Metabolism and non-random occurrence of nonnascent short chains in the DNA of Ehrlich ascites cells

The DNA of Ehrlich ascites cells was labeled with radioactive thymidine using different labeling schedules: Incubation periods between 15 s and 4 h; pulse/pulse-chase experiments with pulses in the range of a few minutes; longtime incubation followed by a longtime chase (both in the range of 1 cell generation). From the purified DNA of the labeled cells a fraction (0.3-0.4%) of short chains was separated and partially fractionated by means of a hydroxyapatite thermochromatography procedure. The evaluation of the labelling patterns of the short chains indicated that less than 5% of them can be regarded as replication intermediates ('Okazaki pieces'). The rest, termed nonnascent pieces, exhibited a slow turnover. The life span of the nonnascent pieces was estimated to be about 1 cell generation. On helical DNA, nonnascent pieces were distributed in a non-random manner. Their preferential localisation was nearby sites which caused binding of the DNA, after purification, to nitrocellulose and which occurred about every 60-80 microns on the nuclear DNA of the cells.     

The secondary structure and nitrocellulose affinity of freshly replicated DNA from Ehrlich ascites cells (author's transl)]

Hydroxyapatite chromatography and isopycnic Cs2SO4 centrifugation normally yield no indications of single-stranded DNA when that fraction of replicating DNA from Ehrlich ascites cells which can be separated by nitrocellulose chromatography is analyzed. Single-stranded DNA is detected by both methods if the DNA is fragmented by ultrasound before the nitrocellulose chromatography. The digestion of this DNA fraction by single-strand-specific nucliase leads to a loss of its binding to nitrocellulose and of the indications of single-stranded DNA. The loss for the affinity to nitrocellulose is also observed when the corresponding fraction separated from unfragmented DNA is digested by endonuclease. It is suggested that replicating DNA is bound to nitrocellulose by means of single-stranded gaps on the replication fork. These gaps are apparently too small to be detected within large, otherwise entirely double-stranded molecules by hydroxyapatite chromatography and Cs2SO4 centrifugation. In the case of nitrocellulose-binding ultrasound fragments, this relation seems to be more favorable because of the separation of most of the residual double-stranded part. It is demonstrated that sonication of helical DNA also generates a small amount of fragments with some single-stranded character. The effects observed with replicating DNA could be distinguished from these artifacts.     

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

C-terminal domains implicated in the functional surface expression of potassium channels

A short C-terminal domain is required for correct tetrameric assembly in some potassium channels. Here, we show that this domain forms a coiled coil that determines not only the stability but also the selectivity of the multimerization. Synthetic peptides comprising the sequence of this domain in Eag1 and other channels are able to form highly stable tetrameric coiled coils and display selective heteromultimeric interactions. We show that loss of function caused by disruption of this domain in Herg1 can be rescued by introducing the equivalent domain from Eag1, and that this chimeric protein can form heteromultimers with Eag1 while wild-type Erg1 cannot. Additionally, a short endoplasmic reticulum retention sequence closely preceding the coiled coil plays a crucial role for surface expression. Both domains appear to co-operate to form fully functional channels on the cell surface and are a frequent finding in ion channels. Many pathological phenotypes may be attributed to mutations affecting one or both domains.     

Controlled Extraction Studies Applied to Polyvinyl Chloride and Polyethylene Materials: Conclusions from the ELSIE Controlled Extraction Pilot Study

The effective management of leachables in pharmaceutical products is a critical aspect of their development. This can be facilitated if extractables information on the materials used in a packaging or delivery system is available to assist companies in selecting materials that will be compatible with the drug product formulation and suitable for the intended use. The Extractables and Leachables Safety Information Exchange (ELSIE) materials working group developed and executed a comprehensive extraction study protocol that included a number of extraction solvents, extraction techniques, and a variety of analytical techniques. This was performed on two test materials, polyethylene (PE) and polyvinyl chloride (PVC), that were selected due to their common use in pharmaceutical packaging. The purpose of the study was to investigate if the protocol could be simplified such that (i) a reduced number or even a single extraction technique could be used and (ii) a reduced number of solvents could be used to obtain information that is useful for material selection regardless of product type. Results indicate that, at least for the PVC, such reductions are feasible. Additionally, the studies indicate that levels of extractable elemental impurities in the two test materials were low and further confirm the importance of using orthogonal analytical detection techniques to gain adequate understanding of extraction profiles.KEY WORDS: extractables, extraction technique, polyethylene, polyvinyl chloride     

Cardiorespiratory effects of heliox using a model of upper airway obstruction]

Heliox is a mixture of Oxygen and Helium. The low density of Helium allows this mixture to flow in a laminar pattern where oxygen, nitrogen or air flow would be turbulent. Therefore the force necessary to move a volume of gas (e.g. Heliox) is greatly reduced in comparison to a turbulent gas flow. In a respiratory loading experiment we investigated the effects which Heliox exerts on hemodynamic as well respiratory variables. 10 volunteers were breathing spontaneously and through three different endotracheal (ET-) tubes (ID 4.0, 4.5, 5.0 mm).The subjects were switched from room air to Heliox and differences in the variables heart rate (HR), blood pressure (BP), stroke volume (SV), stroke index (SI), peripheral vascular resistance (TPRI) and left ventricular work index (LVWI) were measured. Furthermore the (PhAng) between abdomen and thorax was detected using respiratory inductance plethysmography (n=2) and the sense of dyspnoe under the different conditions was assessed by the use of a dyspnea score (DS). The means of BP, SV, SI, TPRI and LVWI did not significantly differ between the resting and the different loading conditions irrespective of the gas that was used. The variability of hemodynamic measures was significant larger during loaded vs. unloaded breathing. Heliox could significantly reduce this degree of variability. In two subjects Heliox could also significantly reduce the PhAng as well as DS. Heliox showed effects on hemodynamic as well as respiratory and subjective variables. These effects can be interpreted as a reduction of the extent of pressure variations in the intrapleural space leading to less impact on hemodynamic variables while breathing Heliox vs. room air in a resistive loading experiment. In the future the combined measurement of hemodynmic variables as well as non-invasive assessment of respiration might shed new light on cardio-respiratory interaction and effects of Heliox during airway obstruction.     

Density-dependent shift from facilitation to competition in a dwarf <Emphasis Type="Italic">Avicennia germinans</Emphasis> forest

The global effort to rehabilitate and restore destroyed mangrove forests is unable to keep up with the high mangrove deforestation rates, which exceed the average pace of global deforestation. Although facilitation theory presents new possibilities for the restoration of heavily degraded mangrove sites, knowledge of tree–tree interactions in stressed mangrove forest ecosystems is too limited to utilize facilitation appropriately. The aim was to determine the mode of local interaction among stressed mangrove trees by investigating the effect of clustering on tree size and crown morphology under contrasting stand densities. The study was conducted in a dwarf Avicennia germinans forest in Northern Brazil, in which tree growth is limited by infrequent inundation and high pore-water salinity. Autoregressive regression, Voronoi tessellation and spatial point pattern statistics were used to address the spatial processes underlying tree interaction. Under low stand density (1.2 trees m^?2) dwarf trees which grew in clustered cohorts of A. germinans had a less stunted crown morphology revealing the dominance of a positive neighborhood influence among plants. In contrast, dwarf trees in the denser forest stand (2.7 trees m^?2) were interacting competitively as indicated by the more negative effect of neighbors on crown morphology and size. The shift from facilitative to competitive interactions is an important feature of mangrove forest regeneration under harsh environmental conditions. If mangrove trees are unable to regenerate naturally on severely degraded sites, intraspecific facilitation could be used to assist regeneration by planting seedlings in clusters and not evenly spaced.     

Expression of human beta-defensins in children with chronic inflammatory bowel disease

Background

Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children.

Methods

Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn''s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative real-time (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha.

Results

Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn''s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon.

Conclusion

Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD, lending further support for their potential role in disease pathogenesis.     

Modes of action and combination effects of polychlorinated biphenyls and gamma-hexachlorocyclohexane on the regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of polychlorinated biphenyls, gamma-hexachlorocyclohexane and the effect of a combination of these substances on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase were investigated. As known from previous investigations polychlorinated biphenyls interfere with the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat liver via enzyme-lipid interaction and at the pretranslational level. In contrast to polychlorinated biphenyls, gamma-hexachlorocyclohexane did not alter the lipid status of the microsomal membrane. Thus the location of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, and consequently the catalytic activity of the enzyme was not changed. As with polychlorinated biphenyls, gamma-hexachlorocyclohexane interacted with enzyme regulation at the pretranslational level. Northern dot hybridization experiments showed a decrease in the level of m-RNA coding for 3-hydroxy-3-methylglutaryl coenzyme A reductase. The effect of combination of gamma-hexachlorocyclohexane and polychlorinated biphenyls was not additive. The gamma-hexachlorocyclohexane effect appeared to play a more important role than that of the polychlorinated biphenyls. The results indicate that the combination effects are as important as the effects of the single compounds when making risk assessments for xenobiotics.