Clinical chemistry of common apolipoprotein E isoforms

Apolipoprotein E plays a central role in clearance of lipoprotein remnants by serving as a ligand for low-density lipoprotein and apolipoprotein E receptors. Three common alleles (apolipoprotein E(2), E(3) and E(4)) give rise to six phenotypes. Apolipoprotein E(3) is the ancestral form. Common apolipoprotein E isoforms derive from nucleotide substitutions in codons 112 and 158. Resulting cysteine-arginine substitutions cause differences in: affinities for low-density lipoprotein and apolipoprotein E receptors, low-density lipoprotein receptor activities, distribution of apolipoprotein E among lipoproteins, low-density lipoprotein formation rate, and cholesterol absorption. Accompanying changes in triglycerides, cholesterol and low-density lipoprotein may promote atherosclerosis development. Over 90% of patients with familial dysbetalipoproteinaemia have apolipoprotein E(2)/E(2). Apolipoprotein E(4) may promote atherosclerosis by its low-density lipoprotein raising effect. Establishment of apolipoprotein E isoforms may be important for patients with diabetes mellitus and several non-atherosclerotic diseases. Apolipoprotein E phenotyping exploits differences in isoelectric points. Isoelectric focusing uses gels that contain pH 4-7 ampholytes and urea. Serum is directly applied, or prepurified by delipidation, lipoprotein precipitation or dialysation. Isoelectric focusing is followed by immunofixation/protein staining. Another approach is electro- or diffusion blotting, followed by protein staining or immunological detection with anti-apolipoprotein E antibodies and an enzyme-conjugated second antibody. Apolipoprotein E genotyping demonstrates underlying point mutations. Analyses of polymerase chain reaction products are done by allele-specific oligonucleotide probes, restriction fragment length polymorphism, single-stranded conformational polymorphism, the primer-guided nucleotide incorporation assay, or denaturating gradient gel electrophoresis. Detection with primers that either or not initiate amplification is performed with the amplification refractory mutation system. Disparities between phenotyping and genotyping may derive from isoelectric focusing methods that do not adequately separate apolipoprotein E posttranslational variants, storage artifacts or faint isoelectric focusing bands.     

A 3.3-mW /spl Sigma//spl Delta/ modulator for UMTS in 0.18-/spl mu/m CMOS with 70-dB dynamic range in 2-MHz bandwidth

A quadrature fourth-order, continuous-time, /spl Sigma//spl Delta/ modulator with 1.5-b quantizer and feedback digital-to-analog converter (DAC) for a universal mobile telecommunication system (UMTS) receiver chain is presented. It achieves a dynamic range of 70 dB in a 2-MHz bandwidth and the total harmonic distortion is -74 dB at full-scale input. When used in an integrated receiver for UMTS, the dynamic range of the modulator substantially reduces the need for analog automatic gain control and its tolerance of large out-of-band interference also permits the use of only first-order prefiltering. An IC including an I and Q /spl Sigma//spl Delta/ modulator, phase-locked loop, oscillator, and bandgap dissipates 11.5 mW at 1.8 V. The active area is 0.41 mm/sup 2/ in a 0.18-/spl mu/m 1-poly 5-metal CMOS technology.     

Synthesis of Hexp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH2)7 CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex = 3-O-methyl-beta-D-Gal, 3-deoxy-beta-D-Gal, 3-deoxy-3-fluoro-beta-D-Gal, 3-amino-3-deoxy-beta-D-Gal, beta-D-Gul, alpha-L-Alt, or beta-L-Gal

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.     

A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a beta-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.     

Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.     

Presupposition failure — A comedy of errors

Presuppositions of utterances are the pieces of information you convey with an utterance no matter whether your utterance is true or not. We first study presupposition in a very simple framework of updating propositional information, with examples of how presuppositions of complex propositional updates can be calculated. Next we move on to presuppositions and quantification, in the context of a dynamic version of predicate logic, suitably modified to allow for presupposition failure. In both the propositional and the quantificational case, presupposition failure can be viewed as error abortion of procedures. Thus, a dynamic assertion logic which describes the preconditions for error abortion is the suitable tool for analysing presupposition.     

Reliability and validity of the measurement of position sense in women with osteoarthritis of the knee

OBJECTIVE: We examined the test-retest reliability and the construct validity of the measurement of knee position sense for describing the functional weightbearing performance of women with osteoarthritis (OA) of the knee. METHODS: For the purpose of this study, position sense was defined as the error occurring when subjects attempted to reproduce a criterion angle in standing with visual cues eliminated. Five such tests were recorded photographically on 3 different occasions. On each occasion the 10 subjects also completed a self-paced walking test over a 13 m indoor walkway. RESULTS: The photographic measurements were reproducible (r = 0.90) and there was no change in positioning accuracy across sessions. There was a significant (p < 0.05) inverse correlation of 0.70 between the standard deviation of the mean individual measurements of position sense (precision of the test) and those of walking speed. CONCLUSION: Our study demonstrates good measurement reliability and a comparable mean angular error with repeated tests. It also suggests the amplitude of the variability of this error is a strong determinant of an individual OA patient's functional performance in walking.     

Analytical and clinical evaluation of new diagnostic tests for myocardial damage

We tested the hypothesis that preventing cyclic GMP degradation with zaprinast, (a selective cyclic GMP-phosphodiesterase inhibitor) would produce a blunted reduction in myocardial O2 consumption in renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy. Four groups of anesthetized open-chest New Zealand white rabbits (n = 26) were utilized. Either vehicle or zaprinast (3 x 10(-3) M) was applied topically to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. The 1K1C rabbits had a greater heart weight-to-body weight ratio (2.94 +/- 0.08 g/kg) than controls (2.58 +/- 0.17). Systolic blood pressure was higher in 1K1C (102 +/- 9 mm Hg) than in controls (86 +/- 3). Zaprinast significantly and similarly increased cyclic GMP in both control (3.90 +/- 0.47 to 4.66 +/- 0.89 pmol/g) subepicardium (EPI) and (5.08 +/- 0.69 to 7.06 +/- 1.36) subendocardium (ENDO) and 1K1C hearts (5.53 +/- 0.61 to 7.48 +/- 1.51 EPI and 6.48 +/- 0.42 to 8.88 +/- 1.08 ENDO). Myocardial O2 consumption (ml O2/min/ 100 g) was significantly lower in controls treated with zaprinast (EPI: 8.8 +/- 0.1; ENDO: 9.5 +/- 1.9) than in controls treated with vehicle (EPI: 13.6 +/- 1.3; ENDO: 16.2 +/- 2.9). This effect was diminished in 1K1C rabbits treated with zaprinast (EPI: 10.3 +/- 2.4; ENDO: 11.2 +/- 2.6) compared with the vehicle-treated 1K1C group (EPI: 13.3 +/- 1.2; ENDO: 14.5 +/- 2.4). There was a similar increase in myocardial cyclic GMP after treatment with zaprinast, but a greater depression of myocardial O2 consumption in control animals than in 1K1C after treatment with zaprinast. This suggested that the reduction in myocardial O2 consumption, related to increases in cyclic GMP caused by cyclic GMP-phosphodiesterase blockade, was less in 1K1C cardiac hypertrophy.