Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.     

A novel process for extraction of edible oils: Enzyme assisted three phase partitioning (EATPP)

Three phase partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t-butanol (1:1,v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t-butanol containing oil and lower aqueous phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme, followed by three phase partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t-butanol which is a safer solvent as compared to n-hexane used in conventional oil extraction process.     

Leptin downregulates ethanol-induced secretion of proinflammatory cytokines and growth factor

Inflammatory mediators, including cytokines and growth factors are associated with the pathology of chronic liver diseases. The aim of our present work was to study the effect of exogenous leptin and/or ethanol on the secretion of TNF-alpha, IL-6 and TGF-beta1 both in vivo and in vitro. Forty eight hours after the exposure to ethanol (500 mM) significantly elevated the secretion of TNF-alpha, IL-6 and TGF-beta1 in the cell-free culture supernatant (HepG2 and mouse HCC cell lines), which were decreased on leptin (31.2 nM) treatment. Similarly, leptin administration to ethanol (6.32 g kg(-1) body weight) fed mice for 45 days significantly lowered the concentration of these cytokines in the circulation; however, leptin alone (230 microg kg(-1) body weight i.p. administered every alternate day for the last 15 days) had no such significant effect on cytokine secretion both in vivo and in vitro conditions. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system in alcoholic liver disease.     

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.     

Substrate specificity and mutational analysis of Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease

tRNA anticodon damage inflicted by the Kluyveromyces lactis γ-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. γ-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 3' of the wobble base of tRNA(Glu(UUC)) to generate a break with 2',3'-cyclic phosphate and 5'-OH ends. Recombinant γ-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA(Glu). A single 2'-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to γ-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 5' of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting γ-toxin's cleavage activity. Whereas either the 5' half or the 3' half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that γ-toxin requires an A-form helical conformation of the anticodon stem. We purified γ-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that γ-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151).     

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.     

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.     

Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.