Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria/host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach which enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared N-Acetyl-D-galactosamine-diphospho-undecaprenyl, the E. coli O86 oligosaccharide repeating unit was assembled via sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy via a chaperone co-expression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of additional substrates revealed a defined mode of recognition for Wzy towards the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well-defined system for further underpinning molecular details of this biosynthetic pathway.

A novel D-galactosyl-β1–3-N-acetyl-D-hexosamine phosphorylase cloned from Bifidobacterium infantis (BiGalHexNAcP) was used with a recombinant E. coli K-12 galactokinase (GalK) for efficient one-pot two-enzyme synthesis of T antigens, galacto-N-biose (Galβ1–3GalNAc), lacto-N-biose (Galβ1–3GlcNAc), and their derivatives.

Glycosyltransferases (GTs) catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. GTs are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new GTs are both difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with analysis by mass spectrometry to screen putative GTs. A total of 14,280 combinations of GT, acceptor and donor in four buffer conditions were screened and led to the identification and characterization of four new GTs. This work is significant because it provides a label-free method for the rapid functional annotation of putative enzymes.

STAT3 is a ubiquitous transcription factor that is indispensable during early embryogenesis. To study the functions of STAT3 postnatally, we generated conditional STAT3-deficient mice. To that end, STAT3 lox/lox mice were crossed with mice expressing Cre under the control of rat insulin II gene promoter (RIP-Cre mice). Immunohistochemical and Western blot analyses showed that STAT3 is deleted from ␤ cells in the islets of Langerhans. Genomic DNA PCR revealed that STAT3 deletion also occurred in the hypothalamus. lox/lox mice also failed to decrease adiposity or to correct other abnormalities in these mice. These data thus suggest that loss of STAT3 in the hypothalamus caused by RIP-Cre action likely interferes with normal body weight homeostasis and glucose metabolism. Signal transducers and activators of transcription (STAT)proteins are a family of latent cytoplasmic transcription factors that are produced in many cell types and that are activated by tyrosine phosphorylation and dimerization in response to a wide variety of extracellular ligands, such as cytokines and growth factors (12,36). One member of this family, STAT3, is expressed ubiquitously and is transiently activated by a large number of ligands, including epidermal growth factor, plateletderived growth factor, interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), oncostatin M, leukemia inhibitory factor, leptin, growth hormone, and prolactin, as well as a number of oncogenic receptor and nonreceptor (Src-like) tyrosine kinases (12). While gene disruption approaches have been used extensively to define the functions of members of the STAT family of transcription factors (18), the knockout of STAT3 results in early embryonic lethality (42). At the cellular level, STAT3 is required in order to maintain the pluripotency of embryonic stem cells, as demonstrated by the reduced ability of cells to undergo undifferentiated clonal growth when the level of STAT3 is reduced (33).The early embryonic lethality of STAT3 knockout mice prevents any type of physiological study (42). To overcome this limitation, many laboratories have employed tissue-specific conditional gene targeting to study STAT3 function in adult mice (2,3,24,39). These efforts have led to the elucidation of the roles played by STAT3 in various aspects of cytokine and growth factor signaling in different tissues and cell types. For instance, in T cells, STAT3 functions to transduce the antiapoptotic function of IL-6 independently from that of Bcl-2 (41); in macrophages and neutrophils, STAT3 is required to suppress the overshooting of inflammatory stimulus-induced proinflammatory response (40); in keratinocytes, loss of STAT3 results in compromised wound healing (19,34,35); in the mammary gland, loss of STAT3 causes delayed mammary gland involution after weaning (9); in the liver, STAT3 is required to mediate the ability of both IL-6-and lipopolysaccharide-induced acute-phase gene expressions (4); in sensory neurons, loss of STAT3 is associated with their enhanced death, which is normally p...