NMR-based metabonomic studies reveal changes in the biochemical profile of plasma and urine from pigs fed high-fibre rye bread

This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLS-DA) on spectra obtained for plasma samples revealed that the spectral region at 3.25 parts per million dominates the differentiation between the two diets, as the WGD is associated with higher spectral intensity in this region. Spiking experiments and LC-MS analyses of the plasma verified that this spectral difference could be ascribed to a significantly higher content of betaine in WGD plasma samples compared with NWD samples. In an identical study with the same diets, urine samples were collected, and 1H NMR spectra were acquired on these. PLS-DA on spectra obtained for urine samples revealed changes in the intensities of spectral regions, which could be ascribed to differences in the content of betaine and creatine/creatinine between the two diets, and LC-MS analyses verified a significantly lower content of creatinine in WGD urine samples compared with NWD urine samples. In conclusion, using an explorative approach, the present studies disclosed biochemical effects of a wholegrain diet on plasma betaine content and excretion of betaine and creatinine.     

Expression, localization, and function of MRP5 (ABCC5), a transporter for cyclic nucleotides, in human placenta and cultured human trophoblasts: effects of gestational age and cellular differentiation

The placenta functions both as site for nutrition and protection of the fetus. Transport proteins, including members of the multidrug resistance protein (MRP)/ABCC subfamily, have been recognized to contribute to the latter function. MRP5 (ABCC5) was identified as transmembrane transport protein for cyclic nucleotides, especially 3',5'-cyclic GMP (cGMP), indicating an additional role in signal transduction and a potential role in placenta development. We therefore studied expression, localization, and function of MRP5 in placenta of different gestational ages. Quantitative real-time polymerase chain reaction revealed expression of MRP5 in all 60 samples from pre-term and term placenta, with a decreasing mean expression with gestational age (MRP5/18S-ratio x 1000; < 32 weeks: 2.91 +/- 0.73, n = 15; 32 to 37 weeks: 2.10 +/- 0.87, n = 15; > 37 weeks: 0.46 +/- 0.08, n = 30; P < 0.01). Immunofluorescence microscopy with an anti-MRP5 antibody indicated localization of MRP5 preferentially in the basal membrane of syncytiotrophoblasts and in and around fetal vessels. ATP-dependent [(3)H]cGMP transport as evidence for MRP5 function could be demonstrated in isolated basal membrane vesicles. Moreover, the influence of cellular differentiation on MRP5 expression was studied in isolated trophoblasts, revealing an increase of the MRP5 expression in parallel with the hCG production (MRP5/18S-ratio x 1000 was 2.4 +/- 0.5 at day 5 of culture and 1.45 +/- 0.5 at day 0 of culture, n = 3 preparations, significant difference with P < 0.05). In conclusion, MRP5 expression depends on gestational age and varies throughout the differentiation process. In view of the important role of cGMP for cellular differentiation, MRP5 may play a role in placental development in context with a specific need for cellular cGMP export.     

Myeloperoxidase-Deficient Polymorphonuclear Leucocytes

Neutrophil myeloperoxidase (MPO) activity was analysed semi-quantitatively both by (i) MPO-scoring of polymorphonuclear leucocytes (PMN) and (ii) counting the MPO-deficient PMN (PMN lacking MPO) in 164 subjects (60 cases of leukaemia and 104 normal humans). The scoring method showed that 10 out of 21 (48%) cases of acute myeloid leukaemia (AML), 2 out of 10 (20 %) cases of chronic myeloid leukaemia (CML), 0 out of 29 cases of lymphoid leukaemia (ALL + CLL), and 1 out of 104 normal humans had decreased MPO scores. These figures correlated well with the more simple counting of PMN lacking MPO in the same groups: 8 out of 21 (37 %) cases of AML, 6 out of 10 (60 %) cases of CML and 0 out of 29 cases of lymphoid leukaemia showing more than 4 % PMN lacking MPO. In cases of otherwise unclassifiable acute leukaemia, a decreased MPO score and an increased number of MPO-deficient PMN suggests the diagnosis of AML and not ALL. Counting the number of PMN lacking MPO was found to be a time-saving and even more reliable method than the semiquantitative scoring of MPO activity in PMN.     

An Active Derivative of Rabbit Antibody Light Chain Composed of the Constant and the Variable Domains Held Together Only by a Native Disulfide Bond

The immunoglobulin light chain is thought to be organized into two structural domains, the constant and the variable regions, linked by a switch region of lesser tertiary organization. Light chains of homogeneous rabbit antibodies to pneumococcal polysaccharides labeled with (125)I were subjected to trypsin digestion at different temperatures. At 43 degrees molecular size (as determined by gel filtration) remained unchanged until the chain was subjected to full reduction and alkylation, when half molecules were demonstrated. One light chain resisted tryptic digestion at 43 degrees . At 50 degrees extensive digestion to small peptides occurred. These experiments confirm the susceptibility of the switch region to enzymatic attack as well as the relative resistance of the constant and variable region domains. They also demonstrate that the third disulfide bridge in rabbit light chain extends between the two structural domains and maintains the integrity of the molecule even when a peptide bond in the switch region is cleaved. When the light chain was subjected to dilute acid hydrolysis [10% acetic acid-pyridine (pH 2.5) in 7 M guanidine-HCl for 24-90 hr] selective cleavage at the switch region was demonstrated in yields up to 90%. The site of cleavage was shown to be the acid labile Asp 109-Pro 110 bond by automated Edman degradation. After cleavage the molecule remained intact until full reduction and alkylation, when half molecules were again demonstrated. A light chain cleaved in this way with its interdomain disulfide bond intact, was able to recombine with a homologous heavy chain and restore antigen binding. This indicates that an intact switch region is not needed for antibody activity. The availability of a functional variable region now allows for planning an examination of the antibody combining site by the technique of peptide synthesis.     

The Effect of Thioridazine and Promazine in Reducing the Potassium Loss from Isolated Perfused Rat Hearts

Electrically stimulated isolated rat hearts were perfused with a Ringer solution containing a low concentration of K⁺. At the end of a control period of 20 min. the cumulative K⁺ loss from the hearts was approximately 30 meq./kg dry weight. This value was used in each experiment as reference (100 per cent) for the total loss found after a subsequent additional test period of 30 min. The addition of promazine and thioridazine to the perfusate at the end of the control period reduced the further K⁺ loss. The values found in the different groups at the end of the test period were: Control group (10 hearts): 154 ± 11.6 per cent; with promazine 10^-5 M added (8 hearts): 103 ± 5.6 per cent; with promazine 2.5 × 10^-5 M added (10 hearts): 74 ± 10.5 per cent and with thioridazine 10^-5 M added (8 hearts): 110 ± 5.8 per cent. In another type of experiments, ouabain 1.5 × 10^-6 M was added at the end of the control period. This drug caused an increased K⁺ loss (181 ± 14.2 per cent) and ventricular fibrillation in 6 out of 8 hearts. Promazine 2.5 × 10^-5 M added before ouabain, prevented the development of fibrillation in another group of 7 hearts, and also markedly reduced the K⁺ loss. It is concluded that phenothiazines reduce the K⁺ permeability of the heart muscle membranes.     

Metastatic Melanoma with Balloon Cell Changes: An Electron Microscopic Study

A case of metastatic balloon cell melanoma is presented. The balloon cells are ultrastructurally characterized by the presence of numerous cytoplasmic vacuoles and abnormal melanosomes, which confirm their melanocytic origin. The study supports the concept that the cytoplasmic vacuoles represent grossly dilated melanosomes. The value of electron microscopic examination is emphasized in order to distinguish these lesions from other malignant clear cell tumors.