Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.     

Cellular events during the primary immune response in the spleen

Summary The cellular events during the primary immune response in T and B cell compartments in the splenic white pulp were analysed in germfree mice immunized with sheep erythrocytes. Light, fluorescence and electronmicroscopic studies revealed that the initial formation of lymphoid blast cells occurs in the thymus-dependent area, i.e. the central periarteriolar lymphatic sheath (central PALS), 2 days after immunization. Lymphoblasts were found in close relation with erythrocyte-containing macrophages and with interdigitating cells. With fluorescence microscopy these blast cells were Ig negative. Lymphoblasts in the central PALS showed many polyribosomes in the cytoplasm, but were virtually devoid of endoplasmic reticulum. The ultrastructure of lymphoblasts in the central PALS, and their relation with interdigitating cells, suggests that these cells are the progeny of antigen-activated T cells.Cells with a positive cytoplasmic fluorescence, plasmablasts, appeared 3 days after immunization in the peripheral part of the PALS. During the progress of the immune response these cells accumulated around branches of the central arteriole, and moved along marginal zone bridging channels towards the red pulp. In the electron microscope plasmablasts showed many polyribosomes, short strands of rough endoplasmic reticulum close to mitochondria, and a few electron-dense bodies. The cell organelles of plasmablasts were frequently gathered in a so called uropod, which is a morphological sign of active cell movement.Germinal center formation started within primary follicles, 4 days after immunization. Blast cells in germinal centers did not show cytoplasmic fluorescence. During the course of the immune response, germinal centers extended in diameter, and fluorescent dendritic cells appeared at the periphery of the germinal center.From the present observations we conclude that: (1) cellular cooperation between different lymphoid and non-lymphoid cell types during the immune response against SRBC takes place in the PALS, (2) the cellular cooperation in the PALS results in the differentiation of B cells into immunoglobulin-producing plasmablasts, (3) the cellular cooperation in the PALS preceeds the formation of germinal centers in primary follicles, hence germinal centers are not involved in early T-B cell cooperation.     

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.     

Measurements of light at night (LAN) for a sample of female school teachers

Epidemiological studies have shown an association between rotating shiftwork and breast cancer (BC) risk. Recently, light at night (LAN) measured by satellite photometry and by self-reports of bedroom brightness has been shown to be associated with BC risk, irrespective of shiftwork history. Importance has been placed on these associations because retinal light exposures at night can suppress the hormone melatonin and/or disrupt circadian entrainment to the local 24-h light-dark cycle. The present study examined whether it was valid to use satellite photometry and self-reports of brightness to characterize light, as it might stimulate the circadian system and thereby affect BC incidence. Calibrated photometric measurements were made at the bedroom windows and in the bedrooms of a sample of female school teachers, who worked regular dayshifts and lived in a variety of satellite-measured sky brightness categories. The light levels at both locations were usually very low and were independent of the amount of satellite-measured light. Calibrated photometric measurements were also obtained at the corneas of these female school teachers together with calibrated accelerometer measurements for seven consecutive days and evenings. Based upon these personal light exposure and activity measurements, the female teachers who participated in this study did not have disrupted light-dark cycles like those associated with rotating shiftworkers who do exhibit a higher risk for BC. Rather, this sample of female school teachers had 24-h light-dark and activity-rest patterns very much like those experienced by dayshift nurses examined in an earlier study who are not at an elevated risk of BC. No relationship was found between the amount of satellite-measured light levels and the 24-h light-dark patterns these women experienced. It was concluded from the present study that satellite photometry is unrelated to personal light exposures as they might affect melatonin suppression and/or circadian disruption. More generally, photometric devices calibrated in terms of the operational characteristics of the human circadian system must be used to meaningfully link LAN and BC incidence.     

Methods to quantify soft-tissue based facial growth and treatment outcomes in children: a systematic review

Context

Technological advancements have led craniofacial researchers and clinicians into the era of three-dimensional digital imaging for quantitative evaluation of craniofacial growth and treatment outcomes.

Objective

To give an overview of soft-tissue based methods for quantitative longitudinal assessment of facial dimensions in children until six years of age and to assess the reliability of these methods in studies with good methodological quality.

Data Source

PubMed, EMBASE, Cochrane Library, Web of Science, Scopus and CINAHL were searched. A hand search was performed to check for additional relevant studies.

Study Selection

Primary publications on facial growth and treatment outcomes in children younger than six years of age were included.

Data Extraction

Independent data extraction by two observers. A quality assessment instrument was used to determine the methodological quality. Methods, used in studies with good methodological quality, were assessed for reliability expressed as the magnitude of the measurement error and the correlation coefficient between repeated measurements.

Results

In total, 47 studies were included describing 4 methods: 2D x-ray cephalometry; 2D photography; anthropometry; 3D imaging techniques (surface laser scanning, stereophotogrammetry and cone beam computed tomography). In general the measurement error was below 1 mm and 1° and correlation coefficients range from 0.65 to 1.0.

Conclusion

Various methods have shown to be reliable. However, at present stereophotogrammetry seems to be the best 3D method for quantitative longitudinal assessment of facial dimensions in children until six years of age due to its millisecond fast image capture, archival capabilities, high resolution and no exposure to ionizing radiation.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.