. EW feeding for 10 days induced significant IL-4 production in PPs, the infiltration of numerous CD4⁺ T-cells into MLNs, and a decrease in CD4⁺ T-cell numbers in spleen. On day 28, CD4⁺ T-cells from all tissues had attenuated responses to ovalbumin, suggesting tolerance acquisition, although MLN CD4⁺ T-cells still maintained IL-4 production with proliferation. In addition, removal of MLNs but not the spleen decreased the severity of enteropathy and PP-disrupted mice showed delayed onset of EW-induced inflammatory responses. Disruption of peripheral lymphoid tissues or of both PPs and MLNs almost completely prevented the enteropathy.

Conclusions

PPs and MLNs coordinately promote enteropathy by generating effector T-cells during the initial and exacerbated phases, respectively; the spleen is dispensable for enteropathy and shows tolerogenic responses throughout EW-feeding. The regulation of PPs may suppress the initiation of intestinal inflammation, subsequently restricting MLNs and inhibiting the progression of food-allergic enteropathy.     

A Multicopper Oxidase-Related Protein Is Essential for Insect Viability,Longevity and Ovary Development

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014     

Design and synthesis of novel androgen receptor antagonists with sterically bulky icosahedral carboranes

Carboranes (dicarba-closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable chemical and thermal stability, and hydrophobic character. These features may allow application of carboranes as a new hydrophobic core structure in biologically active molecules that interact hydrophobically with receptors. Here, we report the design and synthesis of novel androgen antagonists bearing a carborane moiety. These compounds, particularly 8a, 8c, and 9d, exhibited anti-androgenic activity similar to that of the well-known anti-androgen flutamide in reporter gene assay using NIH3T3 cells transfected with a human AR expression plasmid. The carborane cage seems to be a privileged hydrophobic pharmacophore for the expression of AR-antagonistic activity.     

A practical synthesis and biological evaluation of 9-halogenated PGF analogues

A series of 9-halo PGF analogues 1-2 and 5-13 were synthesized and biologically evaluated. Among the compounds, 2 was the best EP2-receptor agonist. A practical method of synthesizing 2 via the Julia olefination of an aldehyde 3 with an optically active sulfone 4, which was prepared by Sharpless asymmetric epoxidation of 15, was developed. Other 9-halogenated PGF analogues were synthesized essentially by the same procedure and evaluated. The absolute configuration of 16-OH of 2 was determined as S by the X-ray analysis of a salt consisting of a 1/1 molar ratio of 2 and L-lysine.     

2-Acylimino-3H-thiazoline derivatives: a novel template for platelet GPIIb/IIIa receptor antagonists

In the course of our research for the low-molecular weight RGD peptide mimics, we have found that a rigid 2-acylimino-3H-thiazoline structure is suitable for the peptide backbone mimics. Introduction of amidinophenyl and beta-alanine moiety as arginine and aspartic acid side-chain surrogates to this backbone mimic resulted in a highly potent fibrinogen receptor antagonist 2-(4-amidinobenzoylimino)-3,4-dimethyl-N-(2-carboxyethyl)-3H-thiazoline-5-carboxamide (7c), namely PS-028 (Ki = 46.5 +/- 5.8 microM).     

Design and synthesis of new orally active inhibitors of human neutrophil elastase

To identify new orally active inhibitors, further modification of 1 (ONO-6818) was performed. Peptidic derivatives 4b, 4c and 4n showed more potent inhibitory activity than nonpeptidic derivatives 3a-c. As a result, a series of peptidic inhibitors, 4a-s and 5a-v, were discovered. Among these N-aryl derivatives 5a-g, 5i, 5m and 5o-v showed oral activity. Their oral activity showed good correlation with their metabolic stability. Compounds 5h and 5j-l, which were extremely metabolically unstable in hamster plasma, did not show oral activity. Oral activity was considered to be determined by a combination of at least two factors: oral absorption and metabolic stability.     

Hsp40 Gene Therapy Exerts Therapeutic Effects on Polyglutamine Disease Mice via a Non-Cell Autonomous Mechanism

The polyglutamine (polyQ) diseases such as Huntington’s disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.     

KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi

Background

Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE) with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi.

Methods

Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA) were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge.

Results

Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4⁺CD62L^lowCCR7^low effector memory T cells pre- and post-sand fly challenge.

Conclusions

This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection.