Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.     

Exercise training attenuates increases in lumbar sympathetic nerve activity produced by stimulation of the rostral ventrolateral medulla.

Exercise training (ExTr) has been associated with blunted activation of the sympathetic nervous system in several animal models and in some human studies. Although these data are consistent with the hypothesis that ExTr reduces the incidence of cardiovascular diseases via reduced sympathoexcitation, the mechanisms are unknown. The rostral ventrolateral medulla (RVLM) is important in control of sympathetic nervous system activity in both physiological and pathophysiological states. The purpose of the present study was to test the hypothesis that ExTr results in reduced sympathoexcitation mediated at the level of the RVLM. Male Sprague-Dawley rats were treadmill trained or remained sedentary for 8-10 wk. RVLM microinjections were performed under Inactin anesthesia while mean arterial pressure, heart rate, and lumbar sympathetic nerve activity (LSNA) were recorded. Bilateral microinjections of the GABA(A) antagonist bicuculline (5 mM, 90 nl) into the RVLM increased LSNA in sedentary animals (169 +/- 33%), which was blunted in ExTr animals (100 +/- 22%, P < 0.05). Activation of the RVLM with unilateral microinjections of glutamate (10 mM, 30 nl) increased LSNA in sedentary animals (76 +/- 13%), which was also attenuated by training (26 +/- 2%, P < 0.05). Bilateral microinjections of the ionotropic glutamate receptor antagonist kynurenate (40 mM, 90 nl) produced small increases in mean arterial pressure and LSNA that were similar between groups. Results suggest that ExTr may reduce increases in LSNA due to reduced activation of the RVLM. Conversely, we speculate that the relatively enhanced activation of LSNA in sedentary animals may be related to the increased incidence of cardiovascular disease associated with a sedentary lifestyle.     

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

Comparison of the uptake of vitamin B12 by Spirometra mansonoides and Hymenolepis diminuta and the functional groups of B12 analogs affecting uptake.

Uptake of 57Co-vitamin B12 (CN-Cbl) by spargana (larvae) of the pseudophyllidean tapeworm, Spirometra mansonoides, was affected by temperature, was saturable with respect to concentration of CN-Cbl in the medium, and was inhibited in the presence of several structural analogs of CN-Cbl. In uptake studies with various analogs it was found that chemical modifications which altered the benzimidazole moiety greatly reduced the ability of the worm to take up these analogs. Modifications in which the amide groups of the propionamide side chains were removed, resulting in carboxylic acid derivatives, showed greatly reduced transport properties. The C-13 epimer in which the e-proprionamide side chain is no longer on the benzimidazole side (lower) of the molecule but is inverted to a position on the upper side was freely taken up. The pharmacological implications of this last observation are discussed. Adult Hymenolepis diminuta did not take up CN-Cbl in vitro, which correlated with the finding that no CN-Cbl was detected in the worm by Ochromonas malhamensis assay.     

Disease activated drugs: a new concept for the treatment of asthma.

Disease activated drugs (DAD) are pro-drugs of one active principle or combinations of two drugs, which have a proven efficacy for the treatment of the target disease. In opposition to pro-drugs, DAD are activated in inflamed but not normal tissues. Due to the disease specific activation, the amount of locally released drug(s) should be related directly to the severity of the inflammation. To test this concept in asthma a PDE4 inhibitor, an isoquinoline derivative, was chemically derivatized into pro-drugs or combined with corticosteroids. These new compounds were more readily cleaved into active PDE4 inhibitor, in bronchoalveolar lavage fluid (BALF) from Brown-Norway rats with lung inflammation than in BALF from rats without airway inflammation. The DAD concept (local selective release and improved therapeutic window) was validated in vivo using the inhibition of methacholine induced bronchoconstriction in guinea pigs with or without ozone induced lung inflammation. An example of DAD hydrolysis (isoquinoline-dexamethasone) was also examined in BALF from asthmatics and healthy volunteers.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.