Exchange of methyl hydrogens in ethanol during incorporation in bile acids in vivo

[2,2,2-2H]Ethanol was administered continuously to bile fistula rats for 72 h, with or without (--)-hydroxycitrate. The deuterium labelling of biliary bile acids was determined by GC-MS and 13C NMR. Difference spectra between 2H,1H- and 1H-decoupled 13C NMR spectra showed the presence of partly deuterated methyl and methylene groups in methyl cholate, indicating exchange of deuterium in [2,2,2-2H]ethanol for protium prior to or during incorporation of acetate into the bile acid. The extent of exchange was 20--30% as calculated from the isotopic composition of a fragment ion containing one methyl and one methylene group derived from C-2 of acetate. The exchange was unaffected by (--)-hydroxycitrate, indicating that it was not due to reversible incorporation of deuterated acetate into citrate. About 60% of the acetyl-CoA serving as precursor of cholic and chenodeoxycholic acids were derived from ethanol. This value was not changed by administration of (--)-hydroxycitrate. The half-life time of cholesterol molecules acting as precursors of both bile acids was about 50 h in the presence of (--)-hydroxycitrate, which is about the same as previously found in the absence of the inhibitor.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Oxygen uptake during running as related to body mass in circumpubertal boys: a longitudinal study

To investigate the effect of endurance training on physiological characteristics during circumpubertal growth, eight young runners (mean starting age 12 years) were studied every 6 months for 8 years. Four other boys served as untrained controls. Oxygen uptake (VO2) and blood lactate concentrations were measured during submaximal and maximal treadmill running. The data were aligned with each individual's age of peak height velocity. The maximal oxygen uptake (VO2max; ml.kg-1.min-1) decreased with growth in the untrained group but remained almost constant in the training group. The oxygen cost of running at 15 km.h-1 (VO2 15, ml.kg-1.min-1) was persistently lower in the trained group but decreased similarly with age in both groups. The development of VO2max and VO2 15 (l.min-1) was related to each individual's increase in body mass so that power functions were obtained. The mean body mass scaling factor was 0.78 (SEM 0.07) and 1.01 (SEM 0.04) for VO2max and 0.75 (SEM 0.09) and 0.75 (SEM 0.02) for VO2 15 in the untrained and trained groups, respectively. Therefore, expressed as ml.kg-0.75.min-1, VO2 15 was unchanged in both groups and VO2max increased only in the trained group. The running velocity corresponding to 4 mmol.l-1 of blood lactate (nu la4) increased only in the trained group. Blood lactate concentration at exhaustion remained constant in both groups over the years studied. In conclusion, recent and the present findings would suggest that changes in the oxygen cost of running and VO2max (ml.kg-1.min-1) during growth may mainly be due to an overestimation of the body mass dependency of VO2 during running.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.     

Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.     

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.     

The refined crystal structure of ribonuclease A at 2.0 A resolution

This paper describes the structure of bovine pancreatic ribonuclease A, refined by a restrained parameter least squares procedure at 2.0 A resolution, and rebuilt using computer graphics. The final agreement factor (formula see text) is 0.159. The positions of the 951 main chain atoms have been determined with an estimated accuracy of 0.17 A. In addition, the model includes a phosphate group in the active site and 176 waters, many of them with partial occupancy. The bond lengths in the refined structure of RNase A differ from the ideal values by an overall root mean square deviation of 0.022 A; the corresponding value for angle distances is 0.06 A. The root mean square deviation of planar atoms from ideality is 0.017 A, and root mean square deviation of the peptide torsion angles from 180 degrees is 3.4 degrees. The model is in good agreement with the final difference Fourier maps. Two active site histidines, His 12 and His 119, form hydrogen bonds to the phosphate ion. His 119 is also hydrogen bonded to the carboxyl of ASp 121 and His 12 to the carbonyl of Thr 45. The structure of the RNase A is very similar to that of RNase S, particularly in the active site region. The root mean square discrepancy of all atoms from residues 1 to 16 and 24 to 123 is 1.06 A and the root mean square discrepancy for the active site region is 0.6 A.     

Ion-exchange high-performance liquid-chromatography steps in peptide purifications

Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected~ ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a con-siderably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix.A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and t h a t it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.     

Focus: Rare Disease: Pulmonary Alveolar Microlithiasis – A Review

Pulmonary Alveolar Microlithiasis (PAM) is a rare genetic disorder causing widespread deposition of calcium-phosphate crystals in the alveolar space. A hallmark of the disease is the discrepancy between perceived symptoms upon diagnosis compared with the extensive, sandstorm-like appearance of the microliths on chest X-ray or HRCT. Caused by a defective sodium-dependent phosphate transport protein due to loss-of-function variants of the SLC34A2 gene, PAM is an autosomal recessive transmitted disorder, and as such has a high correlation to consanguinity. The most common variants of the SLC34A2 gene are single nucleotide biallelic changes, but larger deletions are described. Initial suspicion of PAM on radiological examination should be followed by genetic testing to verify the diagnosis and identify the disease-causing variant. When not available, the diagnosis can be made by means of invasive techniques, such as transbronchial forceps or cryobiopsy, or a surgical lung biopsy. In families with a history of PAM, genetic counseling should be offered, as well as preimplantation/prenatal testing if necessary. As of writing this review, no definitive treatment exists, and PAM may in some cases progress to severe pulmonary disease with respiratory failure and potential death. Patients with PAM should be offered preventative and symptomatic treatments such as vaccinations and oxygen therapy when needed. In some cases, lung transplantation may be required.     

A Highlights from MBoC Selection: A troponin T variant linked with pediatric dilated cardiomyopathy reduces the coupling of thin filament activation to myosin and calcium binding

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.