The inflammatory caspases: key players in the host response to pathogenic invasion and sepsis

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). The inflammatory caspases were among the first to be discovered, but only recently have the mechanisms leading to their activation and inhibition begun to be elucidated. In this review, we examine the biochemistry, substrates, and function of this unique family of inflammatory proteases, highlight the most recent findings regarding their regulatory mechanisms, and discuss what remains to be understood about their roles in health and disease.     

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.     

Remodeling of lipid droplets during lipolysis and growth in adipocytes

Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.     

Selective attenuation of norepinephrine release and stress-induced heart rate increase by partial adenosine A1 agonism

The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10(-8) M (30 μg/l), 6 · 10(-7) M (300 μg/l) or 2-chloro-N(6)-cyclopentyladenosine (CCPA) 10(-6) M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/-87 pmol/g vs. 173+/-18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation-induced NE release in SHR (S2/S1 = 0.90 ± 0.08 with capadenoson 6 · 10(-8) M, 0.54 ± 0.02 with 6 · 10(-7) M), but not in Wistar hearts (S2/S1 = 1.05 ± 0.12 with 6 · 10(-8) M, 1.03 ± 0.09 with 6 · 10(-7) M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [(35)S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/-2% A(1)-receptor stimulation). These results suggest that partial adenosine A(1)-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release.     

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.     

New molecular microbiology approaches in the study of Campylobacter fetus

Campylobacter fetus infection is a substantial problem in herds of domestic cattle worldwide and a rising threat in human disease. Application of comparative and functional genomics approaches will be essential to understand the molecular basis of this pathogen's interactions with various hosts. Here we report recent progress in genome analyses of C. fetus ssp. fetus and C. fetus ssp. venerealis, and the development of molecular tools to determine the genetic basis of niche‐specific adaptations. Campylobacter research has been strengthened by the rapid advancements in imaging technology occurring throughout microbiology. To move forward in understanding the mechanisms underlying C. fetus virulence, current efforts focus on developing suitable in vitro models to reflect host‐ and tissue‐specific aspects of infection.     

Dimethylarginine Dimethylaminohydrolase1 Is an Organ-Specific Mediator of End Organ Damage in a Murine Model of Hypertension

Background

The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension.

Methodology/Principal Findings

Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury.

Conclusion/Significance

Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.     

The Conserved Bud20 Zinc Finger Protein Is a New Component of the Ribosomal 60S Subunit Export Machinery

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C₂H₂-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.     

Rapid Diagnostic Algorithms as a Screening Tool for Tuberculosis: An Assessor Blinded Cross-Sectional Study

Background

A major obstacle to effectively treat and control tuberculosis is the absence of an accurate, rapid, and low-cost diagnostic tool. A new approach for the screening of patients for tuberculosis is the use of rapid diagnostic classification algorithms.

Methods

We tested a previously published diagnostic algorithm based on four biomarkers as a screening tool for tuberculosis in a Central European patient population using an assessor-blinded cross-sectional study design. In addition, we developed an improved diagnostic classification algorithm based on a study population at a tertiary hospital in Vienna, Austria, by supervised computational statistics.

Results

The diagnostic accuracy of the previously published diagnostic algorithm for our patient population consisting of 206 patients was 54% (CI: 47%–61%). An improved model was constructed using inflammation parameters and clinical information. A diagnostic accuracy of 86% (CI: 80%–90%) was demonstrated by 10-fold cross validation. An alternative model relying solely on clinical parameters exhibited a diagnostic accuracy of 85% (CI: 79%–89%).

Conclusion

Here we show that a rapid diagnostic algorithm based on clinical parameters is only slightly improved by inclusion of inflammation markers in our cohort. Our results also emphasize the need for validation of new diagnostic algorithms in different settings and patient populations.     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.