Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies

Assembly of Protein 4.1 during Chicken Erythroid Differentiation

Protein 4.1 is a peripheral membrane protein that strengthens the actin-spectrin based membrane skeleton of the red blood cell and also serves to attach this structure to the plasma membrane. In avian erythrocytes it exists as a family of closely related polypeptides that are differentially expressed during erythropoiesis. We have analyzed the synthesis and assembly onto the membrane skeleton of protein 4.1 and in this paper we show that its assembly is extremely rapid and highly efficient since greater than 95% of the molecules synthesized are assembled in less than 1 min. The remaining minor fraction of unassembled protein 4.1 differs kinetically and is either degraded or assembled with slower kinetics. All protein 4.1 variants exhibit a similar kinetic behavior irrespective of the stage of erythroid differentiation. Thus, the amount and the variants ratio of protein 4.1 assembled are determined largely at the transcriptional or at the translational level and not posttranslationally. During erythroid terminal differentiation the molar amounts of protein 4.1 and spectrin assembled change. In postmitotic cells, as compared with proliferative cells, far more protein 4.1 than spectrin is assembled onto the membrane-skeleton. This modulation may permit the assembly of an initially flexible membrane skeleton in mitotic erythroid cells. As cells become postmitotic and undergo the final steps of maturation the membrane skeleton may be gradually stabilized by the assembly of protein 4.1

Validation of an automated assay for measurement of bovine plasma ceruloplasmin

Ceruloplasmin (Cp) plays an important role in copper transport and iron metabolism, as well as Cp is also an indicator for the health status of dairy cows. The present study reports the validation of an automated assay to assess the plasma Cp in dairy cows. Plasma Cp levels were determined in 40 Holstein cows and intra- and inter-assay precision, accuracy, detection limit, and clinical validation of the assay were determined. Intra- and inter-assay coefficients of variation were < 2% and < 7%, respectively. The results were linear when serial sample dilutions were tested (r = 0.999, P < 0.001). The detection limit was lower than what could be measured in plasma from healthy cows. Increased plasma Cp levels were found in cows with inflammatory diseases. The method validated in this study is precise, simple, and fast and can be easily adapted to biochemical automated analysers. Furthermore, the promising results obtained with this protein will contribute to a wider use of Cp determination in bovine practice

Photon collimator system for the ILC Positron Source

High energy e+e- linear colliders are the next large scale project in particle physics. They need intense sources to achieve the required luminosity. In particular, the positron source must provide about 10E+14 positrons per second. The positron source for the International Linear Collider (ILC) is based on a helical undulator passed by the electron beam to create an intense circularly polarized photon beam. With these photons a longitudinally polarized positron beam is generated; the degree of polarization can be enhanced by collimating the photon beam. However, the high photon beam intensity causes huge thermal load in the collimator material. In this paper the thermal load in the photon collimator is discussed and a flexible design solution is presented.Comment: 22 pages, 19 figures, 8 tables, cross-reference to table 4 fixe

Analytical validation of bovine plasma ceruloplasmin measurement by p-phenylenediamine oxidation and effect of storage time and freezing temperature

Background Determination of ceruloplasmin (Cp) activity in plasma can provide an objective measure of the health of dairy cows as well as it can be used for various diagnostic purposes. The current study was designed to perform an analytical validation of a method for the determination of plasma Cp activity in dairy cows and to evaluate the influences of plasma storage times and temperatures as well as freeze–thaw cycles on the activity of this enzyme. This cohort was carried out on ten cows. For each cow, 24 aliquots of plasma, which were stored at different temperature regimes, were prepared. Both intra- and interassay coefficients of variation (CVs) were determined. The linearity was evaluated using bovine plasma Cp standard. Results The mean values of intra- and interassay CVs were 1.08 and 2.12%, respectively. Results of linearity testing showed a high correlation coefficient (r = 0.998, P < 0.001). After 3 days of storage at room temperature and refrigeration, the plasma activity of Cp was significantly lowered (P < 0.05). Plasma samples kept at freezing for 3 months revealed insignificant changes in the activity of Cp. Repeated freeze–thaw cycles for three times had no significant influence on the activity of Cp. Conclusions The method is easy and may be valid at values of Cp ranging from 100 to 1000 mg/L. It seems that keeping of plasma samples at room temperature and refrigeration longer than 3 days is unsuitable for Cp measurement. In addition, Cp remains stable in plasma samples stored at freezing as well as repeat freeze–thaw cycles

Neuronal microRNA eeregulation in response to Alzheimer's disease Amyloid-β

Normal brain development and function depends on microRNA (miRNA) networks to fine tune the balance between the transcriptome and proteome of the cell. These small non-coding RNA regulators are highly enriched in brain where they play key roles in neuronal development, plasticity and disease. In neurodegenerative disorders such as Alzheimer's disease (AD), brain miRNA profiles are altered; thus miRNA dysfunction could be both a cause and a consequence of disease. Our study dissects the complexity of human AD pathology, and addresses the hypothesis that amyloid-beta (Abeta) itself, a known causative factor of AD, causes neuronal miRNA deregulation, which could contribute to the pathomechanisms of AD. We used sensitive TaqMan low density miRNA arrays (TLDA) on murine primary hippocampal cultures to show that about half of all miRNAs tested were down-regulated in response to Abeta peptides. Time-course assays of neuronal Abeta treatments show that Abeta is in fact a powerful regulator of miRNA levels as the response of certain mature miRNAs is extremely rapid. Bioinformatic analysis predicts that the deregulated miRNAs are likely to affect target genes present in prominent neuronal pathways known to be disrupted in AD. Remarkably, we also found that the miRNA deregulation in hippocampal cultures was paralleled in vivo by a deregulation in the hippocampus of Abeta42-depositing APP23 mice, at the onset of Abeta plaque formation. In addition, the miRNA deregulation in hippocampal cultures and APP23 hippocampus overlaps with those obtained in human AD studies. Taken together, our findings suggest that neuronal miRNA deregulation in response to an insult by Abeta may be an important factor contributing to the cascade of events leading to AD.N.S. is supported by the Human Frontier Science Program. L.I. is supported by the National Health and Medical Research Council (NHMRC) and the Australian Research Council (ARC), and J.G. is supported by grants from the University of Sydney, the National Health and Medical Research Council (NHMRC), the Australian Research Council (ARC), and the J.O. & J.R. Wicking Trust. Postgraduate scholarship support has been provided by the Wenkart Foundation, GlaxoSmithKline and Alzheimer’s Australia

Could job insecurity (also) be a motivator?

"This study tested the idea that there is not only a negative effect of job insecurity on performance but also a positive one. The positive effect can be expected because job insecurity might also motivate people to work hard because good performance might be believed to lessen the chance of being made redundant. We assume that both effects work simultaneously but that the negative effect is stronger than the positive one. Furthermore, we assume that the negative effect is mediated by work attitudes. Job insecurity, performance (in-role behavior and organizational citizenship behaviour), and work attitudes (job satisfaction, commitment, and justice perceptions) data were collected from 132 German nonmanagerial employees. Structural equation modeling provided some evidence for the hypothesized relationships. In addition, our data replicate the finding of Borg & Elizur (1992) that there are two separate dimensions of job insecurity with different correlational patterns: cognitive job insecurity (i.e., the probability estimate of loosing one's job) and affective job insecurity (i.e., being worried about loosing one's job)." (author's abstract

Periodontal status of HIV-infected patients undergoing antiretroviral therapy compared to HIV-therapy naive patients: a case control study

<p>Abstract</p> <p>Background</p> <p>Although severe oral opportunistic infections decreased with the implementation of highly active antiretroviral therapy, periodontitis is still a commonly described problem in patients infected with human immunodeficiency virus (HIV). The objective of the present investigation was to determine possible differences in periodontal parameters between antiretroviral treated and untreated patients.</p> <p>Methods</p> <p>The study population comprised 80 patients infected with HIV divided into two groups. The first group was receiving antiretroviral therapy while the second group was therapy naive. The following parameters were examined: probing pocket depth, gingival recession, clinical attachment level, papilla bleeding score, periodontal screening index and the index for decayed, missed and filled teeth. A questionnaire concerning oral hygiene, dental care and smoking habits was filled out by the patients.</p> <p>Results</p> <p>There were no significant differences regarding the periodontal parameters between the groups except in the clinical marker for inflammation, the papilla bleeding score, which was twice as high (<it>P </it>< 0.0001) in the antiretroviral untreated group (0.58 ± 0.40 versus 1.02 ± 0.59). The participants of this investigation generally showed a prevalence of periodontitis comparable to that in healthy subjects. The results of the questionnaire were comparable between the two groups.</p> <p>Conclusion</p> <p>There is no indication for advanced periodontal damage in HIV-infected versus non-infected patients in comparable age groups. Due to their immunodeficiency, HIV-infected patients should be monitored closely to prevent irreversible periodontal damage. Periodontal monitoring and early therapy is recommended independent of an indication for highly active antiretroviral therapy.</p

24-h variations of blood serum metabolites in high yielding dairy cows and calves

Background Blood profile testing is commonly used to monitor herd health status, diagnose disorders, and predict the risk of diseases in cows and calves, with subsequent optimization the production of dairy herds. By understanding the physiological ranges of serum metabolites relative to age, lactation stage, and the sampling time in healthy cows and calves, the dairy practitioners can accurately diagnose abnormalities with a blood test. The effect of sampling time on the variation of serum metabolites within 24 h were evaluated in 83 cattle. All animals were originated from a dairy herd, where the animals, based on their ages and lactation stages, were classified into eight groups. The blood samples were collected from each animal every 4 h within a day. Results The time of sampling within the day showed significant influences on the serum concentrations of glucose, beta-hydroxybutyric acid (BHBA) and urea. BHBA was the most metabolite that showed day variation among cows' groups. Furthermore, the concentrations of total cholesterol were the most stable metabolite in all groups. The mean values of albumin, total proteins, glucose, non-esterified fatty acids (NEFA), BHBA, total cholesterol, total bilirubin, urea, and creatinine revealed significant variations among the different studied groups. Conclusions A certain suitable time of blood sample collection cannot be recommended. However, care shall be taken for the time of sampling for measurements of glucose, NEFA, BHBA and urea, otherwise the comparative values of these metabolites at different sampling time points may differ significantly from each other's, without a disease cause. It may be recommended, for metabolic assessment of dairy herds, classification the subjects into different groups based on lactation stages and ages of animals

Zur sozialen Determination der kulturellen Funktion der Architektur

Wissenschaftliches Kolloquium vom 24. bis 26. Juni 1986 in Weimar an der Hochschule für Architektur und Bauwesen zum Thema: 'Der wissenschaftlich-technische Fortschritt und die sozial-kulturellen Funktionen von Architektur und industrieller Formgestaltung in unserer Epoche