Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.     

Ferrioxamine-mediated Iron(III) utilization by Salmonella enterica

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA+ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA+ parent strain.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.     

BMP receptor signaling is required for postnatal maintenance of articular cartilage

Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.     

Differential distribution of the cognate and heat-stress-induced isoforms of high Mr cis-trans prolyl peptidyl isomerase (FKBP) in the cytoplasm and nucleoplasm

Wheat root tips express a 73 kDa cognate isoform and a 77 kDa heat-shock-induced isoform of peptidyl prolyl cis-trans isomerase (FK506 binding protein; FKBP) that is part of a chaperone complex with hsp90. The 73 kDa and 77 kDa FKBPs have very similar sequences, differing primarily in the N- and C-terminal 20 amino acids. In order to define the potential functional roles of these proteins, the 73 kDa and 77 kDa FKBPs were localized in root tips using antigen-affinity purified antibodies as a probe. The cognate 73 kDa FKBP is localized in the cytoplasm and appears enriched around the periphery of the early vacuole and vesicles exiting the trans-Golgi. Parallel assays with antibodies directed against tonoplast aquaporin and pyrophosphatase confirmed the association of FKBP with an early vacuole compartment. Sucrose gradient centrifugation analysis of root tip lysates also showed that 73 kDa FKBP is co-fractionated with tonoplast aquaporin and V-ATPase in a light compartment near the top of the gradient. Heat-shock treatment of root tips induces the accumulation of 77 kDa FKBP while the abundance of 73 kDa FKBP remains constant. Quantitative EM immunogold assays of the intracellular distribution of FKBP over an 8 h heat-shock time-course showed that FKBP is initially present in the cytoplasm, but is transported into the nucleus where it accumulates in the nucleoplasm and into specific subnuclear domains. The results of this study show that the intracellular distribution of the high Mr FKBPs in wheat root tips differs at normal and elevated temperatures, indicating different functional roles for the FKBP isoforms.     

Effect of human urotensin-II infusion on hemodynamics and cardiac function

Recent studies have shown that the vasoactive peptide urotensin-II (U-II) exerts a wide range of action on the cardiovascular system of various species. In the present study, we determined the in vivo effects of U-II on basal hemodynamics and cardiac function in the anesthetized intact rat. Intravenous bolus injection of human U-II resulted in a dose-dependent decrease in mean arterial pressure and left ventricular systolic pressure. Cardiac contractility represented by +/-dP/dt was decreased after injection of U-II. However, there was no significant change in heart rate or diastolic pressure. The present study suggests that upregulation of myocardial U-II may contribute to impaired myocardial function in disease conditions such as congestive heart failure.     

Photobleaching of arterial autofluorescence for immunofluorescence applications

Immunohistochemical localization of low-level antigens in the arterial vasculature is complicated by the presence of complex molecules such as collagen, elastin, cholesterol, and fluorescent lipids that exhibit autofluorescence over a wide spectrum of wavelengths. UV irradiation of arterial vasculature has remained ineffective in preparing samples for immunofluorescent staining because of the recovery of the endogenous fluorescence within a short time following treatment. Therefore, we sought to further enhance the signal-to-noise ratio in arteries by optimizing the photobleaching of this tissue. We report here that the use of filtered sunlight significantly reduces arterial autofluorescence compared to standard UV shortwave and longwave irradiation and maintains multiple antigen epitopes suitable for immunohistochemical analysis. Using this method, we localized low-level laminin-5 isoform expression in situ, which was previously indistinguishable from endogenous autofluorescence.     

Addictive potential of cannabinoids: the underlying neurobiology

Drugs that are addictive in humans have a number of commonalities in animal model systems-(1). they enhance electrical brain-stimulation reward in the core meso-accumbens reward circuitry of the brain, a circuit encompassing that portion of the medial forebrain bundle (MFB) which links the ventral tegmental area (VTA) of the mesencephalic midbrain with the nucleus accumbens (Acb) of the ventral limbic forebrain; (2). they enhance neural firing of a core dopamine (DA) component of this meso-accumbens reward circuit; (3). they enhance DA tone in this reward-relevant meso-accumbens DA circuit, with resultant enhancement of extracellular Acb DA; (4). they produce conditioned place preference (CPP), a behavioral model of incentive motivation; (5). they are self-administered; and (6). they trigger reinstatement of drug-seeking behavior in animals behaviorally extinguished from intravenous drug self-administration behavior and, perforce, pharmacologically detoxified from their self-administered drug. Cannabinoids were long considered 'anomalous', in that they were believed to not interact with these brain reward processes or support drug-seeking and drug-taking behavior in these animal model systems. However, it is now clear-from the published data of several research groups over the last 15 years-that this view of cannabinoid action on brain reward processes and reward-related behaviors is untenable. This paper reviews those data, and concludes that cannabinoids act on brain reward processes and reward-related behaviors in strikingly similar fashion to other addictive drugs.     

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.