Insights into the Structure and Metabolic Function of Microbes That Shape Pelagic Iron-Rich Aggregates (“Iron Snow”)

Microbial ferrous iron [Fe(II)] oxidation leads to the formation of iron-rich macroscopic aggregates (“iron snow”) at the redoxcline in a stratified lignite mine lake in east-central Germany. We aimed to identify the abundant Fe-oxidizing and Fe-reducing microorganisms likely to be involved in the formation and transformation of iron snow present in the redoxcline in two basins of the lake that differ in their pH values. Nucleic acid- and lipid-stained microbial cells of various morphologies detected by confocal laser scanning microscopy were homogeneously distributed in all iron snow samples. The dominant iron mineral appeared to be schwertmannite, with shorter needles in the northern than in the central basin samples. Total bacterial 16S rRNA gene copies ranged from 5.0 × 10⁸ copies g (dry weight)⁻¹ in the acidic central lake basin (pH 3.3) to 4.0 × 10¹⁰ copies g (dry weight)⁻¹ in the less acidic (pH 5.9) northern basin. Total RNA-based quantitative PCR assigned up to 61% of metabolically active microbial communities to Fe-oxidizing- and Fe-reducing-related bacteria, indicating that iron metabolism was an important metabolic strategy. Molecular identification of abundant groups suggested that iron snow surfaces were formed by chemoautotrophic iron oxidizers, such as Acidimicrobium, Ferrovum, Acidithiobacillus, Thiobacillus, and Chlorobium, in the redoxcline and were rapidly colonized by heterotrophic iron reducers, such as Acidiphilium, Albidiferax-like, and Geobacter-like groups. Metaproteomics yielded 283 different proteins from northern basin iron snow samples, and protein identification provided a glimpse into some of their in situ metabolic processes, such as primary production (CO₂ fixation), respiration, motility, and survival strategies.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation

Granuloma formation is a form of delayed-type hypersensitivity requiring CD4(+) T cells. Granulomas control the growth and dissemination of pathogens, preventing host inflammation from harming surrounding tissues. Using a murine model of Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) infection we studied the extent of T cell heterogeneity present in liver granulomas. We demonstrate that the TCR repertoire of granuloma-infiltrating T cells is very diverse even at the single-granuloma level, suggesting that before granuloma closure, a large number of different T cells are recruited to the lesion. At the same time, the TCR repertoire is selected, because AND TCR transgenic T cells (Valpha11/Vbeta3 anti-pigeon cytochrome c) are preferentially excluded from granulomas of BCG-infected AND mice, and cells expressing secondary endemic Vbeta-chains are enriched among AND cells homing to granulomas. Next, we addressed whether TCR heterogeneity is required for effective granuloma formation. We infected 5CC7/recombinase-activating gene 2(-/-) mice with recombinant BCG that express pigeon cytochrome c peptide in a mycobacterial 19-kDa bacterial surface lipoprotein. A CD4(+) T cell with a single specificity in the absence of CD8(+) T cells is sufficient to form granulomas and adequately control bacteria. Our study shows that expanded monoclonal T cell populations can be protective in mycobacterial infection.     

A dramatic accumulation of glycogen in the brown adipose tissue of rats following recovery from cold exposure

In a morphological study of brown adipose tissue (BAT) of rats returned after exposure to cold (+5 degrees C) to neutral temperature (+25 degrees C), striking periodic acid Schiff staining was observed, indicating substantial glycogen accumulation. Enzymatic analysis revealed that the glycogen content increased from the 4.05 +/- 0.51 (micromol glucose unit per gram of tissue, mean +/- SE) control value to 57.3 +/- 9.66 when the animals were returned to neutral temperature for 24 h after a 1-week cold period. Glycogen repletion was also observed in liver and skeletal muscle; however, the glycogen levels in these tissues never exceeded the control values. The accumulation of glycogen in the BAT started by the 3rd hour of replacement and peaked by the 24th hour. This glycogen was readily utilized during the next short cold exposure of the animals. The plasma leptin concentration was reduced at the cold temperature. The hexokinase II activity in the BAT increased to 29.3 +/- 1.46 vs the 11.8 +/- 1.06 control (mU/mg protein +/- SE) after a 1-week cold exposure and this level was maintained during the return to neutral temperature. The total glycogen synthetase (GStot) and the glycogen synthetase a activity also increased after a 1-week cold exposure and increased further during the replacement. The level of GStot reached 26.9 +/- 1.39 vs 9.54 +/- 1.43 control by the 24th hour of replacement. At the same time, the glycogen phosphorylase a activity declined during the replacement. The concentration of glucose 6-phosphate (an activator of GS) decreased in the cold but returned to normal during the replacement. These changes in the BAT are in favor of glycogen synthesis.     

Protein-protein docking: is the glass half-full or half-empty?

Are current docking methods capable of building complexes from putative component protein structures? Results of recent computational studies, including those of the CAPRI (Critical Assessment of Protein Interactions) competition, were used to determine the key properties for successful docking and introduce a classification of protein complexes based on docking difficulty. Enzyme-inhibitor complexes could be determined with reasonable accuracy - possibly to within a few alternative structures. Results for antigen-antibody pairs are less predictable, and data for small signaling complexes are generally poor. However, moderate amounts of experimental data can remove uncertainty and the methodology is rapidly improving. Transient complexes with large interface areas undergo substantial conformational change and are beyond the reach of current docking methods. The docking of such complexes might therefore require fundamentally new approaches.     

Effect of lecithin and MgCO3 as additives on the enzymatic activity of carbonic anhydrase encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres

A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme.