Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti‐bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat‐me” signals such as galectin‐8 and ubiquitin that label bacteria as autophagy cargo. Anti‐bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti‐bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella‐associated “eat‐me” signals, including host‐derived glycans and K48‐ and K63‐linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.     

Endodermis-like Sheaths in the Submerged Freshwater MacrophyteRanunculus trichophyllusChaix

Light, fluorescence and electron microscopical analysis of therooted freshwater plantRanunculus trichophyllusrevealed a peculiaranatomical feature. In addition to the true endodermis encirclingthe root stele, endodermis-like sheaths occurred around eachvascular bundle of the leaf segments and of the eustelic stemwith its large central cavity, which assumed an anatomical featureresembling that of some pteridophyte stems. These impermeablesheaths, whose cells differentiate suberized walls, can playa major role in hampering the apoplastic leakage of the pressurizedwater solution which flows throughout the plant in xylem vesselsand contains the mineral nutrients taken up by the roots fromthe sediment. Moreover, these sheaths can function in preventingflooding of the aerenchymatic cavities of the submerged organs.In this way the endodermis-like sheaths preserve the correctcirculation of gas and nutrient solution through the entireorganism and assume great significance as a structural mechanismevolved by this species to survive and grow underwater.Copyright1999 Annals of Botany Company. Ranunculus trichophyllus,freshwater macrophyte, submerged angiosperm, anatomy, endodermis, endodermis-like sheaths, light microscopy, fluorescence microscopy.     

Apoptosis in C3H-10T1/2 cells: Roles of intracellular pH,protein kinase C,and the Na+/H+ antiporter

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na⁺/H⁺ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H⁺ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na⁺/H⁺ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na⁺/H⁺ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na⁺/H⁺ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity. J. Cell. Biochem. 67:231–240, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of road surveys and circuit theory to predict hotspot locations for implementing road-effect mitigation

The mitigation of road-effects on wildlife, especially road mortality and habitat fragmentation, has become increasingly common in the last 20 years. However, exclusion fencing and habitat connectivity structures can be very costly and several questions remain regarding how to best determine locations that will optimize mitigation success. Based on data collected across several years and across multiple landscapes and taxa, we present a comparative analysis of two methods: road surveys and circuit theory, and review their benefits and challenges to better inform decision making. Road surveys were completed in two locations over three years for large mammals and herpetofauna to identify road crossing hotspots. Circuit theory was also applied to these systems to identify crossing hotspots using habitat resistance models. The location, number and width of hotspots were compared between methods. Hotspot distributions were similar between methods for some herpetofauna, but different for Mammals, and road surveys produced a significantly greater number of smaller hotspots compared to circuit theory, implying that road surveys provide better hotspot resolution. As circuit model complexity increased, the number and width of hotspots decreased, diffusing across the landscape. Road surveys were better at predicting optimal crossing structure location at a local scale; however, circuit theory is less costly, and can be useful at large scales. As both methods can offer valuable information, we argue that the combination of these two approaches provides a strong basis for managers and biologists to make informed decisions about costly mitigation measures, optimizing both conservation benefits and limited funding.     

The effect of two teeth resection procedures on the welfare of piglets in farrowing crates. Part 1

Teeth resection is a method of controlling the injurious effects of the aggression displayed when newborn piglets fight to establish a teat order. Recent European legislation discourages the practice. The objective of this study was to assess the effect of clipping and grinding piglets’ needle teeth, compared to leaving them intact, on the welfare of piglets in farrowing crates.

Six days pre-partum, 60 sows were assigned to one of three treatments. Litters had their teeth clipped (C), ground (G) or left intact (I) at birth. The time taken to carry out each procedure was recorded. Piglet weights and facial lesions, which were scored according to severity, were recorded on days 1, 4, 11, 18 and 27. Piglet weights were also recorded at birth. Mouth lesions were recorded on days 1, 4 and 27. Instantaneous scan samples of piglet behaviour were carried out for 30 min post-teeth resection procedure (1 min intervals), and for 6 h on days 1, 4, 8, 14, 21 and 26 (5 min intervals). One male and one female piglet per litter were chosen as focal animals and observed for 5 min each post-procedure and for 10 min each twice per day on days 1, 5, 12, 20 and 26. Mortalities were recorded throughout lactation.

Grinding took significantly longer than clipping the teeth or leaving them intact (F = 638.87, P < 0.001). I piglets had higher facial lesion scores than C and G piglets (F = 10.58, P < 0.001). A smaller proportion of piglets in I litters than C and G litters and a smaller proportion of piglets in G litters than C litters had at least one mouth lesion (F = 4.74, P < 0.001). During 30 min post-procedure, I piglets were active on the heatpad in more observations than C and G piglets (F = 3.49, P < 0.05). During 5 min post-procedure C piglets spent longer chomping than I piglets (F = 5.92, P = 0.05). On day 21, I piglets were active in more observations than G piglets (F = 2.11, P < 0.05). On day 26, G piglets were inactive in more observations than C and I piglets (F = 5.02, P < 0.05). On days 14 and 26, C piglets were sleeping in more observations than G piglets (F = 2.87, P = 0.05). There was a tendency for a larger proportion of I than C piglets to die due to overlying (F = 2.68, P = 0.08).

In conclusion, although all three options were associated with welfare problems, grinding can be recommended in preference to clipping or leaving the teeth intact.     

Surface expression of Bcl-2 in chronic lymphocytic leukemia and other B-cell leukemias and lymphomas without a breakpoint t(14;18)

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.     

SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling

Salmonella enterica serovar Typhimurium ( S. Typhimurium) replicates inside mammalian cells within membrane-bound compartments called Salmonella -containing vacuoles. Intracellular replication is dependent on the activities of several effector proteins translocated across the vacuolar membrane by the Salmonella pathogenicity island 2 (SPI-2)-type III secretion system (T3SS). This is accompanied by the formation in the vicinity of bacterial vacuoles of an F-actin meshwork, thought to be involved in maintaining the integrity of vacuolar membranes. In this study, we investigated the function of the SPI-2 T3SS effector SteC. An steC mutant strain was not defective for intracellular replication or attenuated for virulence in mice. However, the steC mutant was defective for SPI-2-dependent F-actin meshwork formation in host cells, although the vacuolar membranes surrounding mutant bacteria appeared to be normal. Expression of SteC in fibroblast cells following transfection caused extensive rearrangements of the F-actin cytoskeleton. Sequence analysis identified amino acid similarity between SteC and the human kinase Raf-1. A His-tagged SteC fusion protein had kinase activity in vitro and a point mutant lacking kinase activity was unable to induce F-actin rearrangements in vivo . We conclude that SPI-2-dependent F-actin meshwork formation depends on the kinase activity of SteC, which resembles more closely eukaryotic than prokaryotic kinases.