The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5¢-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y 2 receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y 2 nucleotide receptor (P2Y 2 R) expression with siRNA of P2Y 2 R (P2Y 2 R siRNA). UTP also increased the expression in astrocytes of a V b 3/5 integrins that are known to interact directly with the P2Y 2 R to modulate its function. Antia V integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y 2 R/a V interactions mediate the activation of astrocytes by UTP. P2Y 2 R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-a V integrin antibody. These results suggest that P2Y 2 Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders. Keywords: astrocyte migration, astrogliosis, integrins, P2 nucleotide receptors, uridine 5¢-triphosphate. Astrocytes, a type of glial cell in the central nervous system, regulate water and electrolyte transport, local pH and ionic equilibrium, and neurotransmitter uptake (Svendsen 2002). Astrocytes can become reactive under a variety of pathological conditions, a process termed astrogliosis characterized by increased expression of glial fibrillary acidic protein (GFAP) and enhanced cell migration and proliferation (Norton et al. 1992). In cerebral ischemia, reactive astrocytes migrate to the edge of an injured area and form a barrier between damaged and healthy tissue (Ellison et al. 1998). Although there are indications that reactive astrocytes can protect undamaged tissue and limit secondary injury, excessive or chronic accumulation of astrocytes can produce deleterious effects and prevent neuronal regeneration within the damaged area (Rutka et al. 1997;Gahtan and Overmier 1999). Reactive astrogliosis is associated with increased production of cytokines and other pro-inflammatory agents that can damage neurons (McGraw et al. 2001). Release from astrocytes of proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) has been shown to precede neuronal degeneration (Sheng et al. 1996;Loos et al. 2003 Abbreviations used: Akt, protein kinase B; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic pro...

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B 5 ). Both LT-IIb-B 5 and a GD1a binding-defective mutant (LT-IIb-B 5 (T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B 5 induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B 5 -induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.Recent developments in the field of innate immunity support the concept that cellular activation by microbial molecules involves interactions with multiple cooperating host receptors within membrane lipid rafts, whereas single receptor-based interactions may often represent an oversimplified model (1-3). This concept is exemplified by the ability of patternrecognition receptors such as Toll-like receptor 4 (TLR4) 2 or TLR2 to functionally associate with accessory molecules or coreceptors for induction of intracellular signaling. TLR4 alone is not sufficient for inducing a vigorous innate response to lipopolysaccharide (LPS) and requires MD-2 and CD14, although additional components of the LPS recognition complex may play a role in modifying TLR4-mediated signaling (4, 5). TLR2 responds to microbial lipoproteins in association with TLR1 or TLR6 as signaling partners and with CD14 or CD36 as important coreceptors for robust activation of TLR2/1 or TLR2/6 complexes (1, 6). The formation of TLR complexes with coreceptors may serve to generate a combinatorial repertoire for discriminating among the abundant and diverse microbial molecules and thereby to appropriately tailor the host response. However, the mechanisms involved in TLR cooperativity with accessory receptors are currently poorly understood.We have recently shown that the B subunit of type IIb heatlabile enterotoxin of Escherichia coli (designated LT-IIb-B 5 ) activates TLR2 signaling, although the underlying mechanism was not addressed (7). Type IIb and related enterotoxins (types I and IIa) display an AB 5 oligomeric structure in which a toxic A subunit is noncovalently linked to a pe...

Broccoli (Brassica oleracea L., Italica Group) is a good vegetable source of Ca and Mg, two critical minerals in human nutrition. Studies have shown that bioavailability of Ca from broccoli is comparable to that from milk. Thus, broccoli is an important alternative source of Ca in segments of the population that consume limited amounts of dairy products. Essentially nothing is known about the genetic influence on variation in Ca or Mg concentration of broccoli heads. Thus, the goal of this research was to examine variation in Ca, and also Mg concentrations, in a collection of USDA inbreds and commercial F1 hybrids. In 1996 and 1997 field studies, significant differences among inbred entries and among hybrid entries were observed for Ca and Mg concentrations of broccoli heads. With hybrids and inbreds, mean head Ca concentrations were ≈3.0 mg·g-1 dry weight (DW), and entries with lowest and highest Ca concentrations differed >2-fold. Mean Mg concentrations of hybrid heads was 2.3 mg·g-1 DW (range 1.8 to 2.6) and 2.8 mg·g-1 DW (range 2.2 to 3.7) in 1996 and 1997, respectively. Inbred lines had mean head Mg concentrations of 2.0 and 2.6 mg·g-1 DW in the two respective years and ranges in concentration were similar as for hybrids. Analysis of variance indicated significant environment and entry by environment effects for Ca and Mg concentrations of hybrids. With inbreds, a significant entry by environment effect for Ca concentration and environment effect for Mg concentration was also observed. Significant environment and entry by environment effects indicate that the environmental influence on phenotypic expression of Ca and Mg concentrations may complicate genetic improvement of head mineral concentration.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l--β-d--heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes and, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d--β-d--heptose together with GmhA and HldE, two enzymes from the LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.