Structure of a polyisoprenoid binding domain from Saccharophagus degradans implicated in plant cell wall breakdown

Saccharophagus degradans belongs to a recently discovered group of marine bacteria equipped with an arsenal of sugar cleaving enzymes coupled to carbohydrate-binding domains to degrade various insoluble complex polysaccharides. The modular Sde-1182 protein consists of a family 2 carbohydrate binding module linked to a X158 domain of unknown function. The 1.9 Å and 1.55 Å resolution crystal structures of the isolated X158 domain bound to the two related polyisoprenoid molecules, ubiquinone and octaprenyl pyrophosphate, unveil a β-barrel architecture reminiscent of the YceI-like superfamily that resembles the architecture of the lipocalin fold. This unprecedented association coupling oxidoreduction and carbohydrate recognition events may have implications for effective nutrient uptake in the marine environment.     

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Cytokine-stimulated phosphorylation of GSK-3 is primarily dependent upon PKCs, not PKB.

The regulation of glycogen synthase kinase-3 (GSK-3) by phosphorylation at inhibitory sites has been well documented. In many, but not all, cases, the phosphatidylinositol 3-kinase pathway, and particularly the downstream kinase protein kinase B (PKB)/akt, have been shown to be responsible for GSK-3 phosphorylation. Given that no studies have ever reported cytokine-mediated phosphorylation of GSK-3, we investigated the phosphorylation of this kinase in several hemopoietic cell types in response to either interleukin (IL)-3, IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF). Each of the cytokines was able to stimulate phosphorylation of the isoforms GSK-3alpha and GSK-3beta. However, only in the case of IL-4 stimulation was there any dependence on PKB for this phosphorylation. We were clearly able to show that PKB was capable of phosphorylating GSK-3 in these cells, but studies using inhibitors of the protein kinase C (PKC) family of kinases have shown that these enzymes are more likely to play a key role in GSK-3 phosphorylation. Cytokine-mediated generation of diacylglycerol was demonstrated, supporting the possible activation of PKC family members. Thus, cytokine-dependent GSK-3 phosphorylation in hemopoietic cells proceeds primarily through PKB independent pathways.     

How inaccuracies in protein structure models affect estimates of protein-ligand interactions: computational analysis of HIV-I protease inhibitor binding

The influence of possible inaccuracies that can arise during homology modeling of protein structures used for ligand binding studies were investigated with the molecular mechanics generalized Born surface area (MM-GBSA) method. For this, a family of well-characterized HIV-I protease-inhibitor complexes was used. Validation of MM-GBSA led to a correlation coefficient ranging from 0.72 to 0.93 between calculated and experimental binding free energies DeltaG. All calculated DeltaG values were based on molecular dynamics simulations with explicit solvent. Errors introduced into the protein structure through misplacement of side-chains during rotamer modeling led to a correlation coefficient between DeltaG(calc) and DeltaG(exp) of 0.75 compared with 0.90 for the correctly placed side chains. This is in contrast to homology models for members of the retroviral protease family with template structures ranging in sequence identity between 32% and 51%. For these protein models, the correlation coefficients vary between 0.84 and 0.87, which is considerably closer to the original protein (0.90). It is concluded that HIV-I low sequence identity with the template structure still allows creating sufficiently reliable homology models to be used for ligand-binding studies, although placement of the rotamers is a critical step during the modeling.     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

A new mechanism for prolactin processing into 16K PRL by secreted cathepsin D

Cathepsins are lysosomal enzymes that were shown to release the antiangiogenic fragments 16K prolactin (PRL), endostatin, and angiostatin by processing precursors at acidic pH in vitro. However, the physiological relevance of these findings is questionable because the neutral pH of physiological fluids is not compatible with the acidic conditions required for the proteolytic activity of these enzymes. Here we show that cathepsin D secreted from various tissues is able to process PRL into 16K PRL outside the cell. To specifically target extracellular proteolysis, we used tissues from PRL receptor-deficient mice, which are unable to internalize PRL. As assessed by the use of specific inhibitors of proton extruders, we show that the proteolytic activity of cathepsin D requires local acid secretion driven by Na(+)/H(+) exchangers and H(+)/ATPase. Although it is usually assumed that cathepsin-mediated generation of antiangiogenic peptides occurs in the moderately acidic pericellular milieu found in malignant tumors, we propose a new mechanism explaining the extracellular activity of this acidic protease under physiological pH. Our data support the concept that secreted lysosomal enzymes could be involved in the maintenance of angiogenesis dormancy via the generation of active antiangiogenic peptides in nonpathological contexts.     

Analysis of expansion of myeloid progenitors in mice to identify leukemic susceptibility genes

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.     

Sex-based divergence in head shape and diet in the Eastern lubber grasshopper (Romalea microptera)

Biologists are becoming increasingly aware of sex-based differences in ecologically important traits (locomotor performance, feeding morphology, thermoregulatory behavior). Yet, the overwhelming majority of these studies have been performed on vertebrates, despite often striking examples of sexual size dimorphism among the insects. Here, I tested whether adult male and female Eastern lubber grasshoppers differ in size/shape and feeding ecology. The morphological data clearly showed sex-based differences in several aspects of head and body size, with females being significantly larger in all aspects. Moreover, the sexes also differed in two aspects of size-adjusted morphology: head width and pronotum width, with females having relatively wider heads and thoraxes than males. Multiple regression analysis indicated that width of consumed foliage was positively related to pronotum width and foliage thickness was positively related to pronotum width, head width, and size-adjusted head width. Females consumed wider and thicker foliage (primarily smooth cord grass, Spartina alterniflora) than males, which most frequently consumed the narrow-leafed forb hemp sesbania (Sesbania macrocarpa). Therefore, the sex-based differences in size and shape in this grasshopper are correlated with differences in consumed foliage shape.