Phenol oxidase activity and the Lozenge locus of Drosophila melanogaster

We have found that the phenol oxidase activity in 50-hr Drosophila melanogaster pupae is much greater than that of adult flies. The mutants lz and lz ^g have all of the phenol oxidase components present in wild type, whereas the mutant tyr-1 has all of the wild-type components but the activity of each component is greatly reduced in comparison with wild-type activity. The newly discovered lozenge allele, lz ^rfg, lacks all phenol oxidase activity.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation.     

The human vocal cord surface

Summary The effect of phenylthiourea (PTU) on the pigment epithelium and the photoreceptor cells in the developing retina of Haplochromis burtoni was studied by electron microscopy. In the retinal pigment epithelium of 6-day old embryos, both types of melanin granule (spindle-shaped and rod-shaped) are already found. PTU inhibits the biosynthesis of melanin but does not influence the formation of premelanosomes so that in PTU-treated embryos there are no melanosomes, but an abundance of premelanosomes. The structure of the premelanosomes is described. It differs completely from that of all other vertebrates. Other changes: an increase in polysomes, retarded development of the inner segment of the photoreceptor cells and enlargement of the intercellular space between the inner and outer leaflet of the retina, may be due to a toxic effect of PTU.This investigation was supported by grants of the Deutsche Forschungsgemeinschaft     

Growth and maturation of melanosomes in the melanophores of a teleost,Oryzias latipes

Summary Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent hollows in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.     

Alexander Hollaender’s Postwar Vision for Biology: Oak Ridge and Beyond

Experimental radiobiology represented a long-standing priority for the U.S. Atomic Energy Commission (AEC), but organizational issues initially impeded the laboratory progress of this government-funded work: who would direct such interdisciplinary investigations and how? And should the AEC support basic research or only mission-oriented projects? Alexander Hollaender’s vision for biology in the post-war world guided AEC initiatives at Oak Ridge, where he created and presided over the Division of Biology for nearly two decades (1947–1966). Hollaender’s scheme, at once entrepreneurial and system-oriented, made good use of the unique resources provided by the AEC and by Oak Ridge’s national laboratory setting, while at the same time it restructured wartime research practices to better reflect biologists’ own priorities. Because Hollaender offered many academic experimental biologists a way of envisioning military-related patronage as integral – rather than antithetical – to their professional identities, his work provides an important lens through which to examine the early post-war intellectual and institutional development of radiobiology.     

Electron microscope observations of radiation-induced Hela giant cells

Summary An electron microscope study of X-ray produced giant Hela cells is described. The results extend earlier light microscope observations to the sub-microscopic region where clear differences from normal structures are apparent. Of particular interest are intra nuclear inclusions, nucleolar fragments, membrane abnormalities and possible mitochondrial changes.This investigation was supported in part by the U. S. Atomic Energy Commission and in part by a predoctoral fellowship CF 8984 from the National Cancer Institute, Public Health Service.     

Evolution of 5S ribosomal RNA genes in the chromosomes of the virilis group of Drosophila

Drosophila melanogaster 5S ribosomal RNA labeled with ¹²⁵I was used as an in situ hybridization probe to localize complementary sequences in chromosomes of species in the Drosophila virilis group. Whereas virilis, the ancestral species, has two different 5S gene loci, the derived species show only one of these loci; in the two lines that have evolved from virilis it is the opposite locus that is conserved. The possible events leading to such an arrangement are discussed.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with ¹²⁵I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Analysis of an east tennessee oak hickory forest by canonical correlation of species and environmental parameters

Summary A new multivariate analytical technique, canonical group correlation (CGC) is developed to correlate abiotic and biotic characterization of communities. This technique is applied to the plant communities of a 97.5 ha oak-hickory watershed. This analysis has validated inferences drawn in earlier studies which used only species data. We have concluded that the dominant factors discriminating the four distinct types of vegetation which exist in the region being studied are age and slope position. Slope position is inferred to be correlated with a moisture gradient. This information is depicted by the location of the four community types in two canonical spaces. One space is determined by vegetational parameters (species composition), the other by environmental parameters. A linear transformation between the two spaces is derived. This transformation can be used to predict successional development.Research supported by the Eastern Deciduous Forest Biome, US-IBP, funded by the National Sciences Foundation under Interagency Agreement AG-199, 40-193-69 with the Atomic Energy Commission, Oak Ridge National Laboratory. This is Contribution No. 291 from the Eastern Deciduous Forest Biome, US-IBP, and Publication No. 1054, Environmental Sciences Division, Oak Ridge National Laboratory.Nomenclature follows Little (1953).     

Electron microscopy of human melanosomes in unstained,fresh air-dried hair bulbs and their examination by electron probe microanalysis

Summary Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.Supported by a grant from the Ministry of Education of JapanThe author thanks Dr. H. Fujita for his advice and Messrs K. Yonehara, S. Okamura, T. Fukuoka, and T. Asai, Naka Works, Hitachi Ltd., Katsuta, Ibaraki, for use of their X-ray microanalyzer and for their assistance in electron probe analysis     

Stereological Studies of the Effects of α-MSH and cAMP on Melanosomes in Melanoma Cells

The effects of α-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing α-MSH or cAMP harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V_Vpm,cell’V_pm) and mature melanosomes (V_Vmm,cell’V_mm) and the number of mature melanosomes (N_mm). Both stimulators also caused declines in the volume of individual mature melanosomes (_Vimm) the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAME The effect cAMP on some parameters occurred 24 hours prior to α-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enchanced melanogenesis by increasing the V_Vpm,cell’V_Vmm,cell’V_pm, V_mm and N_mm. The melanogenic level did not depend on the _Vimm and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAME It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to α-MSH.     

Systems and organismal aspects of phosphorus remineralization

During a six-month study at Lake George, N.Y., zooplankton contributed an average of 19.4% of the phosphate required for algal photosynthesis. Values ranged from 44.1% prior to the unimodal phytoplankton pulse to 4.6% during the phytoplankton bloom. Copepods accounted for a large percentage (21–68%) of the SRP recycled during the growing season examined, whereas, the cladocera provided only a small percentage of remineralization (6.9%).Contribution No. 315 from the Eastern Deciduous Forest Biome, U. .-IBP, Research supported by the Eastern Diciduous Forest Biome U.S.-IBP, funded by the National Science Foundation under interagency agreement AG-199. DEB 76-00761 with the U.S. Department of Energy — Oak Ridge National Laboratory.     

Approximate bandwidths for π and σ energy states in poly (dA.dT)

Using energy levels of the π and σ orbitals for adenine and thymine obtained by the CNDO method, the widths of those levels for poly (dA · dT) are calculated approximately. The results indicate that the bands are very narrow and that the exciton theory provides the best approximation for these biopolymers. This investigation was supported in part by NIH Fellowship (1F03 CA 5296-01) from the National Cancer Institute. Operated by the Union Carbide Corporation for the U.S. Atomic Energy Commission.     

Spermatogonial exchange involving the X but not the y chromosome inDrosophila Melanogaster

Summary The distal uninverted portion of In(1)sc⁸, which carriesy ⁺ andac ⁺, is occasionally lost during spermatogonial divisions. This is accomplished by exchange between the protion of the proximal heterochromatin that has been removed distally by the inversion and some other heterochromatin in the complement (see alsoLindsley 1955b).. The majority of the recombiants recovered from males carrying In(1)sc⁸ arise through exchange with the Y chromosome (12/15). The majority of the recombinants recovered from males carring In(1)sc^8L, EN^R, which is characterized by a heterochromatic second arm, do not arise through exchange with the Y chromosome (18/22). The absolute frequencies of Y involvement with In(1)sc⁸ (7/105067) and In(1)sc^8L, EN^R,(2/38588), however, are comparable. The heterochromatic constitution of the recombinants examined is consistent with the hypothesis that an observed excess of recombinants recoverred from In(1)sc^8L, EN^R as compared with In(1)sc⁸ is accounted for by Y independent recombinants and is the consequence of exchange between the second heterochromatin arm of In(1)EN^R and the distal heterochromatin of In(1)sc^8L. A maximum of six different regions of exchange between these two regions may be inferred from the constitution of the recombinants. This inference is considered to support the hypothesis that pairing and exchange between heterochromatic regions are not strictly homologous.With 6 Figures in the TextOperated by Union Carbide Nuclear Company for the U.S. Atomic Energy Commission.Part of the material was presented to the Graduate School of the California Institute of Technology in partial fulfillment of requirements for the degree of Doctor of Philosophy supported by an Atomic Energy Commission predoctoral fellowship. Further experimentation has been pursued under a National Research Council postdoctoral fellowship at the University and under a National Science Foundation postdoctoral fellowship at the University of Missouri. Experimentation was completed at Oak Ridge.     

Melanosomal Proteins – Role in Melanin Polymerization

Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific.     

Cycling of organic and inorganic sulphur in a chestnut oak forest

Summary Sulfur (S) cycling in a chestnut oak forest on Walker Branch Watershed, Tennessee, was dominated by geochemical processes involving sulfate. Even though available SO ₄ ^2- was present far in excess of forest nutritional requirements, the ecosystem as a whole accumulated 60% of incoming SO₄–S. Most (90%) of this accumulation occurred by SO ₄ ^2- adsorption in sesquioxide-rich subsurface soils, with a relatively minor amount accumulating and cycling as SO ₄ ^2- within vegetative components. Organic sulfates are thought to constitute a large proportion of total S in surface soils, also, and to provide a pool of readily mineralized available S within the ecosystem.Research sponsored by Division of Biomedical and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with Union Carbide Corporation. Soil ester sulfate work sponsored by contract RP-1813-1 with the Electric Power Research Institute. Publication No. 1990, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830     

Isolation and characterization of pteridines from heads of Drosophila melanogaster by a modified thin-layer chromatography procedure

An improved thin-layer chromatography technique is described for the separation of fluorescent compounds found in extracts of heads of Drosophila melanogaster. Eighteen to twenty fluorescent spots are resolved, two of which are xanthurenic acid and 3-hydroxykynurenine, and the remaining spots are presumably pteridines. Of these, nine have been identified and quantitated directly on the chromatograms with a fluorometer. One of the spots present on the chromatogram apparently has not been described previous to this work. Characteristics of this substance, termed quench spot, are presented, several of which indicate that it may be a pteridine or pteridine derivative.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Nuclear Energy in the Service of Biomedicine: The U.S. Atomic Energy Commission’s Radioisotope Program, 1946–1950

The widespread adoption of radioisotopes as tools in biomedical research and therapy became one of the major consequences of the “physicists’ war” for postwar life science. Scientists in the Manhattan Project, as part of their efforts to advocate for civilian uses of atomic energy after the war, proposed using infrastructure from the wartime bomb project to develop a government-run radioisotope distribution program. After the Atomic Energy Bill was passed and before the Atomic Energy Commission (AEC) was formally established, the Manhattan Project began shipping isotopes from Oak Ridge. Scientists and physicians put these reactor-produced isotopes to many of the same uses that had been pioneered with cyclotron-generated radioisotopes in the 1930s and early 1940s. The majority of early AEC shipments were radioiodine and radiophosphorus, employed to evaluate thyroid function, diagnose medical disorders, and irradiate tumors. Both researchers and politicians lauded radioisotopes publicly for their potential in curing diseases, particularly cancer. However, isotopes proved less successful than anticipated in treating cancer and more successful in medical diagnostics. On the research side, reactor-generated radioisotopes equipped biologists with new tools to trace molecular transformations from metabolic pathways to ecosystems. The U.S. government’s production and promotion of isotopes stimulated their consumption by scientists and physicians (both domestic and abroad), such that in the postwar period isotopes became routine elements of laboratory and clinical use. In the early postwar years, radioisotopes signified the government’s commitment to harness the␣atom for peace, particularly through contributions to biology, medicine, and agriculture.     

Melanosomal proteins--role in melanin polymerization

Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific.     

Molecular and Biological Control of Melanogenesis Through Tyrosinase Genes and Intrinsic and Extrinsic Regulatory Factors

Non-premelanosomal melanogenic compartments and their melanogenesis-controlling functions have been further elucidated. In addition to enzymatic and nonenzymatic controlling factors, we have also been exploring the role of melanogenesis-related genes. Naturally occurring intrinsic melanogenic inhibitors, MW <6,000(α), 6,000-30,000(β), and >30,000(γ), having different modes of action, have been identified within melanoma cells. One of the α-type melanogenic inhibitors of isolated tyrosinase(Ty) nonsuppressive types, later identified as lactic acid, induces depigmentation of cultured B-16 cells by the reduction in Ty activity level due to the inhibition of its mRNA expression. The transfection of Ty cDNA, rather than nuclear DNA-binding master regulatory gene, can induce, within both Ty-deficient amelanotic melanoma cells and also within fibroblasts, melanin polymer formation. This multisequential step occurs not only by the induction of Ty synthesis but also by the induction of other regulatory proteins and factors such as dopachrome tautomerase, DHICA-oxidase, catalase, Ty-glycosylation in GERL, and Ty-transfer by coated vesicles to newly assigned melanogenic vacuoles in which not only eumelanin but also rather pronounced concomitant pheomelanin formation is seen. Investigation of melanin-producing vacuoles in transfected fibroblasts and reexamination of premelanosomes in pigment cells has revealed the following: 1) Melanosomes possess phagocytic ability; 2) melanosomes receive tyrosinase and hydrolases via coated vesicles from GERL; 3) melanosomes possess lysosome-associated membrane protein 1 (LAMP-1); 4) amelanotic melanoma contains lysosome-like vacuoles with myelin figures that acquire typical premelanosome structure after Ty-cDNA transfection. Thus it is proposed that melanosomes are specialized lysosomes in pigment cells. Coated vesicles synthesize melanin monomers such as DHICA and some DHI, and have a monomer-stabilizing system. Thus they can transport them in intact form with Ty to premelanosomes, which subsequently polymerize these monomers by the action of DHICA-oxidase and T₃-Ty. Selective eradication and diagnosis of malignant melanoma using our ¹⁰B-dopa analogue has been successfully performed in human melanoma patients using combined thermal neutron irradiation for the former and positron emission CT for the latter.