Despite the presence of genes that apparently encode NAD salvage-specific enzymes in its genome, it has been previously thought that Mycobacterium tuberculosis can only synthesize NAD de novo. Transcriptional analysis of the de novo synthesis and putative salvage pathway genes revealed an up-regulation of the salvage pathway genes in vivo and in vitro under conditions of hypoxia. [14 C]Nicotinamide incorporation assays in M. tuberculosis isolated directly from the lungs of infected mice or from infected macrophages revealed that incorporation of exogenous nicotinamide was very efficient in in vivo-adapted cells, in contrast to cells grown aerobically in vitro. Two putative nicotinic acid phosphoribosyltransferases, PncB1 (Rv1330c) and PncB2 (Rv0573c), were examined by a combination of in vitro enzymatic activity assays and allelic exchange studies. These studies revealed that both play a role in cofactor salvage. Mutants in the de novo pathway died upon removal of exogenous nicotinamide during active replication in vitro. Cell death is induced by both cofactor starvation and disruption of cellular redox homeostasis as electron transport is impaired by limiting NAD. Inhibitors of NAD synthetase, an essential enzyme common to both recycling and de novo synthesis pathways, displayed the same bactericidal effect as sudden NAD starvation of the de novo pathway mutant in both actively growing and nonreplicating M. tuberculosis. These studies demonstrate the plasticity of the organism in maintaining NAD levels and establish that the two enzymes of the universal pathway are attractive chemotherapeutic targets for active as well as latent tuberculosis.

The palladium-catalyzed cross-coupling reaction of substituted potassium alkyltrifluoroborates with aryl halides and aryl triflates proceeds readily with moderate to good yields. The potassium alkyltrifluoroborates 1, 2, and 3a-e were easily synthesized and obtained as air-stable crystalline solids that can be stored for long periods of time. All of the cross-couplings proceed under the same reaction conditions using PdCl(2)(dppf).CH(2)Cl(2) as catalyst in THF-H(2)O in the presence of 3 equiv of Cs(2)CO(3) as base.

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Collagen is the structural framework for a wide range of animal connective tissues. It is known to induce cell proliferation and differentiation by direct epitope binding and by serving as a reservoir for growth factors and signaling molecules. [1] In the past, collagen mimetic peptide (CMP), a synthetic peptide composed of a collagen-like repetitive amino acid sequence, has played a central role in elucidating the triple-helical structure and thermal stability of natural collagens. [2][3][4][5][6][7] Herein, we show for the first time that a biochemically inert but highly helicogenic CMP, (Pro-HypGly) 7 (Hyp = hydroxyproline), binds preferentially to the gap regions within the surface of intact type I collagen fibers. This binding behavior was demonstrated by synthesizing CMPconjugated gold nanoparticles (NPs) that are colloidally stable under a wide range of aqueous conditions and by transmission electron microscopy (TEM) observation of their attraction to type I collagen fibers under physiological conditions. The results suggest that such binding affinity may exist in other natural proteins/peptides that contain collagen-like sequences.[8] The study also demonstrates the potential use of nanoparticle-labeling techniques in detecting structurally unstable domains in collagen fibers which are related to many debilitating human diseases. [9] The collagen triple helix is composed of three poly-(proline)-II strands that are held together by interchain hydrogen bonds. [4,10] This structure is similar to the doublehelical structure of DNA. Both collagen and DNA exhibit melting transitions that reflect the stability and strength of the multiplex assemblies. Strand invasion in DNA by short DNA strands or peptide nucleic acids is well documented. [11] Although collagens are known to incorporate thermally unstable domains where small segments of the triple helix are thought to be partially unraveled, [12] strand invasion by other collagen molecules or collagen analogues have not been reported to date.We previously presented a method of collagen modification that uses CMP as a collagen-specific molecular "hitchhiker".[13] Our results suggested that the CMP, (Pro-HypGly) x binds to a film composed of type I collagen molecules (in nonfibrous form) through a process that involves both strand invasion and assembly of a triple helix. However, the binding location and physical state of the collagen molecules that interact with CMP have not been studied in detail.In nature, type I collagen exists not as individual molecules but instead forms fibrous bundles of high tensile strength. Under TEM, collagen fibers exhibit characteristic banding patterns that indicate the structural integrity of the collagen molecules and the regularity of their assembly. The banding patterns also provide approximate position markers along the length of collagen molecules. As CMP is invisible under TEM, we prepared CMP-conjugated gold nanoparticles to use as an electron-dense TEM marker for the location of CMPs. We investigated binding between type I ...