Microanalytical Approach Research Articles

N-Methyl-d-aspartate Receptor Stimulation Activates Tyrosinase and Promotes Melanin Synthesis in the Ink Gland of the Cuttlefish Sepia officinalis through the Nitric Oxide/cGMP Signal Transduction Pathway: A NOVEL POSSIBLE ROLE FOR GLUTAMATE AS PHYSIOLOGIC ACTIVATOR OF MELANOGENESIS

The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway.

The Institutions and Governance of Economic Development and Reform

Why are the ambitions of economic development practitioners and reformers so often disappointed? One answer is that development policymakers and reformers are congenital optimists. Another answer is that good plans are regularly defeated by those who occupy strategic positions. An intermediate answer is that institutions are important, yet are persistently neglected in the planning process. The article takes a bottom-up, microanalytical approach to economic development and reform. It examines the governance of contract, investment, and private ordering through the lens of transaction-cost economizing. And it assesses the efficacy of the de facto (as against the de jure) institutional environment with respect to credible commitments. In effect, institutional economists are cast in the role of archaeologists of economic reform and development - with the task of unpacking the lessons of the past to inform choices and programs for the future.

Intraepidermal distribution of free amino acids in porcine skin

The study describes the vertical distribution of free amino acids in the porcine epidermis as compared to the human integument, using a micro-determination TLC method based on the reaction of amino acids with dansyl chloride. This microanalytical approach demonstrated 22 free amino acids, with the relatively largest amounts being present for acidic amino acids and their amides. It was obvious that the relative amounts of certain amino acids (alanine, proline, valine, glutamine, histidine, glycine, threonine) decreased, whereas acidic amino acids (glutamic acid, aspartic acid) increased from the stratum basale up to the stratum corneum. This distributional pattern could be verified for the dorsal and lateral body regions of the pig breeds used, and for man. The results obtained are discussed in view of the development of epidermal keratinization.

Characterization of aggregating proteoglycans from the proliferative, maturing, hypertrophic, and calcifying zones of the cartilaginous physis.

The introduction of a new microanalytical approach has made it possible to investigate the molecular structure and content of proteoglycans within the extracellular matrix of the physis when the volumes of the matrix and the cells change before mineralization of the extracellular matrix. By using extracts of serial frozen sections with a combination of gel chromatography and chemical, radioimmunoassay, and morphometric analyses, we demonstrated that, in the bovine fetal physis, the content of large aggregating proteoglycans reaches a maximum in the extracellular matrix of the hypertrophic zone, in the region where calcification begins, at the time when calcification starts. Morphometric analyses revealed that, at this time, the volume of matrix is reduced to a minimum. There was no evidence that the remaining proteoglycans were reduced in size, nor was aggregation changed at the time when calcification was initiated. Thus, contrary to earlier reports, there is no indication that these molecules need to be degraded and removed for calcification to begin, although a progressive loss of proteoglycans clearly occurs as the volume of the matrix is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

A microanalytical approach to plant tissue characterization: A comparative study of healthy and fungus-infected carnation by pyrolysis-mass spectrometry

Minute samples of xylem from healthy and Fusarium oxysporum f.sp. dianthi-infected stems of carnation cultivars differing in degree of resistance to F. oxysporum were investigated by in-source pyrolysis low voltage EI mass spectrometry and by pyrolysis under ammonia CI conditions. Healthy xylem of both cultivars showed PY-MS spectra with markers for cellulose, hemicelluloses, phenolic acids, and a mixed guaiacyl-syringyl lignin. At different stages of infection visibly infected, brown coloured, xylem was separated under the microscope from healthy-looking parts. The PY-MS spectra of the diseased xylem of susceptible ‘Lena’ were entirely different from those of healthy(-looking) areas, showing a drastic change in lignin pyrolysis product distributions. In most cases practically all syringyl constituents, both mono- and dimeric, had disappeared. In some cases also an overall decrease in relative intensity of the lignin pyrolysis products was observed. Invasion of the fungus thus induced a major change of cell wall composition as a result of lignin degradation along different pathways: demethoxylation of syringyl units, the major pathway in ‘Lena’, and propenyl side chain oxidation. The PY-MS spectra of infected ‘Novada’ generally showed the overall oxidation of lignin and less demethoxylation. The ‘Novada’ spectrum also showed m/z 239, 287, and an increase of 257 and 271, which are molecular ions indicative of the phytoalexins dianthalexin and the dianthramides methoxydianthramide S, hydroxydianthramide B and methoxydianthramide B. The compounds appeared comparatively volatile and therefore could be desorbed as a thermal extract before other pyrolysis fragments appear. The compounds were practically absent in adjacent, healthy looking, xylem. Low voltage EI MS of methanol extracts of about 2 mm 3 of infected ‘Novada’ xylem confirmed these data and in a single case also showed evidence for hydroxyanthranilic acid ( m/z 153) which is attributed to degradation of dianthramine. Spectra of thermal and methanolic extracts were quite similar.

Coupled transport in cultured heart cells: A microanalytical approach

Coupled transport in cultured heart cells: A microanalytical approach

Peptidergic pathways in the central nervous system.

Before detailed studies of the physiology and pharmacology of central peptidergic neurons can be undertaken, the location of these neurons must be determined and the mechanism(s) by which they synthesize their peptide products must be explored. In the previous paper, Dr Hökfelt described his elegant immunohistochemical studies, which are designed to answer the questions: Where are peptidergic perikarya?; Where do these perikarya send their processes?; Do these processes branch extensively and innervate several structures?; and Do peptidergic cells contain more than one active product? By studying the effects of lesions on peptide levels in microdissected tissue samples, immunocytochemical data can be confirmed and extended. The microanalytical approach also allows one to determine the nature of the immunologically active substances in a tissue extract, and in vivo or in vitro pulse--chase studies provide the ultimate validation of immunohistological localization of peptidergic perikarya and new information about the biosynthesis of peptides and regulation of this biosynthetic process. Our recent studies of central proopiocortin- and neurophysin/vasopressin-producing neurons will illustrate the above points.

Physicochemical characterization of lead in urban dusts. A microanalytical approach to lead tracing

Scanning electron microscopy associated with energy dispersive x-ray analysis is used to identify sources of lead-containing particles in urban dust. Identities of individual particles are established on the basis of characteristic particle morphology and chemical composition. The combined use of individual particle analysis and determination of lead in physically fractionated dust samples is used to quantify contributions of specific sources. Automobile exhaust particles are the main contributors of lead to roadway dusts and also contribute substantially to dusts collected near buildings that have lead-painted trim and are situated at some distance from a roadway. The effectiveness of the microscopic approach to lead tracing is assessed. (1 diagram, 12 photos, 27 references, 3 tables)

The Theory and Practice of Innovation in the Private Insurance Industry: Comment

Murray's article on innovation in the private insurance industry is interesting and provocative.1 One interesting aspect of the article is the combination, for analytical purposes, of the life and non-life insurance industries. Such a macro-approach is a good approach for presenting broad outlines, but this telescopic view did not draw attention to the important details that a micro-analytical approach might have provided. One purpose of this comment, therefore, is to provide some microscopic detail about the innovative practices of the life insurance industry. Specifically I wish to focus attention on a section of Murray's article entitled Inhibitors of Innovation in Insurance. Murray emphasizes the retarding aspects of regulatory activities. In addition to this inhibitor, he also mentions distrust as another factor which inhibits innovation in insurance. Neither of these problems, in this writer's opinion, is nearly as serious as the problem of consumer ignorance about life insurance including the rights and duties created by life insurance contracts. (I suspect this observation holds true in non-life insurance as well.) The problem with consumer ignorance is well recognized in economic literature. In essence, the consumer who is uninformed about insurance (uninformed is perhaps a more polite and less inflamatory designation than ignorant) cannot distinguish between an improved product and one which is changed but unimproved. Given the generally accepted notion that the typical consumer of life insurance is uninformed about the product/ service being purchased, an insurer (assuming little or no social responsibility motivation) has little or no incentive to produce an improved product (from the consumer's standpoint) since it likely would not be distinguished from an unimproved product with a new name, or even from the unimproved product of a competitor. Because of the serious economic problems caused by consumer ignorance of life insurance, problems which go far beyond the area of product innovation, this author has long felt that a genuine consumer education program would be one of the most essential new innovations the life insurance industry could produce.

Electron probe analysis of cryosections of epiphyseal cartilage

Conventional methods for preparing specimens for analysis of the chemical composition of mineralizing tissues at the electron microscopic level can produce artefacts during fixation, dehydration, embedding and wet-sectioning. Cryoultramicrotomy provides a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Two procedures are described (a) for a micro-analytical approach and (b) for a morphological study. By using a negative staining procedure (b) cytoplasmic details in terms of subcellular organelles and extracellular, membrane-bound, matrix vesicles could easily be identified in relatively large sections of epiphyseal cartilage. Unfixed, dry, unstained cryosections obtained by procedure (a) showed dense mineral granules (50–120 nm in diameter) in the mitochondria of chondrocytes and electron probe analysis of these gave a mean Ca/P mass ratio of 1.20. Adjacent cytoplasmic areas showed negligible levels of calcium. In the longitudinal septa matrix, vesicle-like particles could be identified with characteristic crystal needle clusters. Microanalysis of these structures showed them to have a mean Ca/P mass ratio of 2.15. Non-vesicular matrix appeared to be very low in calcium and phosphorus. The combination of cryoultramicrotomy and electron probe analysis of different areas in the same cryosection has provided some information on the coexistence of amorphous calcium phosphate in the mitochondria and of crystalline apatite in matrix vesicles a few micrometers away. These findings may thus explain the sequence of hydroxyapatite formation in epiphyseal cartilage.