Protein phosphorylation: technologies for the identification of phosphoamino acids

Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.     

A potential role for angiotensin II-induced vascular endothelial growth factor expression in the pathogenesis of diabetic nephropathy?

The relationship between sensory quality of carrots and their contents and composition of essential oils and total sugars as influenced by nitrogen fertilization was investigated. Carrots (Daucus carota L.) of the variety 'Nanthya' F1 (S&G Sandoz Seeds) were grown in Weihenstephan 1996 with three levels of inorganic nitrogen fertilization (3 levels in 4 replications). Medium- and higher-boiling flavour-components were extracted as essential oils and separated gas-chromatographically (GC-FID). Lower-boiling flavour-components were taken from the headspace and analysed chemosensorially. The human sensory assessments were performed by an untrained panel of 300 people (students and employees of the TU München)--these results were compared with those of the chemical analyses. Carrots with lower nitrogen application were found to taste more intensive, more fruity, sweeter and better and at the same time less bitter and less earthy. They had higher contents of total sugar and a higher percentage of dry matter. Fertilization with nitrogen does not only affect the quantity but also the composition of the essential oils. The taste intensive was positively correlated with the quantity of essential oils, the taste sweet was positively correlated with the content of total sugars. It was possible to differentiate the carrots from each other by chemo-sensorial headspace analyses according to their N-fertilization levels.     

Radioimmunoscintigraphy with In-111-labeled capromab pendetide predicts prostate cancer response to salvage radiotherapy after failed radical prostatectomy

PURPOSE: We investigated the ability of In-111-capromab pendetide to separate patients who have failed radical prostatectomy into categories of those who would versus those who would not respond to salvage radiotherapy. METHODS: Prostate-specific antigen (PSA) levels in 32 men with prostate cancer who had failed radical prostatectomy and had undergone a whole-body In-111-capromab pendetide scan were followed-up for 13 months (median) after salvage radiotherapy to the pelvis. A logistic regression model was used to determine whether the scan findings, as well as other clinical variables, were associated with a durable complete response (DCR), a nondurable response (NDR), or no response (NR). RESULTS: Sixteen of 23 (70%) men with a normal scan outside the prostatic fossa achieved a DCR after salvage radiotherapy versus two of nine (22%) who had a positive scan outside the prostate fossa and pelvis (P = .0225, Fisher's exact test). Predicted probability (95% confidence interval [CI]) that a DCR would be obtained with a normal scan was 0.88 (0.55 to 0.98); for men with a positive scan limited to the prostatic fossa it was 0.62 (0.42 to 0.79); and for men with a positive scan outside the pelvis it was 0.27 (0.09 to 0.58). No other variables before radiotherapy showed a significant association with the DCR rate. CONCLUSION: Salvage radiotherapy is statistically more likely to lead to a durable complete PSA response in men with prostate cancer who have failed radical prostatectomy and have a negative In-111-capromab pendetide scan outside the pelvis as compared with those who have a positive scan.     

In search of a dose-response relationship with radiotherapy in the management of recurrent rectal carcinoma in the pelvis: a systematic review

BACKGROUND: Research on the subject of insight has been hampered by difficulties in definition and reliable measurement. METHODS: We compared several rating scales to measure insight on a group of 33 psychotic patients as well as assessing patients' psychopathology, clinical characteristics and cognitive functioning. RESULTS: Most currently used scales showed a high degree of inter-correlation. Measures of insight related strongly to the presence of delusions; grandiosity (inversely), and depression (positively). Higher insight scores correlated with indices of treatment compliance and inversely with substance abuse. Measures of pre-morbid IQ and impaired executive functioning, including the Wisconsin Card Sorting Test were not associated with poor insight. CONCLUSIONS: The study highlights aspects of psychopathology and clinical variables particularly related to insight and supports the continued use of standardized scales in further research in this area.     

Z/I and A-band lattice spacings in frog skeletal muscle: effects of contraction and osmolarity

A-band and Z-line/I-band lattice spacings were measured by small-angle X-ray diffraction from relaxed and isometrically-contracting whole frog sartorius muscles with lattice spacings reduced or swollen by changing the osmolarity of the bathing solution. A-band spacing increased by approximately 3% upon isometric contraction at reduced lattice spacings (245-356 mOsm) and decreased by approximately 1% at swollen spacings (172 mOsm), similarly to the behaviour of skinned muscles upon changing from the relaxed state to rigor. The Z/I lattice underwent a significant lattice expansion (3-8%) upon isometric contraction at all osmolarities, in qualitative agreement (but quantitative disagreement) with results from electron microscopy on mammalian skeletal muscle. Lattice areas calculated for the Z/I and A-band lattices indicate a barrel-shaped sarcomere in the resting state, which may provide a partial explanation for how longitudinal forces produced in the A-band can produce a radial expansive force in the Z-line during contraction. The radial component of cross-bridge stiffness was calculated from the A-band data for contracting muscle, using a lattice stability model incorporating structural, osmotic and electrostatic forces. The calculations gave a radial cross-bridge stiffness during contraction of about 9 x 10(5) N m-2, and outward radial force per thick filament in normal Ringer's solution of 6 x 10(-9) N, corresponding to a radial force per cross-bridge of 10(-11) N.     

Information technology and regulatory reformThe interorganisational effects of a technological innovation

This paper examines whether information technology can play a strategic role in supporting regulatory reforms aimed at achieving voluntary cooperation with government regulation. Analysis of the case of electronic tax return lodgment in Australia suggests that the effects of IT are contextual, and identifies positive effects of the new IT system on voluntary cooperation. However, in this case, divergence between private and public interests and uncertainty and lack of participation in regulatory decision-making appear to restrict the development of voluntary cooperation as a primary basis for government regulation.     

Sanger DNA-sequencing reactions performed in a solid-phase nanoreactor directly coupled to capillary gel electrophoresis

A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.