Intravascular heavy chain-modification of hyaluronan during endotoxic shock

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1β stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric Cα structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

PHYSIOLOGICAL AND OPTICAL PROPERTIES OF RHIZOSOLENIA FORMOSA (BACILLARIOPHYCEAE) IN THE CONTEXT OF OPEN-OCEAN VERTICAL MIGRATION1

Cultures of Rhizosolenia formosa H. Peragallo were studied to assess whether or not physiological and optical characteristics of this large diatom were consistent with the ability to migrate vertically in the open ocean. Time-course experiments examined changes in chemical composition and buoyancy of R. formosa during nitrate (N)–replete growth, N starvation, and recovery. Cells could maintain unbalanced growth for at least 53 h after depletion of ambient nitrate. Increases in C:N and carbohydrate: protein ratios observed during N starvation reversed within 24 h of reintroduction of nitrate to culture medium. Buoyancy was related to nutrition: Upon N depletion, the percentage of positively buoyant cells decreased to 4% from 11% but reverted to 9% within 12 h of nitrate readdition. Cells took up nitrate in the dark. Nitrogen-specific uptake rates averaged 0.48 d^?1; these rates were higher than N-specific growth rates (0. 15 d^?1), indicating the potential for luxury consumption of nitrate, which can be stored for later use. Measurements of photosynthesis vs. irradiance, chlorophyll-specific absorption (a_ph*(λ)), and pigment composition showed that cells may be adapted for growth under a wide range of irradiances. Values of a_ph*(λ) were lower for N-depleted cells than for N-replete cells, and N-depleted cells had higher ratios of total carotenoids to chlorophyll a. Aggregation of chloroplasts was more pronounced in N-depleted cells. These are possibly photoprotective mechanisms that would be an advantage to N-depleted cells in surface waters. Compounds that absorb in the ultraviolet region were detected in N-replete cells but were absent in N-depleted cultures. Overall, these results have important implications for migrations of Rhizosolenia in nature. Cells may survive fairly long periods in N-depleted surface waters and will continue to take up carbon; then they can resume nitrate uptake and revert to positive buoyancy upon returning to deep, N-rich water. Uncoupled uptake of carbon and nitrogen during migrations of Rhizosolenia is a form of new production that may result in the net removal of carbon from oceanic surface waters.     

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.