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Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ERalpha (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ERalpha. Downregulation of ERalpha using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ERalpha ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ERalpha ligands. We also detected ERalpha binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ERalpha-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ERalpha-dependent development of malignant diseases.

Wool fabrics treated for shrinkage control by applying a novel two-step ARS process3 involving an activated peroxide bleach followed by enzyme treatment were dyed at lower temperatures within shorter dyeing times than conventional dyeing with acid dyes which require 90 ° C or higher for 60 minutes or longer. The shrinkage control process involved bleaching pretreatment with dicyandiamide in alkaline hydrogen peroxide and with gluconic acid additive at 30 ° C (86 ° F) for 30 minutes followed by sulfite-assisted serine protease treatment for biopolishing and shrinkage prevention at 45 ° C (113 ° F) for 40 minutes. Dye uptake with time over the temperature range of dyeing showed that untreated fabrics and pretreated fabrics exhibited sigmoidal dyeing behavior with exhaustion within 55–70 minutes at 55–60 ° C. Fabrics pretreated and subsequently treated with enzyme exhibited exponential dyeing behavior with exhaustion within 20–30 minutes at 30–55 ° C. We attributed low temperature dyeing with reduced dyeing times to changes in wool morphology and chemical structure as documented by both scanning electron and confocal fluorescent microscopy. The ARS process provides shrinkage control with greater ease of bleaching and dyeing.

In this manuscript we present a modified kinetic model for pathogen capture efficiency (E) variation with mixing time (Tmix) which is applicable for most combinations of immuno-magnetic bead (IMB)-target organism concentrations (e.g., [IMB] ˜ 8 × 106 IMB mL-1; Salmonella enterica serovar Enteritidis, [SE] ˜ 102-108 CFU mL-1). We found that as the ratio of [IMB] to [SE] was decreased from more than 105 to ca. 10-1, the average number of cells captured per IMB SE complex increased from ca. 1 to 4 ± 1. Concurrently, using a modified kinetic model for fitting E variation with Tmix, the maximum possible level of capture (§) was observed to decline from ˜ 100% (§˜ 1; [IMB] > > [SE]) to 23% (§˜ 0.23; [IMB]:[SE] ˜ 0.1). Using a purely kinetic approach we also found that an empirical mass transport term (yobs= (3.1 ± 1.2) × 10-9 mL IMB-1 min 1; averaged across all [SE] tested: [IMB]:[SE] ˜ 200000, 200, 3, and 0.1) did not differ much from yobs reported in earlier work (3.35 ± 0.2 × 10-9 mL min-1 IMB-1) at extremely low [SE] (e.g., ± 100 CFU mL-1). Upon inserting the classically-derived equation for γ into the kinetic model and solving for the effective IMB radius (rIMB), we found that the rIMB was 2.3 ± 0.16 μ (§ S; averaged across all [IMB]:[SE]) which was only 0.5–0.9 μ greater than the rIMB reported by the manufacturer (1.4 ± 0.2 μ). These results support previous work which showed a similar small rIMB deviation possibly due to the hydro-dynamic behavior of the IMBs in relatively dilute buffer suspensions ([IMB ˜ 8 × 106 IMB mL-1) and argues that IMB-based cell capture is almost exclusively a function of mass transport.

A method for estimating immobilized enzyme reaction progress curves, using simultaneous non-linear regression analysis of 2–3 substrate concentrations with time, is presented. These facile procedures involve using nested Gauss–Newton curve fitting algorithms on a Microsoft EXCEL spreadsheet. We refer to our technique as "nested" because the analysis consists of two or three mutually parameter-dependent sets of computations associated with bi- or termolecular enzyme-catalyzed reactions, respectively. We have applied the method to immobilized glucose oxidase-catalyzed reactions ([ d -glucose] and [O 2 ] with time) and found that the kinetic parameters from initial velocity data were similar to those determined by the nested curve fitting method discussed herein.

The endothelial cell-specific mitogen vascular-endothelial growth factor (VEGF) plays a key role in both physiological and pathological angiogenesis. The up-regulation of VEGF expression in response to reduced oxygen tension occurs through transcriptional and post-transcriptional mechanisms. To investigate the molecular mechanisms of transcriptional activation by hypoxia (1% oxygen), fine mapping of a hypoxia-responsive region of the human VEGF promoter was carried out using luciferase reporter-gene constructs in C6 glioma cells. Here, we report that the binding site of hypoxia-inducible factor 1 (HIF1) is crucial for the hypoxic induction of VEGF gene expression. However, an enhancer subfragment containing the HIF1 binding site was not sufficient to confer full hypoxia responsiveness. Addition of upstream sequences restored the full sensitivity to hypoxia induction. This potentiating effect is due to activator protein 1 binding. The ‘potentiating’ sequences are unable to confer hypoxia responsiveness on their own. Our results strongly suggest that in C6 glioma cells a complex array of trans-acting factors facilitates full transcriptional induction of VEGF gene expression by hypoxia.

There is a need for new strategies for noninvasive imaging of pathological conditions within the human body. The approach of combining the unique physical properties of noble-metal nanoparticles with their chemical specificity and an easy way of conjugation open up new routes toward building bio-nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal. When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity, without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising candidate for noninvasive imaging of cancer cells.

Size distributions of extracts derived from starch were investigated to aid in elucidating structure-function relationships of these polymers in water. Starch granules derived from waxy maize and amylomaize VII were dissolved in water by microwave heating in a high pressure vessel. Transmission electron microscopy of starch deposited from dilute solution and rotary shadowed with platinum, revealed that amylopectin imaged from waxy maize could be broadly classified as about 28% circular space filling patches containing branched clusters and 72% asymmetric linear containing branched clusters. Lengths of asymmetric linear amylopectin components ranged from about 37 to 980 nm whereas the diameter of circular amylopectin components ranged from about 44 to 200 nm. Although the starch in amylomaize VII is about 70% amylose, its narrow asymmetric structure when visualized by microscopy enabled us to image amylose even though amylopectin was present. Lengths of components ranged from about 46 to 254 nm. After smoothing and curve fitting, we found that all size distributions investigated could be treated as if they were multimodal in nature. The most abundant amylose component had a linear density of 8.2 × 103 molar mass units/ nm. This value could be explained if amylose had an aggregation number of about 5.9.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.

Vascular endothelial growth factor (VEGF), an essential regulator of angiogenesis during early development as well as during the growth of solid tumours, bears an unusually large 5′ untranslated region (5′-UTR) in the mRNA of over 1000 nucleotides. We found that the VEGF 5′-UTR, despite being GC-rich and containing an upstream short open reading frame, promotes efficient translation of a luciferase reporter. The VEGF 5′-UTR also allowed translation of luciferase from a dicistronic mRNA when placed between the two cistrons, demonstrating that it contains an internal ribosome entry site. Deletion analysis indicated that the IRES resides towards the 3′ end of the 5′-UTR.

Angiogenesis, the sprouting of capillaries from pre-existing vessels, is of fundamental importance during embryonic vascular development and during certain physiological processes in the adult organism, for example menstruation, pregnancy, or wound healing. Moreover, angiogenesis plays a major role in the pathogenesis of several diseases, such as proliferative retinopathy, psoriasis, or solid tumor growth (Folkman, 1995). The growth of solid tumors is largely dependent on the supply of oxygen and nutrients from the blood stream. Small tumors of less than 1–2 mm in diameter are not vascularized, and they can be nourished by simple diffusion. Such avascular tumors do not induce neovascularization, and they rarely metastasize. However, further tumor growth is dependent on a vascular network that is able to fulfill the demands of the growing tumor for oxygen and nutrients. Vascularized tumors induce host vessels to extend vascular sprouts. They have the potential to expand their cell population, and they may eventually metastasize (for review, see Folkman, 1995).

Tissues respond to hypoxia with several compensatory mechanisms to maintain a microenvironment in which the cells can survive. These compensatory mechanisms include the formation of new blood vessels from preexisting vessels, a process termed angiogenesis, which restores the reduced oxygen supply. Hypoxiainduced angiogenesis plays an important role in physiological and pathological situations such as embryogenesis, diabetic retinopathy, and solid tumor growth. Increasing evidence suggests that upregulation of the expression of vascular endothelial growth factor (VEGF) is a key step in hypoxia-induced angiogenesis. Investigation into this hypoxic induction of VEGF should therefore lead to elucidation of the molecular mechanisms involved in hypoxia-induced angiogenesis. Using C6 glioma cells, which generate a highly vascularized glioma in vivo, the nuclear run-on assay and determination of mRNA half-life demonstrate that hypoxic induction of VEGF is the result of both transcriptional activation and increased mRNA stability. Reporter gene studies revealed that the hypoxia-responsive transcription-activating element was present in the 5′-flanking region of the VEGF gene. This element contains a consensus binding site for hypoxia-inducible factor-1 (HIF-1), which was originally found to bind the hypoxia-responsive enhancer sequence in the erythropoietin gene.

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

The approach of combining different mechanisms of antibacterial action by designing hybrid nanomaterials provides a new paradigm in the fight against resistant bacteria. Here, we present a new method for the synthesis of silver nanoparticles enveloped in the biopolymer chitosan. The method aims at the production of bionanocomposites with enhanced antibacterial properties. We find that chitosan and silver nanoparticles act synergistically against two strains of Gram-positive Staphylococcus aureus (S. aureus). As a result the bionanocomposites exhibit higher antibacterial activity than any component acting alone. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of the chitosan-silver nanoparticles synthesized at 0 °C were found to be lower than those reported for other types of silver nanoparticles. Atomic force microscopy (AFM) revealed dramatic changes in morphology of S. aureus cells due to disruption of bacterial cell wall integrity after incubation with chitosan-silver nanoparticles. Finally, we demonstrate that silver nanoparticles can be used not only as antibacterial agents but also as excellent plasmonic substrates to identify bacteria and monitor the induced biochemical changes in the bacterial cell wall via surface enhanced Raman scattering (SERS) spectroscopy.

The structural organization of alkaline soluble peach pectin was investigated over size ranges extending from micrometers to tenths of nanometers. Analysis was by electron microscopy and high-performance anion-exchange chromatography (HPAEC). Superimposed and individual circular microgels in the micrometer size range were isolated from mesocarp tissue of cell walls and visualized by rotary shadowing. Dilute NaCl and 50% aqueous glycerol disaggregated these microgels into rods, segmented rods, and kinked rods, which collectively comprised the internal gel network of the microgels. Image analysis of the shadowed specimens before and after disaggregation followed by curve fitting of the smoothed distributions revealed a multimodal distribution of lengths. HPAEC revealed that the multimodal aggregates were stable for the most part to further dissociation by increasing ionic strength.

A direct mass spectrometric measurement method is developed for the determination of 13C enrichment and chemical purity of a labelled compound. The labelled compound is mixed with various amounts of unlabelled analogue to provide a series of mixtures containing different but comparable quantities of labelled and unlabelled species. Intensity ratios of selected isotopically anologous ions from these two species are determined using selected ion monitoring. Equations are established to relate measured intensity ratios to sample mixture weight ratios, chemical purities of the labelled and unlabelled compounds, ion composition and isotopic abundance of the enriched and unenriched carbons, A non-linear regression procedure, based on the measured intensity ratio, the mixture weight ratio and the known chemical purity of the unlabelled compound, is used to resolve the chemical purity and the isotopic abundance yielding results with standard errors of 1.0 and 0.4%, respectively. This procedure is suitable for compounds labelled with more than 60% of an isotope.

High-performance, size-exclusion chromatography columns were calibrated in average root-mean-square radii of gyration (μ R gz ) by a combination of commercial “narrow” pullulan and “broad” dextran standards. The nonlinear calibration-curves were fitted by a computer-aided, iterative, least-squares procedure. Values of (μ R gz ), obtained from a point-by-point transformation of the respective pullulan and dextran chromatograms by utilizing universal calibration, were compared with inputted μ R gz calibration values. For standards ranging in μ R gz value from 20.1 to 389 A, the accuracy ranged from 1 to 15.3%. Furthermore, from relationships in the literature, μ R gz values were transformed to μ M w . These values of μ M w were comparable to, but generally less accurate than, μ M w values from direct, molecular-weight calibration.

Laser-Raman spectra of L-α-dimyristoylphosphatidylcholine (DMPC) liposomes in the spectral range 1000–1200 cm −1 were obtained as a function of temperature from −80 to +50°C. The triplet found in this spectral region was resolved into Lorentzian components by means of an iterative computer program. The peak intensities, band widths, and band areas of the resolved 1062 cm −1 and 1130 cm −1 bands, assigned to CC stretching vibrations of trans segments, were evaluated as a function of temperature. While the peak intensities of the bands decrease substantially with temperature, the band widths show a considerable increase. The change in band areas is therefore smaller than the change in peak heights. Experiments with all trans carboxylic acids showed that in these compounds the area of the Raman bands at 1062 cm −1 and 1130 cm −1 is proportional to the number of trans bonds. The variation with temperature of the number of trans and gauche bonds in the studied phospholipid is reflected by the change of the area of the 1130 cm −1 Raman band.

Instrumental parameters and possible correction procedures for hydrogen-transfer phenomena are studied, with emphasis on the improvement of quantitative measurements in selected ion monitoring mass spectrometry. The intensity ratio of the m/z 387 and 386 ions, derived from cholesterol, are measured by an HP-5995 mass spectrometer using different parameters, including calibration peak width, mass filter window size and ion monitoring dwell time. The intrinsic ratio of these two ions is calculated with a correction procedure accounting for the [MH]+ and [M2H]+ hydrogen-transfer phenomena.

Using 25 accurately known bond-stretching force constants and bond lengths for 23 molecules, a relation of the form: F = 37.3 r −5.35 has been obtained for carbon—nitrogen bonds. The root-mean-square deviation between the force constant values reported in the literature and those calculated from the equation above is 4.09 %. Fourteen of the molecules studied have a rotational symmetry axis that coincides with the carbon-to-nitrogen bond. The remaining molecules have lower symmetry. The observed force constant—bond length relationship gives quite satisfactory results for both groups of molecules.

Cephalothin significantly suppressed in vitro DNA and total protein synthesis in human peripheral blood lymphocytes stimulated by antigens or mitogens. However, similar concentrations of this antibiotic enhanced streptokinase-streptodornase-stimulated production of the lymphokine leukocyte migration-inhibition factor (LMIF) and directly stimulated production of this lymphokine by otherwise unstimulated lymphocytes from 10 of 12 normal human subjects. Penicillin did not appear to produce these effects. Cephalothin did not interfere directly with neutrophil migration or the interaction of preformed LMIF with neutrophils. Stimulation of LMIF production by cephalothin required viable lymphocytes and was inhibited by puromycin. These results suggest that cephalothin is capable of inducing lymphokine production by human lymphocytes in a manner that appears to be nonspecific in nature. This type of effect could be the basis of some apparently immunologic reactions to this antibiotic.

The effect of chloramphenicol on the in vitro function of human peripheral blood lymphocytes was studied in assays of lymphocyte transformation and lymphokine production. When lymphocytes were stimulated by phytohemagglutinin, concanavalin A, or pokeweed mitogen in the presence of various concentrations of chloramphenicol, only minimal effects on blastogenesis were noted. However, suppression by chloramphenicol of blastogenesis induced by candida antigen or streptokinase-streptodornase was greater in magnitude and was dose-dependent; blastogenesis was suppressed to 25%--30% or normal levels by concentrations of chloramphenicol of 25--50 microgram/ml. Chloramphenicol had little effect on the production of the lymphokine leukocyte migration inhibition factor by lymphocytes stimulated either by candida antigen or by concanavalin A, whereas puromycin at a concentration of 5 microgram/ml significantly suppressed this response. Thus chloramphenicol appears to suppress antigen-induced lymphocyte blastogenesis significantly but not lymphokine production by stimulated lymphocytes.

Angiogenic growth factors and their endothelial receptors are thought to function as major regulators of blood vessel formation. Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2), as well as Angiopoietin-1 and its receptor, Tie-2, represent key signal transduction systems involved in the regulation of embryonic vascular development. The expression of these molecules correlates with phases of blood vessel formation during embryogenesis. Inactivation of any of the genes encoding these molecules in mouse embryos results in defective vascular development and embryonic lethality around mid-gestation. In addition, the VEGF signal transduction system has been implicated in the regulation of pathological blood vessel growth during certain angiogenesis-dependent diseases that are often associated with tissue ischemia, such as proliferative retinopathy or solid tumor growth. This hypothesis is substantiated by experiments, in which the inhibition of VEGF signal transduction resulted in the the inhibition of neovascularization in these diseases. Thus, the VEGF signal transduction system represents a useful target for an anti-angiogenic therapy.