Muscarinic receptors in the prenatal mouse embryo. Comparison of M35-immunohistochemistry with [3H]quinuclidinyl benzylate autoradiography

Muscarinic cholinergic receptors are widespread in nervous tissue and smooth muscsle or paracrine epithelial cells of various organs. In the embryo, muscarinic receptors are transitorily expressed in the early blastoderm and later on in blastemic tissues during morphogenesis. Recently, a monoclonal antibody (M35) against muscarinic receptor from calf brain became available. In the present study the use of M35-immunohistochemistry is compared to autoradiographic localization of muscarinic binding sites in the mouse embryo. The aim of the study is to test the suitability of the antibody for localization of muscrinic receptors in embryonic tissues. For autoradiography whole-body sagittal cryostat sections of the 17- and 18-day mouse embryo were covered with LKB-Ultrofilm after incubation with the radioactive ligand [³H] quinuclidinyl benzylate (QNB). For immunohistochemistry cryostat sections of formalin fixed tissues were used. In general, all tissues exhibiting ligand binding were also recognized by the antibody. M35-immunohistochemistry resulted in higher spatial resolution of receptor localization than [³H]QNB autoradiography. Definitive muscarinic receptors were observed in smooth muscle and the epithelial lining of the vascular, intestinal, respiratory and urinary system, in the brain, spinal cord and peripheral nerves. The embryonic type of the muscarinic receptor was detected in the mesothelium of lung and liver, in the nephrogenic blastema of the metanephros, and in lung mesenchyme. A large amount of embryonic muscarinic receptors was found in the remnants of the notochord and in the nucleus pulposus of the developing vertebral column. A function in morphogenesis is discussed of the embryonic muscarinic receptor.     

The evolution of primate communities and societies in Madagascar

During their 120 to 165 million years of isolation, the flora and fauna of Madagascar evolved, to a large extent, independently of the African mainland.¹ In contrast to other oceanic islands, Madagascar is large enough to house the major components of tropical ecosystems, allowing tests of evolutionary hypotheses on the level of complete communities. Taking lemurs, the primates of Madagascar, as an example, evolutionary hypotheses correctly predict the organization of their community structure with respect to ecological correlates. Lemur social systems and their morphological correlates, on the other hand, deviate in some respects from those of other primates. Apparently, lemur social systems are influenced by several selection pressures that are weak or rare in other primates. These include variable activity patterns and avoidance of infanticide. The interspecific variation in lemur social systems therefore offers a unique opportunity for a comprehensive study of the determinants of primate social systems.     

Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.     

Ventilatory effects of hypercapnic end-tidal PCO2 clamps during aerobic exercise of varying intensity

Nine subjects performed a sequence of sustained and randomised changes between 40 W and 100 W on a cycle ergometer while the end-tidal PO2 was kept close to 17.3 kPa (130 mm Hg) by means of a dynamic forcing technique (reference experiment). In a second series inspiratory CO2 was additionally manipulated so as to hold end-tidal PCO2 (PETCO2) near 6.5 kPa (49 mm Hg; 'CO2-clamp' experiment). By this forcing PETCO2 oscillations were attenuated and more evenly distributed over the frequency range. Ventilation (VT) responded to this manoeuvre with an upward trend that could not be ascribed to a slow CO2-response component, changes in metabolic rate or a dissociation of end-tidal and arterial PCO2. VT differences between reference and CO2-clamp experiments were abolished within a 3-min period following the termination of the external CO2 control. The present results suggest that the CO2-H+ stimulus plays a major role in adjusting ventilation when exercise intensity is decreased. The underlying CO2 effect appears to be neither additive nor bi-directionally symmetrical.     

Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor.

The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.     

General characteristics of the sigmoidal model equation representing quasi-static pulmonary P-V curves.

A pulmonary pressure-volume (P-V) curve represented by a sigmoidal model equation with four parameters, V(P) = a + b[1 + exp[-(P - c)/d]](-1), has been demonstrated to fit inflation and deflation data obtained under a variety of conditions extremely well. In the present report, a differential equation on V(P) is identified, thus relating the fourth parameter, d, to the difference between the upper and the lower asymptotes of the volume, b, through a proportionality constant, alpha, with its order of magnitude of 10(-4) to 10(-5) (in ml(-1). cmH(2)O(-1)). When the model equation is normalized using a nondimensional volume, (-1 < < 1), and a nondimensional pressure, (=(p/c) - 1), the resulting - curve depends on a single nondimensional parameter, Lambda = alphabc. A nondimensional work of expansion/compression, (1-2), is also obtained along the quasi-static sigmoidal P-V curve between an initial volume (at 1) and a final volume (at 2). Six sets of P-V data available in the literature are used to show the changes that occur in these two parameters (Lambda defining the shape of the sigmoidal curve and (1-2) accounting for the range of clinical data) with different conditions of the total respiratory system. The clinical usefulness of these parameters requires further study.     

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

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.