Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol - Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.     

黄土丘陵沟壑区水平梯田改土培肥增产技术措施体系

黄土丘陵沟壑区水平梯田改土培肥增产技术措施体系刘东海赵廷宁（宁夏西吉县农业局７５６２００）（北京林业大学水土保持学院１０００８３）赵国杰（宁夏西吉县农业技术推广站７５６２００）ＴｅｃｈｎｉｑｕｅｓｏｆＳｏｉｌＩｍｐｒｏｖｅｍｅｎｔａｎｄＹｉｅｌｄＩｎ...     

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function,in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 μM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 μM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

A direct observation technique for studies on rhizoplane and rhizosphere colonization

Summary A slide incubation chamber was described which allowed small plants to be grown from seed and the root systems to be observed microscopically. A fluorescence stain, the ammonium salt of 8-anilino-1-naphthalene sulfonic acid, was applied to the soil in which the roots were growing and the stained microorganisms on the roots and in the rhizosphere were counted. A statistical pattern analysis technique, the two-within-four randomization test, was used to analyze the data obtained from quadrats on the roots. Distinct colonization patterns and colony growth, especially of bacteria, were easily distinguished with the technique.     

Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.)

Summary In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl₂ iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.For the preparation of tritiated proctolin we thank Dr. S. Reißmann, WB Biochemie, Sektion Biologie, FSU JenaThe authors wish to thank G. Schörlitz, Film- und Bildstelle, FSU Jena, for photographs of whole mount preparations and Ms. A. Zinßer and Mrs. B. Cosack for excellent technical assistance.     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

Early events in calcium-induced liposome fusion

Calcium-induced interaction of liposomes composed of pure phosphatidylserine (PS) has been studied using a rapid-mixing, rapid-freeze device. Freeze-fracture electron microscopy of this material revealed that liposomes react very rapidly after addition of calcium ions. After only 10 ms (the resolution of the technique) vesicle fusion was apparent. At the same time, however, vesicles also collapsed, and appeared as aggregates of flattened membranes. This may explain controversies which have arisen over vesicle fusion studied with more indirect methods.