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Simultaneous biodetoxification of S, N, and O pollutants by engineering of a carbazole-degrading gene cassette in a recombinant biocatalyst.

The gene cassette encoding enzymes responsible for degrading carbazole to anthranilic acid was introduced into a dibenzothiophene degrader. The resultant strain, Rhodococcus erythropolis XPDN, could simultaneously transform the model pollutants dibenzothiophene, carbazole, and dibenzofuran to nontoxic metabolites and may have an application potential for bioremediation.

Benzofurans↗

Escherichia coli hemoglobin protease autotransporter contributes to synergistic abscess formation and heme-dependent growth of Bacteroides fragilis.

Intra-abdominal infections (IAI) continue to be a serious clinical problem. Bacterial synergism is an important factor that influences the shift from contamination to IAI, leading to the development of lesions and abscess formation. Escherichia coli and Bacteroides fragilis are particularly abundant in IAI. The underlying molecular mechanisms of this pathogenic synergy are still unclear. The role of the hemoglobin protease (Hbp) autotransporter protein from E. coli in the synergy of IAI was investigated. Hbp is identical to Tsh, a temperature-sensitive hemagglutinin associated with avian pathogenic E. coli. Clinical isolates from miscellaneous extraintestinal infections were phenotypically and genotypically screened for Hbp. The presence of Hbp was significantly associated with E. coli isolated from IAI and other extraintestinal infections. In a murine infection model, Hbp was shown to contribute to the pathogenic synergy of abscess development. Mice immunized with Hbp were protected against mixed infections and did not develop abscess lesions. Furthermore, an E. coli wild-type strain that did not induce abscess formation in the synergy model was transformed with a plasmid encoding the hbp gene, and mixed infections with this strain lead to increased growth of B. fragilis and induction of abscess lesions. Growth-promoting studies showed that purified Hbp is able to deliver heme to B. fragilis strain BE1. In conclusion, results suggest the synergy of abscess formation by E. coli and B. fragilis can be partly explained by the capacity of B. fragilis to intercept Hbp and iron from heme to overcome the iron restrictions imposed by the host.

Abscess↗

Drosophila chorion gene amplification requires an upstream region regulating s18 transcription.

A cluster of Drosophila melanogaster chorion genes at locus 66D on the third chromosome amplifies 60-fold in the ovarian follicle cells prior to the onset of gene expression. A 3.8-kilobase (kb) region of the gene cluster can induce tissue-specific amplification in transformants. Previous models postulated that amplification is activated in follicle cells by transcription of one of the two chorion genes (s15 and s18) located within the 3.8-kb essential region. In this study, we showed that neither s15 nor s18 chorion gene transcription was required for amplification. However, a 510-bp region upstream from s18 contained sequences essential for both amplification and s18 transcription. No other region within the 3.8-kb fragment was required for amplification. We propose that upstream transcription control elements rather than transcription per se are involved in controlling amplification during development.

Animals↗

[Methods of chemical immobilization of an enzyme on a solid support].

Carbonic anhydrase was immobilized on a polymer thermoplastic to promote gaseous CO2 hydration into bicarbonate ions. Catalyst immobilization was realized through a series of chemical reactions enabling enzyme covalent binding to polyamide support. Different initial enzyme concentrations of 0.25, 0.50, 0.75, 1, and 2 mg/ml were verified. Different techniques were developed to assess enzyme attachment. The amount of bound protein was determined using Bradford dosage of proteins remaining in solution following CA II incubation with solid support. ELISA has given a qualitative evaluation of the protein, enabling a follow up of enzyme binding robustness as a function of time. p-nitrophenyl acetate hydrolysis and CO2 hydration were assessed by spectrophotometry and electrometry, respectively. The percentage of active enzyme following immobilization was measured using an esterase linear model. Catalytic transformation rates for gaseous CO2 hydration were calculated for each type of immobilization.

Bicarbonates↗

Estimation of doubly labeled water energy expenditure with confidence intervals.

Bivariate regression is used to estimate energy expenditure from doubly labeled water data. Two straight lines are fitted to the logarithms of the enrichments of oxygen-18 and deuterium simultaneously as a bivariate regression, so that the correlations between the oxygen and deuterium regression coefficients can be estimated. Maximum likelihood methods are used to extend bivariate regression to unbalanced situations caused by missing observations and to include replicate laboratory determination from the same urine samples, even if one of the replicates is missing. Use of maximum likelihood allows the determination of a confidence interval for the energy expenditure based on the log likelihood surface rather than use of the propagation of variance methods for nonlinear transformations. The model is extended to include the subject's deviations from the two lines as a bivariate continuous-time first-order autoregression to allow for serial correlation in the observations. The analysis of data from two subjects, one without apparent serial correlation and one with serial correlation, is presented.

Deuterium↗

Contrast sensitivity functions to stimuli defined in Cartesian, polar and hyperbolic coordinates.

Recent electrophysiological studies indicate that cells in the LGN, V1, V2, and V4 areas in monkeys are specifically sensitive to Cartesian, polar and hyperbolic stimuli. We have characterized the contrast sensitivity functions (CSF) to stimuli defined in these coordinates with the two-alternatives forced-choice paradigm. CSFs to Cartesian, concentric, and hyperbolic stimuli have had similar shapes, with peak sensitivity at approximately 3 c/deg. However, the Cartesian CSF peak sensitivity has been at least 0.1 log units higher than that to stimuli in any other coordinate system. The concentric-Bessel CSF has a low-pass shape, peaking at 1.5 c/deg or below. The radial CSF has a bell shape with maximum sensitivity at 8 c/360 degrees. Only the concentric-Bessel CSF could be explained in terms of the components of maximum amplitude of the Fourier transform. Neural models, which in previous studies predicted the responses to Cartesian and polar Glass patterns, failed to account for the full CSFs data.

Contrast Sensitivity↗

Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata.

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.

Autoanalysis↗

Update on HER-2 as a target for cancer therapy: alternative strategies for targeting the epidermal growth factor system in cancer.

The epidermal growth factor (EGF) family of ligands and receptors interact to influence cell division, differentiation and motility. Much evidence supports their importance in causing and sustaining cell transformation in model systems and in human cancer. The exact mechanism by which this is achieved varies in different tumour types and from case to case. The EGF system is a target for new types of targeted chemotherapy. The choice of strategy will depend on the mechanism involved, however, and several approaches are under development or evaluation in clinical trials. Each will have a different spectrum of side effects and the potential for development of drug resistance.

Antineoplastic Agents↗

Physicochemical stability of cimetidine amorphous forms estimated by isothermal microcalorimetry.

The effect of humidity on the physicochemical properties of amorphous forms of cimetidine was investigated using differential scanning calorimetry, isothermal microcalorimetry, and x-ray diffraction analysis. Amorphous forms were obtained by the melting (amorphous form M [AM]) and the cotton candy (amorphous form C [AC]) methods. Thermal behaviors of AM and AC with or without seed crystals were measured using an isothermal microcalorimeter under various conditions of relative humidity (RH) and temperature, respectively. The crystallization kinetics of amorphous solids was analyzed based on 10 kinds of solid-state reaction models. AM transformed into form A at 11% RH, 50 degrees C but transformed into a mixture of form A and monohydrate at 51% and 75% RH at 25 degrees C. The mean crystallization times (MCTs) of the heat flow curve of AM and AC at 11% RH, 50 degrees C were 47.82 and 32.00 hours, respectively, but at 11% RH, 25 degrees C both were more than 4320 hours. In contrast, AC transformed into form A under all storage conditions. The MCTs of AC at 51% and 75% RH were 29.61 and 11.81 hours, respectively; whereas the MCTs of AM were 46.79 and 15.52 hours, respectively. The crystallization of amorphous solids followed the three-dimensional growth of nuclei (Avrami equation) with an induction period (IP). The IP for AM at 11% RH, 50 degrees C was more than 2 times that for AC, but the difference in the crystal growth rate constant (CR) between AC and AM was within 10%. The IP for AM at 75% RH, 25 degrees C was reduced to only 10% of the IP at 51% RH with increasing humidity, but the CR did not change significantly. In contrast, the IP for AC was slightly reduced at 75% RH compared with 51% RH, but the CR was about 5 times greater. At 75% RH, 25 degrees C, the IP and CR of AM were about one-fourth the values of AC. This result suggests that the crystallization process consists of an initial stage during which the nuclei are formed and a final stage of growth.

Calorimetry, Differential Scanning↗

Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition.

Epithelium-to-mesenchyme transitions (EMTs) are characterized by morphological and behavioral changes in cells. During an EMT, E-cadherin is downregulated while N-cadherin is upregulated. The goal of this study was to understand the role cadherin switching plays in EMT using a classical model system: transforming growth factor beta1 (TGF-beta1)-mediated EMT in mammary epithelial cells. We showed that stress fibers and focal adhesions are increased, and cell-cell junctions are decreased in response to TGF-beta1. Moreover, these changes were reversible upon removal of TGF-beta1. Downregulation of E-cadherin and upregulation of N-cadherin were both transcriptional. Neither experimental knockdown nor experimental overexpression of N-cadherin interfered with the morphological changes. In addition, the morphological changes associated with EMT preceded the downregulation of E-cadherin. Interestingly, TGF-beta1-induced motility in N-cadherin-knockdown cells was significantly reduced. Together, these data suggest that cadherin switching is necessary for increased motility but is not required for the morphological changes that accompany EMT.

Adherens Junctions↗

Chemical induction of oncogene mutations and growth factor activity in mouse skin carcinogenesis.

The goal of understanding the molecular basis of human tumor development has been greatly facilitated by the use of animal model systems in which the etiology of tumor development can be carefully controlled. Environmental chemicals, either naturally occurring or artificially produced, are thought to make a major contribution to the human tumor burden. Many of the concepts of multistage carcinogenesis have been developed and refined using the mouse skin model system and the work described in this article has been carried out in an attempt to analyze the molecular changes that are associated with the initiation of tumor development, the selection of initiated cells to form papillomas, or the progression of premalignant tumors to carcinoma. We have analyzed a number of skin tumors induced in mice by a two-stage initiation and promotion protocol and have detected a high frequency of c-ras oncogene mutations in this system. The mutation found in each case correlates well with the known reactivity of the carcinogens used. It has also been shown that where ras activation occurs this represents an early event in the tumor model system. Transforming growth factor beta is induced in mouse skin by tumor promoter treatment and may therefore play a role in the selection of initiated cells to form papillomas. Additional events, some of which involve the loss of normal ras alleles and possibly tumor suppressor genes, appear to take place at a later stage of carcinogenesis.

Animals↗

Theory of spatiochromatic image encoding and feature extraction.

We consider how to interpret, filter, and cross-correlate complex-value color (hue and saturation) images by using a single discrete Fourier transform: the spatiochromatic discrete Fourier transform. The model defines new types of spatiochromatic oriented sinusoidal gratings, termed rainbow gratings, which encode the variation of color over space. We demonstrate how color-opponent detectors observed within the vertebrate visual system can be easily defined by linear filters within this representation. This model also allows us to filter and detect both spatial and chromatic patterns in images by using a single cross-correlation procedure. In doing so, we explore a new form of the Cauchy-Schwartz inequality applied to complex-valued scalar products. Results demonstrate the power of this form of spatiochromatic matched filtering in detecting signals embedded in such a significant amount of noise that they are not visible to the unaided human eye.

Animals↗

Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Amino Acid Sequence↗

Adjustment in conditions with short stature: a conceptual framework.

A major justification of extended indications for GH therapy in conditions with short stature is based on the objective of preventing or alleviating suspected psychosocial maladjustment. Despite more sophisticated research, results are still controversial concerning the actual seriousness of adjustment difficulties in various conditions with short stature, thus making it difficult to determine a patient's need for treatment. This paper discusses different concepts and assessment strategies of "adjustment" as a major source of the apparent heterogeneity among study findings, conclusions and treatment recommendations. A concise framework is developed that identifies and differentiates three hierarchical levels of research on adjustment in conditions with growth retardation: (1) stress exposure due to short stature, (2) quality of coping responses, and (3) occurrence of psychopathology. Choosing a particular research level and its corresponding measures may imply whether a high, medium or very low impact of short stature on adjustment is found, and the need for treatment that is inferred. The integration of these different research approaches within an integrative model may help to resolve apparent inconsistencies among empirical studies and reveal sources of confoundation due to other, short stature independent risk factors associated with the particular underlying growth disorder. The key terms of the suggested model are transformed into a clinical guideline for psychological assessment in patients with growth retardation and into three key criteria for the decision making process on extended indications for GH treatment with respect to the improvement of psychosocial adjustment.

Adaptation, Psychological↗

Heparin-binding EGF-like growth factor mRNA is upregulated in the peri-infarct region of the remnant kidney model: in vitro evidence suggests a regulatory role in myofibroblast transformation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent fibroblast and epithelial cell mitogen that may be important in wound healing. The aim of this study was to determine its distribution and possible function in segmental renal infarction. At day 1 postinfarction, in situ hybridization showed that HB-EGF mRNA was markedly increased by tubular epithelial cells bordering the infarcted zone. At day 3, typical myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA) were present in large numbers at the peri-ischemic border and, over succeeding days, were also seen within the infarcted area. Some of these cells expressed HB-EGF mRNA by in situ hybridization suggesting possible autocrine stimulation. Endothelial cells appeared to be more resistant to ischemia than tubules because some capillaries at the periphery of the infarct, surrounded by infarcted tubules, also expressed HB-EGF mRNA. The staining intensity of HB-EGF mRNA in individual tubules and endothelial cells was maximal at day 5 after infarction, although Northern blots of tissue from the peri-infarct area only showed significantly increased expression of HB-EGF mRNA at days 1 and 3, perhaps reflecting a smaller area of greater intensity of expression at day 5. Because tubular cells expressing high levels of HB-EGF mRNA were directly apposed to myofibroblasts, an attempt was made to determine whether HB-EGF contributed to upregulation of alpha-SMA by human fibroblasts. Although stimulation of the fibroblast cell line MRC-5 with transforming growth factor-beta1 (TGF-beta1) increased alpha-SMA, HB-EGF reduced expression. HB-EGF also strongly inhibited the increased expression of alpha-SMA due to TGF-beta1. Because HB-EGF is a potent fibroblast mitogen and TGF-beta is usually antiproliferative, this study suggests that HB-EGF may contribute to a local balance between fibroblast proliferation and differentiation into myofibroblasts during scarring.

Actins↗

Staffing and worker injury in nursing homes.

OBJECTIVES: We examined the relationship between nursing home staffing levels and worker injury rates in 445 nursing homes in 3 states. METHODS: We obtained First Reports of Injury and workers' compensation data from 3 states (Ohio, West Virginia, and Maryland) for the year 2000. We then linked these data to Medicare's Online Survey, Certification and Reporting system to obtain nursing home staffing details and organizational descriptors. We used ordinary least squares and log-transformed regression models to examine the association between worker injury rate and nursing home staffing and organizational characteristics. RESULTS: Total nursing hours per resident day were significantly associated with worker injury rates in nursing homes after we adjusted for organizational characteristics and state dummy variables (P=.0004). CONCLUSIONS: Our findings suggest that nursing home staffing levels have an important impact on worker health. These findings were supported for multiple facilities across different states; therefore, policies and resources that increase staffing levels in nursing homes are warranted.

Data Collection↗

Variable stoichiometry with thermodynamic control in ADM1.

The effect of a variable stoichiometry of the carbohydrate fermentation process in the IWA Anaerobic Digestion Model No.1 (ADM1) is investigated. Most existing anaerobic digestion models including ADM1 consider a fixed-stoichiometry for their conversion processes. The ADM1 model was first transformed into an only mol based model to remove some errors derived from the mixed COD-mol based standard model and to allow for implementation of the variable stoichiometry. Consequently, the values of the butyrate and acetate catabolic yields of carbohydrate fermentation are made dependent on the hydrogen concentration and the reactor pH according to the predictions of a recently developed mixed culture fermentation model based on thermodynamic considerations. The simulation results obtained showed no significantly different responses in terms of effluent quality and system robustness between the standard and the variable stoichiometry ADM1 under overload conditions, and for both single- and two-step anaerobic digestion configurations. This behaviour is explained by the non-limiting acetogenic activity that compensated for the changes in the acidogenic products, typical behaviour for serial processes close to equilibrium. Based on the results obtained, thermodynamic rather than kinetic control for these conversions is suggested. Depending on the objectives to be met, lumping of carbohydrate fermenters and oxidative acetogens into a single biomass group with a variable stoichiometry is proposed for further consideration.

Anaerobiosis↗

Professionalism: rise and fall.

Historically, the early professionalization movements in medicine and the law appear as organizational projects which aspire to monopolize income and opportunities in markets of services or labor and to monopolize status and work privileges in occupational hierarchies. Their central task is to standardize training and link it to actual or potential markets of labor or services, a linkage that is structurally effected in the modern university. The second wave of professionalization has different protagonists than the older "market professions": placed in a different structural situation, the bureaucratic professions transform the model of profession (which they adopt as a strategy of collective ascension) into an ideology. The import of the ideology of professionalism is examined in relation to two issues: the relationships between professional occupations and bureaucratic organizations; and the position of professional occupations within the larger structure of inequality. Analysis of the first point requires consideration of the distinctions between professional occupations in the public and private sectors, the use of professional knowledge and the image of profession in bureaucratic organizations, and the specific characteristics of professions that produce their own knowledge. In the discussion of the second point, professional occupations and their ideology are examined in relation to other occupations and to the possibilities of political awareness generated by uncertain professional statuses.

Certification↗