3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.     

Chromogenic redox assay for beta-lactamases yielding water-insoluble products. II. Heterogeneous sandwich assay for hCG.

A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described. The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label. The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan. The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically. The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C. Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.     

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Photobleaching in the unicellular green alga Dunaliella parva 19/9

The change in the pigment composition of the unicellular alga Dunaliella parva 19/9 during exposure to high light (4000 mol m^-2 s^-1) has been investigated. During photobleaching the carotenoids were lost at a greater rate than the chlorophylls. In these photoinhibitory conditions, -carotene and especially the minor carotenes, - and -carotene, were more susceptible to oxidative destriction than the xanthophylls. Lutein, the major carotenoid present, was the most stable of the carotenoids in these conditions. In addition to the direct photobleaching of carotenoids and chlorophylls, high light treatment induced the de-epoxidation of violaxanthin to antheraxantin and zeaxanthin. Small amounts of zeaxanthin were present in cells prior to illumination but the amount increased 2.4 fold following high light treatment. The effects of extremes of temperature during exposure to high light intensities were also investigated. The destruction of chlorophylls was found to be more temperature sensitive than that of the carotenoids. The general pattern of loss for the individual carotenoids was similar to that found at 25°C, i.e., the carotenes were more readily degraded than the xanthophylls.     

Hwabyung: The construction of a korean popular illness among korean elderly immigrant women in the United States

The cultural construction of Hwabyung, a Korean culture-bound syndrome, is explored among a sample of 20 elderly Korean immigrant women in the United States. Hwabyung results when distressed emotions associated with the specifically Korean way of perceiving and reacting to intolerable and tragic life situations cause bodily symptoms by interfering with the harmony of Ki (vital energy). Korean elderly immigrants report a broad range of symptoms associated with Hwabyung; they less frequently report the epigastric mass, which had been considered the cardinal symptom by cosmopolitan and traditional medical writers. Hwabyung is treated holistically with psychosocial support from family, spiritual comfort, home and popular remedies, traditional Korean medicine, and biomedical treatments. Hwabyung provides a way of conceptualizing and resolving emotional distress through somatization among Korean elderly immigrant women.     

The Athena semi-automated karyotyping system

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.     

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na⁺ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na⁺ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na⁺, we decided to use whole-cell patch clamp techniques to investigate whether the Na⁺ conductance in salivary duct cells is also regulated by extracellular Na⁺. Using Na⁺-free pipette solutions, we observed that the whole-cell Na⁺ conductance increased when the extracellular Na⁺ was increased, whereas the whole-cell Na⁺ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na⁺ conductance on extracellular Na⁺ could be described by the Michaelis-Menten equation with a K _m of 47.3 mmol/1 and a maximum conductance (G _max) of 2.18 nS. To investigate whether this saturation of the Na⁺ conductance with increasing extracellular Na⁺ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na⁺ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na⁺ solutions. We found that Na⁺ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na⁺. On the other hand, the single-channel current saturated with increasing extracellular Na⁺ and, consequently, whole-cell Na⁺ permeability declined. In other words, the decline in Na⁺ permeability in salivary duct cells with increasing extracellular Na⁺ concentration is due simply to saturation of the single-channel Na⁺ conductance rather than to inactivation of channel activity. Received: 27 July 1995/Revised: 7 December 1995