Maynard M. Dewey

Intercellular Connection between Smooth Muscle Cells: the Nexus

High-resolution electron microscopy has revealed that the regions of contact between smooth muscle cells from dog intestine are areas of fusion of adjacent cell membranes. For morphological and functional reasons this type of contact between excitable cells has been termed a nexus.

Electron microscope and low-angle X-ray diffraction studies on outer segment membranes from the retina of the frog

Ordered arrays of particles about 40 A in diameter within the outer segment membranes of retinal receptor have been observed by electron microscopy, and low-angle X-ray diffraction studies are in agreement with this finding. Surface-on views of outer segment membranes negatively-stained with phospho-tuhgstate or fixed with permanganate show square arrays of particles with a non-polar core 40 A in diameter. Light diffraction patterns of a mask of holes simulating electron micrographs showed first and second orders of a uniformly square array with a unit cell side of about 70 A. Low-angle X-ray diffraction patterns obtained from ultracentrifugal pellets of partially dried (unstained and unfixed) isolated outer segment membranes gave reflections consistent with a square array of particles within the membranes with a unit cell side of 70 A. The intensity ratios of the reflections are consistent with a spherical particle 40 A in diameter. Low-angle X-ray scattering, which corresponded to a mean center-to-center distance between the particles, was obtained from the moist membranes with a half-hour minimum exposure time. The birefringence characteristics of pellets of the moist membranes remained constant during this interval and were identical with those of intact outer segments of retinal receptors. Furthermore, low-angle X-ray diffraction periodicities of the membrane layering of such pellets were identical to those of the intact outer segment of the retinal receptor. Thus, it can be argued that ordered arrays of particles are present in these membranes.

Localization of rhodopsin antibody in the retina of the frog

Abstract A specific immune serum against frog rhodopsin has been developed in rabbits. The specificity of the antirhodopsin serum was established using the indirect Coons antibody technique. Absorption of the serum with electrophoretically purified rhodopsin abolished its ability to stain sections of retina or isolated retinal receptor disk membranes. Using fluorescein-labeled sheep anti-rabbit γ-globulin on sections of retina treated with antirhodopsin γ-globulin, fluorescence was observed in all retinal receptor outer segments and in the myoids and ellipsoids of retinal receptor inner segments. In addition, it was localized in cytoplasmic structures of the pigmented epithelium. Dissected retinas with intact outer segments showed fluorescence in the outer segments when the retinas were treated with antirhodopsin immune serum, washed thoroughly, incubated in fluorescein-labeled sheep anti-rabbit γ-globulin and again washed thoroughly. This is suggestive of a diffusion pathway into the outer segments. Isolated whole rod outer segments and isolated disk membranes stained uniformly by the indirect technique. From these observations we conclude that the photopigment is indeed part of the disk membranes of the outer segments and that the external membrane of the outer segment also contains photopigment. Use of the specific antirhodopsin immune serum has allowed the description of the molecular arrangement of photopigment in retinal receptor disk membranes (Blasie, Worthington & Dewey, 1968; Blasie & Worthington, 1969).

Starch gel electrophoresis of lactic dehydrogenase from rat kidney.

Summary 1. An improved method for demonstration of lactic dehydrogenase after starch gel electrophoresis is described. This method employs a more sensitive tetrazolium and phenazine methosulfate. 2. Rat kidney contained 5 lactic dehydrogenase fractions which varied in cofactor requirements. DPN diaphorase was not demonstrable in any of these fractions, when reduced DPN was employed as the substrate, but was demonstrated in 2 additional bands. 3. The possibility is discussed that at least certain of the lactic dehydrogenase fractions from rat kidney exhibit electrophoretic heterogenity because of their association with various components of the respiratory system. We are deeply indebted to Mrs. Julia Ternak for technical assistance.

Subfractions of lactate dehydrogenase of the rat.

A survey of the lactate dehydrogenase (LDH) isozymes of 20 organs and tissues of the rat revealed a total of nine fractions exhibiting LDH activity after starch gel electrophoresis. Of the nine LDH fractions, eight were found in testis and lymph node, while the ninth fraction was unique to kidney. The occurrence of LDH fractions in excess of the usual five LDH isozymes is accounted for by two alternate proposals. One proposal is an extension of the present tetramer postulate of LDH structure, while the other is a consideration of the significance of heterozygosity in the albino rats employed in the study. Both hypotheses are plausible and should be considered in future studies of isozymes.

ON THE HETEROGENEITY OF LACTIC DEHYDROGENASE

Yasummsni and! Ichikawa (J. Lab. & Clin. Med. 41: 296, 1953) recommmnusendcd the ninhydnin-Schiff reaction mis a histochemicmol test for proteins. Yet, in moo!el cxpenimssents smith various proteins only ali)umims svas colored (Burstone: J. Histocheni. 3: 32, 1955). When the nirshydnin-Schiff reaction sm-as applied to hunusan autopsy mmsatenial the intensity of staining wmus vanimuble and difficult to reproduce. The intensity of time reaction varied also with the hnnomuo! of ninshys!nin (Fisher, Emtstmams, Dajac). It wmos therefore deenned desirmobie to review penti nenst chenmuical literature to obtain i nfonmmsation whether or not the ninhydnin-Schiff ummethod can i)e coussic!ered suitai)le for the histochennical identificatious of proteinms. Accorc!insg to Virtmunen (Z. physiol. Chem. 266: 195, 1940), Van Slyke and coworkers (J. Biol. Cheat. 141: 627, 1941), and Greenstein and Winitz (Chemistry of the Amino Acids, Vol. 2, John Wiley & Sons, Inc., New York, 1961) the ninhydnin reaction is specific for free amino acids in that it requires the presenuce ins the free unconjugated state, of both the carboxyl and the neighboring ammmino group. Neither monsirso acids with a blocked cari)oxyl on mu i)iocked nunminso grommp react mmith ninhydnium. Pepticles and proteins also do not foruss noblehytles, I)ilt are isydrolyzed by nuinshydnium. Timcse chcmicmul dmutmo ins!icate thmot aldehydes s!eummonsstrmutec! by the mmininydnin-Schiff reactions more s!crivet! eitiser fromus free muusmiuuoacids still prcsenst ins paraffin-enusbedc!es! tissunes, or froums ausmi nso mucis!s split off the proteins ciumuinus during tneatusuennt mvith ni uuhvo!ri muTisoingh usmost ansuinno nocids are onuly sligint-ly soleble ins alcohol, time s-ornesponding nnext--lowcr noldehmydes derivet! fronsm nolmunsine, vnolinme, lemncine, isoleucimse mmmd l)imens lmulmunsi ne are very sob mimic Sunpportcs! l)Y USI HS Grant#R(’ 7303 ammo! i\Ieo!icmo! College of Gs-orgimo Professionunsl Ho-search Fmnnush (‘,ranmt #51 -79. (Hodgnmmanm et al. : Handbook of (‘hemistry and Physics. Chemical Hmubben Ponbi. Co., Cleveland, 1955). It sccnss therefore prol)noble that diffusion occurs during treatment of tissune sectionss -smiths ninhydnin in alcoholic solution for 16 hours or longer. Furthernusore, glyci ne , proli ne monsd hsydnoxyproline do not yield aidehydes (Van Slyke mind commorkers). Thus, these ausmino mocis!s, mm-iuich consstitute approximately 50% of the anuino acids in coilagen, can not be visualized mm-ith the ninhydnin-Schiff nsethod. Yet connective tissmne is often strongly colored. Landucci et at. (Recent -ldvances in Gelatin an(l Glue Resea,-ch, p. 62-67, Pengaumson Press, NewYork, 1958) reported thmut aldehydes are built into peptide chainss of collageus and other proteins. I)onning degradation of proteins these bonds betsveen momssino acids and nuldehvdes are i)roken and sommse of the resulting chsnoinus carry terumsinal alo!ehvde groimps . Under hmistos-isenmicmol condit ions coilagcns-svithout pretreat msmenmt-does not react with Schiff’s reagenut, but gelmuhine is mmtcnsseiv colored. However, it is nsot possible on the basis of these expenimmmcnts to cleterumminue whether or muot prefornued aldehydes l)lnu3 a role ins time nninmhydninu-Scimiff nemmction. It is commclmms!ec! that because of its ol)scinrc chenmicmi! bnosis, t hue irsherenut insmpossibihi ty to slensmonustrnote glycinme, proline minus! Isyo!roxypro!imme-t-hmot is, nspproxiussatcly 5(Y;: of the munsmimnonucioLs mmo-s)Ilmugemsmons! the probability of s!ifTusions norhifnocts, the uuinshvo!rinm-Ss,hiff nmmethoo! cnunn nuot i)c conmsicleresl a rehinuble iuistochemmmicnol mumet-huos! for the study of pnot-ei mutt.

Elevation of proteolytic activity in the pancreas of hypophysectomized rats by hormonal therapy.

Summary Hypophysectomy of the rat failed to reduce consistently the concentration of PA in the pancreas when computed on the basis of wet weight. When related to mg of nitrogen per g of pancreas the amount of PA was increased. Treatment with STC restored body weight, increased pancreatic weight and total PA. Except when insulin was added to the hormonal regimen, PA did not reach the level observed in the nonhypophysectomized controls.

Localization of rhodopsin in isolated, osmotically intact rod outer segment discs.

Localization of rhodopsin and its position in the membrane has been the subject of numerous studies. Most recently, immunocytochemical techniques have been employed to localize the opsin component of the molecule in in situ rod outer segments. Due to the problems inherent in localization procedures (penetration and mechanical interference) we have utilized isolated, osmotically intact rod outer segment discs in this study. Specific antibodies to chromatographically pure rhodopsin were prepared and enzymatically digested to their Fab components. The univalent Fab antibodies were conjugated to horseradish peroxidase and used to label the isolated rod outer segment discs. Discs treated with anti-opsin conjugate stained uniformly and heavily on their interdisc surfaces. Reaction product was also present on the intradisc surface in a thinner but still uniformly distributed layer. Controls treated with preimmune Fab - horseradish peroxidase conjugate showed no deposition of reaction product.