Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter⁻¹. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter⁻¹) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m⁻² s⁻¹ at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m⁻² s⁻¹ for photosynthetic ¹⁴CO₂ fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr^−/− mice reconstituted with Akt1^−/− fetal liver cells (Akt1^−/−→Ldlr^−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr^−/−). In contrast, Akt2^−/−→Ldlr^−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr^−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2^−/−→Ldlr^−/− mice had smaller aortic lesions compared with WT→Ldlr^−/− and Akt1^−/−→Ldlr^−/− mice. Importantly, Akt2^−/−→Ldlr^−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C^hi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2^−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.     

Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1 ^−/− RhoB ^−/− mice are fertile and develop normally. Relative to age-matched Drf1 ^−/− RhoB ^+/− mice, the age of myelodysplasia onset was earlier in Drf1 ^−/− RhoB ^−/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1 ^−/− RhoB ^−/− mice relative to Drf1 ^−/− RhoB ^+/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells.     

Rag2-deficient IL-1 Receptor Antagonist-deficient Mice Are a Novel Colitis Model in Which Innate Lymphoid Cell-derived IL-17 Is Involved in the Pathogenesis

Il1rn^−/− mice spontaneously develop arthritis and aortitis by an autoimmune mechanism and also develop dermatitis by an autoinflammatory mechanism. Here, we show that Rag2^−/−Il1rn^−/− mice develop spontaneous colitis with high mortality, making a contrast to the suppression of arthritis in these mice. Enhanced IL-17A expression in group 3 innate lymphoid cells (ILC3s) was observed in the colon of Rag2^−/−Il1rn^−/− mice. IL-17A-deficiency prolonged the survival of Rag2^−/−Il1rn^−/− mice, suggesting a pathogenic role of this cytokine in the development of intestinal inflammation. Although IL-17A-producing T cells were increased in Il1rn^−/− mice, these mice did not develop colitis, because CD4⁺Foxp3⁺ regulatory T cell population was also expanded. Thus, excess IL-1 signaling and IL-1-induced IL-17A from ILC3s cause colitis in Rag2^−/−Il1rn^−/− mice in which Treg cells are absent. These observations suggest that the balance between IL-17A-producing cells and Treg cells is important to keep the immune homeostasis of the colon.     

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2^−/−Msh6^−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice are indistinguishable from Msh2^−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2^−/−, Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2^−/−Msh6^−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Identification of the leaf vacuole as a major nitrate storage pool

Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO₃⁻ storage sink. α-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, α-mannosidase being the more suitable. Nitrate and α-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO₃⁻ than α-mannosidase during a single washing. Empirically determined corrections were used to account for NO₃⁻ movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO₃⁻ contained 58% of the total cellular NO₃⁻ and therefore represent a major NO₃⁻ storage pool.     

Deletion of Nardilysin Prevents the Development of Steatohepatitis and Liver Fibrotic Changes

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1^+/+ and Nrd1^−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1^+/+ and Nrd1^−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1^−/− mice was lower than that in Nrd1^+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1^−/− mice than in Nrd1^+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1^+/+ and Nrd1^−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1^−/− mice was significantly less than that in Nrd1^+/+ mice, indicating the decreased TNF-α shedding in Nrd1^−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1^−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.     

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1^−/−) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1^−/− mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1^−/− mice had decreased trabecular bone volume/total volume and that both male and female Wisp1^−/− mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1^−/− mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1^−/− mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1^−/− mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1^−/− bone marrow stromal cells had reduced expression of β-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1^−/− mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

Investigation on Crude and High-Temperature Heated Coffee Oil by ATR-FTIR Spectroscopy along with Antioxidant and Antimicrobial Properties

The coffee oil has a promising potential to be used in food industry, but an efficient use, especially in products that required high-temperature heating, depends on its chemical composition and the changes induced by processing. Since there is little information on this topic, the aim of our study was to investigate the crude green and roasted coffee oil (GCO, RCO) and heated (HGCO, HRCO) for 1 h at 200°C, by Fourier Transform Infrared (FTIR) spectroscopy and in terms of antioxidant and antimicrobial properties. The results of FTIR spectroscopy revealed that no statistically significant differences (one-way ANOVA, p>0.05) in the oxidative status of GCO and RCO were found. The coffee oils heating induced significant spectral changes in the regions 3100–3600 cm^–1, 2800–3050 cm^–1 and 1680–1780 cm^–1 proved by the differences in the absorbance ratios A 3009 cm⁻¹/A 2922 cm⁻¹, A 3009 cm⁻¹/A 2853 cm⁻¹, A 3009 cm⁻¹/A 1744 cm⁻¹, A 1744 cm⁻¹/A 2922 cm⁻¹. These alterations were related to the reduction of the unsaturation degree due to primary and secondary oxidation processes of the lipid fraction. The radical scavenging ability of oils investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay revealed that the IC50 value of GCO was significantly lower than of RCO (p<0.05). The IC50 values of crude coffee oils were lower than those of heated samples. The antioxidant activity of oils was attributed to both antioxidant compounds with free-radical scavenging capacity and to lipids oxidation products generated by heating. In the first 6 h of incubation, the inhibitory activity of crude oils against E. coli and E. faecalis was not significantly different to the control (p>0.05). Also, HGCO and HRCO showed significantly different inhibitory potential related to the control (p<0.05). The heating induced statistically significant decreases in the effectiveness of coffee oils against the tested bacteria. GCO proved to be the most effective among investigated coffee oils against the tested bacteria.     

Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.     

Cyp1b1 Mediates Periostin Regulation of Trabecular Meshwork Development by Suppression of Oxidative Stress

Mutation in CYP1B1 has been reported for patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1^−/−) mice. Cyp1b1^−/− mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue along with increased oxidative stress and decreased levels of periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited TM tissue ultrastructural abnormalities similar to those of Cyp1b1^−/− mice. Administration of the antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1^−/− mice. In addition, TM cells prepared from Cyp1b1^−/− mice exhibited increased oxidative stress, altered adhesion, and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1^+/+ TM cells resulted in a Cyp1b1^−/− phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax^−/−Bak^−/− cells and better than either Bid^−/− or dominant-negative caspase-9-expressing cells. Only Bim^−/− and Bax^−/−Bak^−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid^−/− cells, it readily did so in Bim^−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1^−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X_L with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1^−/− mice, cellular infiltration into infected TLR2^−/−, TLR4^−/−, and MD-2^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4^−/− mice, but not TLR2^−/− or MD-2^−/− mice. We also found that TRIF^−/− and TIRAP^−/− mice exhibited no fungal-killing defects, but that MyD88^−/− and IL-1R1^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.