IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

Sensitivity of tibio-menisco-femoral joint contact behavior to variations in knee kinematics

Use of computational models with kinematic boundary conditions to study the knee joint contact behavior for normal and pathologic knee joints depends on an understanding of the impacts of kinematic uncertainty. We studied the sensitivities of tibio-menisco-femoral joint contact behavior to variations in knee kinematics using a finite element model (FEM) with geometry and kinematic boundary conditions derived from sequences of magnetic resonance (MR) images. The MR images were taken before and after axial compression was applied to the knee joint of a healthy subject. A design of experiments approach was used to study the impact of the variation in knee kinematics on the contact outputs. We also explored the feasibility of using supplementary hip images to improve the accuracy of knee kinematics. Variations in knee kinematics (0.25mm in medial-lateral, 0.1mm in anterior-posterior and superior-inferior translations, and 0.1 degrees in flexion-extension and varus-valgus, 0.25 degrees in external-internal rotations) caused large variations in joint contact behavior. When kinematic boundary conditions resulted in close approximations of the model-predicted joint contact force to the applied force, variations in predictions of contact parameters were also reduced. The combination of inferior-superior and medial-lateral translations accounted for over 70% of variations for all the contact parameters examined. The inclusion of hip images in kinematic calculations improved knee kinematics by matching the femoral head position. Our findings demonstrate the importance of improving the accuracy and precision of knee kinematic measurements, especially when utilized as an input for finite element models.     

Lipid-derived aldehydes accelerate light chain amyloid and amorphous aggregation

Antibody light chain (LC) aggregation in vivo leads to the systemic deposition of Ig light chain domains in the form of either amyloid fibrils (AL-amyloidosis) or amorphous deposits, light-chain deposition disease (LCDD), in mainly cardiac or renal tissue and is a pathological condition that is often fatal. Molecular factors that may contribute to the propensity of LCs to aggregate in vivo, such as the protein primary structure or local environment, are intensive areas of study. Herein, we show that the aggregation of a human antibody kappa-(kappa-MJM) and lambda-(lambda-L155) light chain (1 mg/mL) can be accelerated in vitro when they are incubated under physiologically relevant conditions, PBS, pH 7.4 and 37 degrees C, in the presence of a panel of biologically relevant lipid-derived aldehydes, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), glyoxal (GLY), atheronal-A (KA), and atheronal-B (ALD). Thioflavin-T (ThT) and Congo Red (CR) binding assays coupled with turbidity studies reveal that this aldehyde-induced aggregation can be associated with alteration of protein secondary structure to an increased beta-sheet conformation. We observed that the nature of the conformational change is primarily dependent upon the lipidic aldehyde studied, not the protein sequence. Thus, the cholesterol 5,6-secosterols, KA and ALD, cause an amorphous-type aggregation which is ThT and CR negative for both the kappa-MJM and lambda-L155 light chains, whereas 4-HNE, MDA, and GLY induce aggregates that bind both ThT and CR. TEM analysis revealed that amyloid fibrils were formed during the 4-HNE-mediated aggregation of kappa-MJM and lambda-L155 light chains, whereas ALD-induced aggregates of these LCs where amorphous in nature. Kinetic profiles of LC aggregation reveal clear differences between the aldehydes, KA and ALD, causing a classic nucleated polymerization-type aggregation, with a lag phase (of approximately 150 h) followed by a growth phase that plateaus, whereas 4-HNE, MDA, and GLY trigger a seeded-type aggregation process that has no lag phase. In-depth studies of the 4-HNE-accelerated aggregation of kappa-MJM and lambda-L155 reveal a clear aldehyde concentration dependence and a process that can be inhibited by the naturally occurring osmolyte trimethylamine N-oxide (TMAO). Given these data, we feel our recently discovered paradigm of inflammatory aldehyde-induced protein misfolding may now extend to LC aggregation.     

Choline transport specificity in animal cells and tissues

1. Beta carbolines inhibit choline transport in rat brain. 2. The aziridinium ring on the nitrogen of mustard analogs of choline causes irreversible binding to the carrier in rat brain. 3. The uptake system in rat brain is stereoselective, requires a quaternary nitrogen, and prefers analogs with a nitrogen-oxygen distance of about 3.26 A. 4. In mouse brain troxonium derivatives inhibit choline transport. 5. In cuttlefish optic lobes and torpedo electric organ pyrene derivatives potently inhibit choline transport. 6. In guinea pig placenta, the affinity of the choline carrier remains high even when this molecule lacks one or two methyl groups.     

An isolation method of DNA from Pneumocystis carinii: a quantitative comparison to known parasitic protozoan DNA

1. A method for isolating DNA from Pneumocystis carinii is described. 2. The DNA content per nucleus is 0.22-0.34 pg. 3. This finding is consistent with other parasitic protozoa DNA content per nuclei.