X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: Detection of heterozygotes by selective medium

Skin fibroblasts from five unrelated males with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency and from their families have been exposed to medium containing 6-thioguanine. This purine analogue selects against cells with normal hypoxanthine-guanine phosphoribosyl transferase activity and therefore permits detection of mutant cells in heterozygous populations. The results of these studies are compared to those obtained by autoradiography of single-cell clones of skin fibroblasts from the same subjects. In each case, the results of the selective method are similar to those obtained by clonal analysis. The use of selective medium therefore provides a sensitive means to detect heterozygosity at this locus and may provide a general method to select cells with X-linked markers from heterozygous populations.This work was supported by grants from the U.S.P.H.S. (#HD 00486), The Joseph P. Kennedy, Jr., Memorial Fluid Research Fund, and the National Foundation for Neuromuscular Diseases, Inc.     

Autoactive molluscan neuron: Reflex function and synaptic modulation during feeding in the terrestrial slug,Limax maximus

Summary The salivary burster (SB) is an autoactive motoneuron to the salivary duct of the terrestrial slugLimax maximus. The SB is electrically coupled to protractor motoneurons and inhibited by the metacerebral giant cell. During feeding these synaptic inputs cause SB activity to be phase-locked to the protraction-retraction cycle. The SB can be used as a clear and reliable monitor of feeding motorprogram activation.We thank Dr. Joseph Jin Chang for invaluable assistance with some of this work. Support was provided by NSF grant BMS74-15217 to D.J.P., NSF grant BMS74-03572 to A.G., and a grant from the Spencer Foundation.     

What price medical malpractice insurance?

The Medical Review and Advisory Board has been established as a committee of the Commission on Professional Welfare of the California Medical Association to make studies and recommendations toward solution of the growing problems of professional liability insurance and malpractice actions in California. The members of the Board are: Joseph F. Sadusk, Jr., Oakland, Chairman; Wilbur Bailey, M.D., Los Angeles, vice-chairman; Howard W. Bosworth, M.D., Los Angeles; H. I. Burtness, M.D., Santa Barbara; Paul W. Frame, Jr., M.D., Sacramento; Verne G. Ghormley, M.D., Fresno; Carl M. Hadley, M.D., San Bernardino; Joseph J. O'Hara, M.D., San Diego; William F. Quinn, M.D., Los Angeles; Rees B. Rees, M.D., San Francisco; and Bernard Silber, M.D., Redwood City; Mr. Rollen Waterson, 564 Market Street, San Francisco 4, is executive secretary, and Mr. Howard Hassard is legal counsel.     

The fine structure of photoreceptors in a marine flatworm

Summary The ocelli or eyes of the marine polyclad turbellarian Notoplana acticola are clustered on the paired dorsal nuchal tentacles and in two longitudinal bands lateral to the cerebral ganglion. The ocelli, studied by electron microscopy, were characterized as rhabdomeric and non-ciliary in origin. There are 60 to 80 ocelli per animal each enclosed in a fibrous capsule to which muscle fibers may attach. An ocellus consists of a pigmented eyecup into which 30 to 50 photoreceptor cells send dendritic processes through interruptions in or among pigment cell projections across the eyecup opening. The dendritic processes terminate in numerous long intertwined microvilli which fill the eyecup. The nucleated cell body of each photoreceptor cell lies outside the eyecup and projects an axonal process to the cerebral mass. Within the dendritic processes are observed mitochondria, ribosomes, neurotubules, multivesicular bodies, vesicles and vacuoles. The cell body contains smaller mitochondria, endoplasmic reticulum, ribosomes, vesicles and prominent Golgi complexes.After dark adaptation, there are some structural alterations in terms of swelling of microvilli, increased numbers of vacuoles associated with the microvilli and dendritic processes, and changes in the pigment cell projections.This work was supported by Grant No. GM 10292 from the U.S. Public Health Service to Professor Richard M. Eakin, Department of Zoology at the University of California, Berkeley, U.S.A., where this investigation was conducted during the author's sabbatical leave of absence from the University of Illinois, and by Grant No. 1 SO 1 FR 5369 from the U.S. Public Health Service to the University of Illinois at the Medical Center.I express appreciation to Professor Eakin for interesting discussions and generous hospitality to me as a guest in his laboratory, and to the John Simon Guggenheim Memorial Foundation for the Fellowship which I held during 1964–65. I thank Dr. John P. Marbarger, Director of the Aeromedical Laboratory for the electron microscope facilities used at the University of Illinois.     

On lipid droplets in renal interstitial cells

Summary By examining Epon sections of the renal papilla, it is demonstrated that a brief period of salt repletion in salt-depleted rats induces a considerable increase in the number of lipid droplets in the renal interstitial cells. It is supposed that this difference indicates that the lipid droplets contain substances which are physiologically important for the kidneys. Possibly, these substances are prostaglandins, which have been demonstrated previously by other authors in extracts from the renal medulla.This work was supported by a grant from the Danish State Research Foundation.The author wishes to acknowledge the support and inspiration given by E. Bojesen, M.D., and P. Leyssac, M.D., the Institute of Experimental Medicine.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

A new trematosphaeria from roots of rhizophora racemosa

Summary A new species ofTrematosphaeria Fuck.,T. mangrovis Kohlm. sp. nov., is described. The fungus occurred as perthophyte in roots ofRhizophora racemosa Meyer in West Africa (Liberia) and is classified as a marine species. The taxonomical situation of the Ascomycete is discussed and some ecological considerations are given.Support in part by grant GB 5587 of the National Science Foundation is gratefully acknowledged. Mrs.Erika Kohlmeyer kindly prepared the drawings.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology

Summary This is the last in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxyribonucleate)-3-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article discusses the use of the nuclease as a model system for the study of the mechanisms and energetics of the folding-unfolding reaction in proteins and for the study of the interrelationships between amino acid sequence and three-dimensional structure.This article is the last in a series of four devoted to the staphylococcal nuclease. The previous articles have discussed its isolation and enzymology, the chemical and enzymological studies of its active site and the high-resolution crystallographic work. The third article also put forth a mechanism for the enzymatic reaction based on the nuclease's known chemical and structural properties. Work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, N.I.H. and the Robert A. Welch Foundation to F. A. Cotton and from the Welch Foundation to E. E. Hazen, Jr.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology

Summary This is the third in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxribonucleate)-3-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article describes the structure of the nuclease and of a nuclease-inhibitor complex as determined by x-ray crystallography. The crystal structures are correlated with some of the known chemical and enzymological properties of the enzyme, and the three areas combined to propose a mechanism of action.This article is the third in a series of four devoted to the stapholoccal nuclease. Reviews concerning its isolation and enzymology (1) as well as the features of its ligand binding site (2) have appeared in previous issues. Work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, NIH and from the Robert A. Welch Foundation to F. A. Cotton and E. E. Hazen, Jr.     

A randomized clinical trial of remission induction,consolidation, and chemo-immunotherapy maintenance in adult acute myeloblastic leukemia

Summary The Southeastern Cancer Study Group conducted a randomized clinical trial in acute myeloblastic leukemia and the blastic phase of chronic myelocytic leukemia to compare: Two induction programs (Schedule A) cytosine arabinoside and 6-thioguanine or (Schedule B) cytosine arabinoside, 6-thioguanine and daunorubicin; two consolidation programs (Schedule C) continuation of induction programs at a reduced dose or (Schedule D) a combination of cyclophosphamide, methotrexate and vincristine; and two maintenance programs — (Schedule E) 1 month of BCG, followed by methotrexate or (Schedule F) methotrexate. Over a 3 year period 372 patients were entered and 295 were judged evaluable. None of 11 patients with blastic phase of chronic myelocytic leukemia responded. There were no significant differences between the schedules in the number of patients with acute myeloblastic leukemia achieving complete remissions (37%, Schedule A vs. 41% Schedule B). The relapse rates on consolidation were similar (43%, Schedule C and 39%, Schedule D). BCG significantly prolonged the duration of first remission following consolidation (P<0.05) from 13.0–23.9 weeks. Survival was not significantly prolonged (92.7 weeks vs. 71.7 weeks). There were no serious complications from BCG therapy. Contributors. The following members of the Southeastern Cancer Study Group participated in this study: John T. Carpenter, John R. Durant, Richard Gams, William J. Hammack, George A. Omura, Gayle Roberts, University of Alabama School of Medicine, Birmingham, Alabama; Harold Silberman, Donald S. Miller, Duke University School of Medicine, Durham, North Carolina; William B. Kremer, Durham Veterans Administration Hospital, Durham, North Carolina; Evert A. Bruckner, Lawrence E. Cooper, Charles C. Corley, Joseph E. Hardison, Charles M. Huguley, Jr., James Keller, Mason G. Robertson, John D. Schmale, Charles Vogel, W. R. Vogler, William H. Whaley, E. F. Winton, Emory University School of Medicine, Atlanta, Georgia; Chan Kon Chin, Guy Faguet, Claude-Starr Wright, Medical College of Georgia, Augusta, Georgia; Y. S. Ahn, Howard E. Lessner, University of Miami School of Medicine, Miami, Florida; Dov Gorshein, Scott Murphy, Presbyterian University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; William E. Barry, Sharon P. Fischer, Rosaline R. Joseph, Richard V. Smalley, Temple University School of Medicine, Philadelphia, Pennsylvania; Virgil Loeb, Jr., Cary Presant, Edward Reinhard, Shabbir H. Safdar, Washington University School of Medicine, St. Louis, Missouri; Norman Maldonado, Enrique Velez-Garcia, University of Puerto Rico School of Medicine, San Juan, Puerto Rico; S. A. Gregory, William H. Knospe, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois; Stephen Krauss, University of Tennessee Memorial Research Center, Knoxville, Tennessee; Karl Tornyos, New Orleans Veterans Hospital, New Orleans, Louisiana; W. B. Forman, R. W. Kellermeyer, A. Rassiga, Case Western Reserve University School of Medicine, Cleveland, Ohio; William R. Arrowsmith, George Porter, Donald M. Samples, Ochsner Clinic, New Orleans, Louisiana; Lois W. Dow, Charles L. Neely, University of Tennessee School of Medicine, Memphis, Tennessee; G. O. Broun, Jr., St. Louis University School of Medicine, St. Louis, Missouri     

The subcellular fractionation of the electric organ of Torpedo

Summary The acetylcholine-rich electric organ of Torpedo has been submitted to subcellular fractionation in an attempt to isolate nerve endings and synaptic vesicles derived from cholinergic neurones. Fractions containing small vesicles and granules as their only morphologically identifiable components also contained appreciable amounts of bound acetylcholine; however, it was not possible to demonstrate a specific enrichment of any one fraction with respect to bound acetylcholine as has been possible in brain. The tissue proved difficult to homogenize and few detached nerve endings (synaptosomes) were formed. A low-speed fraction rich in Na, K- activated adenosine triphosphatase contained numerous membrane fragments with tubular appendages derived from the non-innervated surface of electroplaques. Homogenization in media isotonic with elasmobranch plasma (e.g. 0.5 M sucrose + 0.33 M urea) was essential to preserve the structure of osmotically sensitive organelles (e.g. mitochondria).We wish to express our gratitude to Dr. R. D. Keynes who arranged the supply of Torpedos and to Mr G. H. C. Dowe and Miss L. Swales for skilled technical assistance. The electron microscopic facilities were provided by a grant from the Wellcome Trust and the work was supported by a grant no. NB-03928-02 (to V.P.W.) from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. During the period of this work Dr. Sheridan held a Postdoctoral Fellowship of the U.S. Public Health Service and Dr. Israël was an Exchange Scholar of the Medical Research Council.We are also most grateful to Professor Sir Bryan Matthews, C.B.E., Sc. D., F.R.S., for providing aquarium facilities in the Physiological Laboratory of Cambridge University.     

Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

Action ofl-acetylcarnitine on different cerebral mitochondrial populations from hippocampus and striatum during aging

The maximum rates (V_max) of some mitochondrial enzyme activities related to energy transduction (citrate synthase, malate dehydrogenase, NADH cytochrome c reductase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase) were evaluated in non-synaptic (free) and synaptic mitochondria from rat hippocampus and striatum. Three types of mitochondria were isolated from control rats aged 4, 8, 12, 16, 20 and 24 months and treated ones withl-acetylcarnitine (100 mg·kg^–1, i.p., 60 min). Enzyme activities of non-synaptic and synaptic mitochondria are different in hippocampus and striatum., confirming that a different metabolic machinery exists in various types of brain mitochondria. During aging, enzyme activities behave quite similarly in both areas. In vivo administration ofl-acetylcarnitine decreased the enzyme activities related to Krebs' cycle mainly of synaptic mitochondria, suggesting a specific subcellular trigger site of action. The drug increased cytochrome oxidase activity of synaptic and non-synaptic mitochondria, indicating the specificity of molecular interaction with this enzyme.     

Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation

Synapsin I (protein I) is a neuron-specific phosphoprotein, which is a substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases. In two accompanying studies (De Camilli, P., R. Cameron, and P. Greengard, and De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354 and 1355-1373) we have shown, by immunocytochemical techniques at the light microscopic and electron microscopic levels, that synapsin I is present in the majority of, and possibly in all, nerve terminals, where it is primarily associated with synaptic vesicles. In the present study we have prepared a highly purified synaptic vesicle fraction from rat brain by a procedure that involves permeation chromatography on controlled-pore glass as a final purification step. Using immunological methods, synapsin I concentrations were determined in various subcellular fractions obtained in the course of vesicle purification. Synapsin I was found to copurify with synaptic vesicles and to represent approximately 6% of the total protein in the highly purified synaptic vesicle fraction. The copurification of synapsin I with synaptic vesicles was dependent on the use of low ionic strength media throughout the purification. Synapsin I was released into the soluble phase by increased ionic strength at neutral pH, but not by nonionic detergents. The highly purified synaptic vesicle fraction contained a calcium-dependent protein kinase that phosphorylated endogenous synapsin I in its collagenase-sensitive tail region. The phosphorylation of this region appeared to facilitate the dissociation of synapsin I from synaptic vesicles under the experimental conditions used.     

Synaptic relationships in the plexiform layers of carp retina

Summary The synaptic contacts made by carp retinal neurons were studied with electron microscopic techniques. Three kinds of contacts are described: (1) a conventional synapse in which an accumulation of agranular vesicles is found on the presynaptic side along with membrane densification of both pre- and postsynaptic elements; (2) a ribbon synapse in which a presynaptic ribbon surrounded by a halo of agranular vesicles faces two postsynaptic elements; and (3) close apposition of plasma membranes without any vesicle accumulation or membrane densification.In the external plexiform layer, conventional synapses between horizontal cells are described. Horizontal cells possess dense-core vesicles about 1,000 Å in diameter. Membranes of adjacent horizontal cells of the same type (external, intermediate or internal) are found closely apposed over broad regions.In the inner plexiform layer ribbon synapses occur only in bipolar cell terminals. The postsynaptic elements opposite the ribbon may be two amacrine processes or one amacrine process and one ganglion cell dendrite. Amacrine processes make conventional synaptic contacts onto bipolar terminals, other amacrine processes, amacrine cell bodies, ganglion cell dendrites and bodies. Amacrine cells possess dense-core vesicles. Ganglion cells are never presynaptic elements. Serial synapses between amacrine processes and reciprocal synapses between amacrine processes and bipolar terminals are described. The inner plexiform layer contains a large number of myelinated fibers which terminate near the layer of amacrine cells.This work was supported by an N.I.H. grant NB 05404-05 and a Fight for Sight grant G-396 to P.W. and N.I.H. grant NB 05336 to J.E.D. The authors wish to thank Mrs. P. Sheppard and Miss B. Hecker for able technical assistance. P.W. is grateful to Dr. G. K. Smelser, Department of Ophthalmology, Columbia University, for the use of his electron microscope facilities.     

Plastids of sieve tube members in the stamen vascular bundle ofSorghum bicolor (Gramineae)

Summary The plastids in sieve tube members of the stamen vascular bundles ofSorghum bicolor, fixed in glutaraldehyde with postfixation in osmium tetroxide, are of the P-type containing cuneate crystalloids of a proteinaceous nature surrounded by a double envelope. Secondary inclusions are present in these P-type plastids. P-type plastids inSorghum often remain intact in the mature sieve tube members.This work was supported by a grant from the Iowa Agricultural and Home Economics Experiment Station, Ames, U.S.A. Project No. 1740 toHarry T.Horner, Jr., andNels R.Lersten and Project No. 1914 toHarry T.Horner, Jr. of the Department of Botany and Plant Pathology, Iowa State University, Ames, U.S.A. This work was completed by the author as part of the Ph. D. dissertation research.     

Non-competitive antagonists for derivatives of p-aminobenzoic acid

Summary Shive's method of “inhibition analysis” was applied to the growth inhibition ofE. coli by some derivatives of p-aminobenzoic acid. In accordance with the findings ofWinkler andde Haan methionine, xanthine, serine, thymine, and valine were shown to be non-competitive antagonists of sulfanilamide forE. coli strain Bray β. It is highly probable that for this bacterium glycine should be added to this series. Methionine is the only non-competitive antagonist of the 2-chloro, 2-bromo, and 2-amino derivatives of P.A.B., provided their action is investigated at no higher concentrations than can be antagonised by P.A.B. The same complete series of non-competitive antagonists of sulfanilamide was shown to be active against the bacteriostatic action of the 2-methyl, 3-methyl, and 3-amino derivatives of P.A.B. The hypothesis is proposed that these derivatives are not competing directly with P.A.B. for an essential enzyme but that they are used by the bacterium in the formation of derivatives of pteroylglutamic acid; these may be competitive antagonists of co-enzymes, related to pteroylglutamic acid. There is some evidence in favour of this supposition. Further investigations are needed, however, before any judgment regarding its value can be given.     

Juxtanuclear changes during the early spermatogenesis in Lebistes reticulatus (Guppy)

Summary The sperm cell of Lebistes reticulatus during Spermatogenesis is studied. The nuclear differentiation correlate to the flagellar outgrowth. Differential distribution in the chromatin condensation corresponds to a new membrane synthesis at the centriolar side of the nucleus. The fan-shaped microtubular bundle in Juxtanuclear position is described and its function as a stabilizing entity is discussed. At the centriolar complex satellites and an intercentriolar lamellated body are investigated. In submicroscopic details the sperm cell of Lebistes distinctively differs from other species previously described.The present investigation was supported by a grant from the Pehr Oscar Klingendahl Foundation.The authors wish to thank Mr. Mauri Nyholm, M. Sc., for his technical advices and Miss Maria Sjöstedt for preparing the specimens.     

Regulation of aspartokinase activity in the genus Beneckea and marine,luminous bacteria

Summary The pattern of allosteric regulation of aspartokinase activity was determined in species of Beneckea and in the marine, luminous bacteria. The results indicated that these organisms have at least three isofunctional aspartokinases of which the first is inhibited by l-threonine, the second is inhibited by l-lysine, and the third is unaffected by either l-threonine or l-lysine. The homoserine dehydrogenase activity is clearly separable from the l-lysine-sensitive aspartokinase, that may be associated with one (or both) of the other isofunctional aspartokinases.The species of Beneckea and luminous bacteria studied varied in the relative levels of the three isofunctional aspartokinases. B. parahaemolytica, B. alginolytica, B. pelagia, B. neptuna, B. harveyi, B. nigrapulchrituda, B. natriegens, and Photobacterium fischeri had predominant levels of the l-lysine-sensitive activity; B. campellii, P. phosphoreum, and P. mandapamensis had predominant levels of the l-threonine-sensitive activity; while in B. nereida these two activities were approximately equivalent. Species of Beneckea could be distinguished from P. fischeri on the basis of the sensitivity of their aspartokinase activities to inhibition by l-lysine. In P. fischeri 10.3×10^-5 M l-lysine was required for a 50% inhibition of the l-lysine-sensitive enzyme, while in species of Beneckea 0.5–2.7×10^-5 M l-lysine was required to achieve the same level of inhibition.Non-standard abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - GC guanine plus cytosine     

The effects of adenohypophysectomy on the hypothalamic-hypophysial neurosecretory system and the adrenal gland of the toad Bufo arenarum Hensel

Summary The effects of adenohypophysectomy were studied on the hypothalamichypophysial neurosecretory system and the adrenal glands of Bufo arenarum Hensel. An increase in vascularization of the pars intermedia was found and the neurosecretory material (NSM) in the glandular region of the median eminence disappeared. Its reappearance later was accompanied by hypertrophy of the pars intermedia; differentiated chromophil cells appeared in the pars intermedia around the vessels and, at the same time, the adrenal reverted to normal. These findings are interpreted as hypertrophy and differentiation of the pars intermedia with replacement, at least partly, of the functions of the pars distalis. The probable mechanism of differentiation and the functions of chromophil cells of the pars intermedia are discussed.This paper was presented at the VI Reunión de la Asociación Latinoamericana de Ciencias Fisiológicas (A.L.A.C.F.), Viña del Mar, Chile, 1964. It was carried out under the auspices of the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and the Rockefeller Foundation (School grant RF-58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.The authors wish to thank Prof. M. H. Burgos for his constant interest, Prof. B. A. Houssay, Prof. H. Heller and Dr. J. H. Tramezzani for their criticisms, Miss B. Rodriguez and Mr. L. Castro for their technical assistance.