NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2 ؊/؊ ) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (10 7 bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2 ؊/؊ and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2 ؊/؊ mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P ‫؍‬ 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2 ؊/؊ and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2 ؊/؊ mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2 ؊/؊ mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1␤, IL-4, IL-6, and IL-10) were similar in Nod2 ؊/؊ and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.

We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.

A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold specificity. Recent studies have revealed a significant bias in unstructured peptides toward the polyproline II (P(II)) conformation, even when no prolines are present in the sequence. This indicates that the P(II) conformation is a dominant component of the denatured states of proteins, although a quantitative description of the component enthalpy and entropy functions associated with this conformation (i.e., the thermodynamic mechanism) has thus far proven elusive. An experimental system has been designed that, when analyzed with high-precision isothermal titration calorimetry, provides direct access to the residue-specific thermodynamics of the P(II) structure formation in disordered proteins and peptides. Here, it is shown that the P(II) bias is driven by a favorable and significant enthalpy (Deltah) of -1.7 kcal mol(-1) residue(-1), which is partially offset by an unfavorable entropy (TDeltas) of -0.7 kcal mol(-1) residue(-1), relative to the ensemble of disordered conformations of the molecule. In addition to impacting dramatically the interpretation of thermal denaturation experiments, these experimental values form the framework of a quantitative energetic description of the denatured states of proteins.

The glycodepsipeptide antibiotic ramoplanin A2 is in late stage clinical development for the treatment of infections from Grampositive pathogens, especially those that are resistant to first line antibiotics such as vancomycin. Ramoplanin A2 achieves its antibacterial effects by interfering with production of the bacterial cell wall; it indirectly inhibits the transglycosylases responsible for peptidoglycan biosynthesis by sequestering their Lipid II substrate. Lipid II recognition and sequestration occur at the interface between the extracellular environment and the bacterial membrane. Therefore, we determined the structure of ramoplanin A2 in an amphipathic environment, using detergents as membrane mimetics, to provide the most physiologically relevant structural context for mechanistic and pharmacological studies. We report here the X-ray crystal structure of ramoplanin A2 at a resolution of 1.4 Å. This structure reveals that ramoplanin A2 forms an intimate and highly amphipathic dimer and illustrates the potential means by which it interacts with bacterial target membranes. The structure also suggests a mechanism by which ramoplanin A2 recognizes its Lipid II ligand.glycolipodepsipeptide ͉ mechanism ͉ vancomycin resistance

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