Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-N-acetylglucosaminyltransferase II

A novel recombinant baculovirus expression vector was used to produce His-tagged human transferrin in a transformed insect cell line (Tn5beta4GalT) that constitutively expresses a mammalian beta-1,4-galactosyltransferase. This virus encoded the His-tagged human transferrin protein in conventional fashion under the control of the very late polyhedrin promoter. In addition, to enhance the synthesis of galactosylated biantennary N-glycans, this virus encoded human beta-1,2- N-acetylglucosaminyltransferase II under the control of an immediate-early (ie1) promoter. Detailed analyses by MALDI-TOF MS, exoglycosidase digestion, and two-dimensional HPLC revealed that the N-glycans on the purified recombinant human transferrin produced by this virus-host system included four different fully galactosylated, biantennary, complex-type glycans. Thus, this study describes a novel baculovirus-host system, which can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans.     

Too much of a good thing: mechanisms of gene action in Down syndrome

The molecular mechanisms underlying the specific traits in individuals with Down syndrome (DS) have been postulated to derive either from nonspecific perturbation of balanced genetic programs, or from the simple, mendelian-like influence of a small subset of genes on chromosome 21. However, these models do not provide a comprehensive explanation for experimental or clinical observations of the effects of trisomy 21. DS is best viewed as a complex genetic disorder, where the specific phenotypic manifestations in a given individual are products of genetic, environmental and stochastic influences. Mouse models that recapitulate both the genetic basis for and the phenotypic consequences of trisomy provide an experimental system to define these contributions.     

Parenteral nutrition with lipid or glucose suppresses liver growth and response to GH in adolescent male rats

Our aim was to investigate the effects of modifying the carbohydrate-to-lipid ratio of parenteral nutrition (PN) on body composition and the anabolic actions of insulin-like growth factor I (IGF-I) and growth hormone (GH). Adolescent male Sprague-Dawley rats were randomized to receive 7 days of GH, IGF-I (3.5 mg. kg(-1). day(-1) for both) or placebo while receiving high-carbohydrate PN (CHO-PN), high-lipid PN (L-PN), or an oral diet (chow) (the PN protocols were isonitrogenous and isocaloric). PN impaired muscle growth, which was reversed by GH in the CHO-PN group only (P < 0.03). PN increased carcass lipid (P < 0.02), the effect being greater in the L-PN than in the CHO-PN group (P < 0.001). Visceral lean tissue growth was significantly impaired by PN (P < 0.001). IGF-I reversed this impairment, but GH had no effect. PN impaired the normal increase in hepatic protein and DNA (P < 0.001) and produced liver steatosis (P < 0.001). However, this steatosis was less in L-PN than in CHO-PN (P < 0.001). Serum IGF-I and the acid-labile subunit (ALS) were decreased by PN (P < 0.001) and were not affected by GH during PN treatment. However, GH significantly increased serum ALS concentrations in the chow-fed rats (P = 0.032). In conclusion, modifying the CHO-to-L ratio of PN had no significant effect on IGF-I action, but CHO-PN increased the peripheral effect of GH. L-PN increased carcass lipid significantly and decreased hepatic steatosis. Nevertheless, PN caused significant liver steatosis and profound impairment of hepatic cell growth, which was associated with relative hepatic GH resistance.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Architectural and physiological heterogeneity within the synflorescence of the pseudoviviparous grass Poa alpina var. vivipara L

Many biotypes of the northern-hemisphere Arctic-Alpine grass Poa alpina L. reproduce asexually via prolification of the spikelet axis to produce dehiscing shoots. Although capable of photosynthesis, the source-sink characteristics of these synflorescence systems are unknown, including the degree to which plantlets from different regions of the synflorescence are capable of providing for their own carbon requirements, or contributing to other sinks. Photosynthetic rates within the paracladial zone, as determined by infrared gas analysis (IRGA), exceeded respiratory rates by 3-4-fold. (14)CO(2) tracer studies determined that the paracladial zone was not only as efficient at fixing carbon as the youngest fully expanded leaf (per unit dry weight), but that both organs exported carbon mainly basipetally (cf. extensive acropetal export from this leaf in seminiferous grasses). Distal plantlets of the paracladial zone fixed approximately 20% more (14)CO(2) than did proximal plantlets. This was by virtue of their greater dry weight. At dehiscence, 'distal' plantlets were more likely to become established, and possessed relative growth rates more than 10 times those of 'proximal' plantlets. Paracladial heterogeneity was also apparent as an increased proportion of aborted spikelets on proximal paracladia. The possible causes of this heterogeneity are discussed.     

Interaction of sodium molybdate with highly purified glucocorticoid receptor

Sodium molybdate can affect the properties of the glucocorticoid receptor in relatively crude preparations. To obtain more information as to whether these effects are due to direct interactions of the ion with the receptor or with other components present in the receptor-containing mixtures, the effects were examined of sodium molybdate on glucocorticoid receptors purified 3000-5000-fold to about 10% homogeneity from rat liver cytosol. The ion was found to: (1) increase the stability of the purified receptor at either 0 or 20 degrees C, although the effect was more pronounced at 20 degrees C (2) induce an apparent dimerization of the receptors as judged by sephadex G-150 gel filtration and sucrose density gradient sedimentation and (3) decrease the ionic strength required for elution of the purified receptor from DEAE-cellulose columns. Although, it is conceivable that each of these observed effects is due to indirect actions of the ion on contaminants in the preparations, it is more likely that the ion exerts its effects through direct interactions with the receptor.     

Vitamin D Prevents Hypoxia/Reoxygenation-Induced Blood-Brain Barrier Disruption via Vitamin D Receptor-Mediated NF-kB Signaling Pathways

Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)₂D₃ on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)₂D₃ treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF—kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )₂D₃ with the vitamin D receptor (VDR) was inhibited by pyridoxal 5’-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )₂D₃ against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells.     

Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins

A molecular test for Alzheimer's disease could lead to better treatment and therapies. We found 18 signaling proteins in blood plasma that can be used to classify blinded samples from Alzheimer's and control subjects with close to 90% accuracy and to identify patients who had mild cognitive impairment that progressed to Alzheimer's disease 2-6 years later. Biological analysis of the 18 proteins points to systemic dysregulation of hematopoiesis, immune responses, apoptosis and neuronal support in presymptomatic Alzheimer's disease.