RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na⁺-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-³H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10^-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells.     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

Regression of hypoxic hypertension-induced changes in the elastic laminae of rat pulmonary arteries

Liu, S. Q. Regression of hypoxic hypertension-inducedchanges in the elastic laminae of rat pulmonary arteries.J. Appl. Physiol. 82(5):1677-1684, 1997.The elastic laminae of the pulmonary arteries(PAs) undergo a progressive structural change in hypoxic hypertension.This study focused on the reversibility of altered PA elastic laminaeof the rat due to hypoxic hypertension. The structure andcross-sectional area of the PA medial elastic laminae were examined byusing electron-microscopic and image-analytic approaches duringrecovery from 12 h and 10 days of hypoxic hypertension. At 12 h ofhypoxic hypertension, the elastic laminae, which appeared homogeneousin normal control animals, were reorganized into structures composed ofrandomly oriented filaments, with an increase in the cross-sectionalarea of 70%. At 10 days of hypoxic hypertension, the elastic laminaeappeared homogeneous in structure and normal in cross-sectional areadespite continuous exposure to hypoxia. During recovery from 12 h ofhypoxic hypertension, the medial elastic laminae regained theirhomogeneous structure and normal cross-sectional area afterday 2. During recovery from 10 days ofhypoxic hypertension, the medial elastic laminae changed from homogeneous to filamentous structures, with a progressively altered cross-sectional area that increased by 89% from recoveryday 0 to day10 and returned to the normal level onday 30. These changes were associatedwith alterations in the PA wall tensile stress. These results indicatedthat structural changes in the PA elastic laminae were reversible andthat the regression process depended on the duration of exposure tohypoxia, the state of the elastic laminae, and possibly the tensilestress level in the PA wall.

     

Electron microscopic radioautography of intramitochondrial RNA synthesis of HeLa cells in culture

Summary HeLa cells were cultivated in vitro and incubated in a medium containing ³H-uridine (20 c/ml) for 30 minutes 1, 2, 4, 8 and 18 hours, doubly fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, embedded in Epon or hydroxypropyl methacrylate, radioautographed with Sakura NR-H2 emulsion, exposed for 40 days, developed with gold-latensification and elon-ascorbic acid developer.As the results, silver grains appeared not only in the chromatin, nucleoli, endoplasmic reticulum and ribosomes but also in the mitochondria. Most of the silver grains found in the mitochondria were localized on the mitochondrial matrix, while some others were on the cristae and the mitochondrial membranes. These silver grains were removed with RNase digestion. The percentage of labeled mitochondria increased in accordance with the time of incubation. Almost all the mitochondria were labeled within 18 hours.From the results, it was concluded that the mitochondria synthesized RNA at their matrices.     

On the axonal migration of catecholamines in constricted sciatic nerve of the rat

Summary The radioautographic technique has been used to study the axonal migration of catecholamines in sympathetic fibres of the sciatic nerve of rats after ligature. A first series of experiments aimed at ascertaining the capacity of the proximal portion of adrenergic fibres to take up and store exogenous tritiated catecholamines (³H-DOPA; ³H-DA and ³H-NA) 3 to 22 hours after ligation. The results are qualitatively similar in rats pretreated and non-pretreated with IMAO, but the intensity of the radioautographic reaction is lower in the latter cases. Most of the labeled axons are filled mainly with vesicular and tubular profiles of endoplasmic reticular origin, large dense bodies (probably lysosomes) and/or mitochondria. The silver grains are generally superimposed on the vesicular and/or the tubular profiles, but in some cases on the large dense bodies, suggesting that exogenous catecholamines can be stored in lysosomes. The question whether SGV specifically store catecholamines also in the modified adrenergic fibres has been investigated in KMnO₄ fixed material. These results show that beside a large number of fibres in which there is a strict correlation between labeling and SGV, some fibres containing SGV do not retain the ³H-NA. Conversely some fibres which contain mainly agranular vesicles display radioautographic reaction. Therefore, in case of ligated fibres, SGV cannot be considered the specific organelles for storage of catecholamines.The axonal migration of labeled catecholamines has been studied in animals pretreated with IMAO. A moderate, but selective, labeling is present in the proximal portion of sciatic fibres of rats in which administration of labeled catecholamine preceeded of 2 hours the ligature and this was performed 22 hours before fixation.From these combined types of experiments, it is concluded that despite the presence of all the structures necessary for the storage of a high amount of catecholamines in the modified adrenergic fibres, only a small fraction of catecholamines accumulated above the ligature has been transported by axonal migration. Therefore, the axonal migration of catecholamines appears as an epiphenomenon related to the distal migration of enzymatic and storage proteins from the perikaryon.Part of this work was presented to the 84th Annual Session of American Association of Anatomists (Sotelo and Taxi, 1971).Some of the electron microscopic observations were made at the Unité 106 I.N.S.E.R.M.     

Protein metabolism in an astroglial primary culture

Protein metabolism was studied in astroglial primary cultures, grown for different time periods. Removal of fetal calf-serum for two days led to a morphological differentiation consisting of retraction of cell soma and extension of processes. There was a prominent decrease in total soluble protein and a decrease in [³H]valine incorporation into soluble protein. Dibutyrylcyclic-3-5-adenosine monophosphate (dB-cAMP)-treatment for two days also changed morphology in a similar way, but had no effect on [³H]valine incorporation into protein. After addition of soluble brain extract to the cultures an increased [³H]valine incorporation into soluble protein was seen together with a morphological differentiation, more pronounced in the presence than in the absence of fetal calf-serum. Proteins were secreted from the cells into the incubation medium and studied by electrophoresis. The more prominent protein bands had m.w. in the region of 10,000–100,000 daltons. The amount of newly synthesized proteins released into the medium was unchanged (or decreased slightly in 14 and 16 day old cultures) after addition of dB-cAMP or soluble brain extract, and was much reduced after removal of fetal calf-serum.     

Growth regulation of Eurasian watermilfoil with flurprimidol

Studies were conducted in 55-liter aquariums under controlled environment conditions to evaluate growth regulator effects of flurprimidol [-(1-methylethyl)--[4-(trifluoromethoxy) phenyl]-5-pyrimidinemethanol] on Eurasian watermilfoil (Myriophyllum spicatum L.). Treatments included flurprimidol concentrations ranging from 0 to 500 g L^-1, with exposure times varying from 0.25 to 28 days. Extending the flurprimidol contact time increased the growth inhibitory response. Flurprimidol-treated shoots were 14–64% shorter than untreated plants at 14 DAT (days after treatment). Growth inhibition persisted 56 DAT for plants exposed to 25 and 100 g L^-1 flurprimidol for 28 days or 200 g L^-1 flurprimidol for 10 days. Growth-inhibited plants accumulated starch in shoots and roots, whereas plants showing little or no growth suppression utilized available assimilate for growth. Treatments that most effectively suppressed shoot length accumulated up to 68% more total nonstructural carbohydrate compared with untreated plants. Shoot and root dry weight biomass were unaffected by flurprimidol.Abbreviations PGR(s) plant growth regulator(s) - TNC total non-structural carbohydrate - DAT days after treatment - PVC polyvinyl chloride - DW dry weight - BOD biological oxygen demand - DMSO dimethyl sulfoxide - LSD least significant difference     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Auflichtphotometrische Untersuchungen zur Grösse der Koinzidenz in der Autoradiographie mit Tritium

Zusammenfassung Auf Grund abgestufter Lichtschwärzungen an Strippingfilm-Präparaten wird ermittelt, daß bis zu einer Zahl von rund 70 Silberkörnern pro 10 ²-Fläche eine quantitative auflichtphotometrische Silberkornzählung möglich ist. An den Basalzellen des Vormagens der Maus wird nach ³H-Thymidin-Gabe das Verhältnis von vorhandener und zu erwartender Silberkornzahl in Abhängigkeit von der Expositionszeit ermittelt. Daraus ergibt sich der Koinzidenz-Koeffizient in Abhängigkeit von der Silberkorndichte. Es wird darauf hingewiesen, daß bei größeren Silberkorndichten und bestimmten quantitativ-autoradiographischen Fragestellungen die Koinzidenz der -Teilchen zu berücksichtigen ist.

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Summary Based on tests with graduated light densities on stripping film preparations it is shown that up to a number of approximately 70 silver grains/10 ² a quantitative determination of the silver grains is possible by incident light microscopy. The proportion of the number of silver grains present to the number of silver grains to be expected in relation to the exposure time is determined on the basal cells of the forestomach in mice after the administration of ³H-thymidine. The result is a coincidence-coefficient which depends on the silver grain density. It is pointed out that in cases of a high silver grain density and in certain quantitative autoradiographic problems the coincidence of -corpuscles has to be taken into consideration.

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Studie im Rahmen der Assoziation Hämatologie Euratom-GSF.

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Ich danke Herrn Prof. Dr. F. Wachsmann, Institut für Strahlenschutz der Gesellschaft für Strahlenforschung m.b.H., für Diskussion und Ratschläge während des Entstehens der Arbeit, sowie Herrn Prof. Dr. W. Oehlert, Pathologisches Institut der Universität Freiburg i. Br., für die Durchsicht des Manuskriptes.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Alterations in structure of elastic laminae of rat pulmonary arteries in hypoxic hypertension

Liu, S. Q. Alterations in structure of elastic laminaeof rat pulmonary arteries in hypoxic hypertension. J. Appl. Physiol. 81(5): 2147-2155, 1996.The effectof hypoxic hypertension on the remodeling process of the elasticlaminae of the rat hilar pulmonary arteries (PAs) was studied byelectron microscopy. Rats were exposed to hypoxia (10%O₂) for periods of 0.5, 2, 6, 12, 48, 96, 144, and 240 h. Changes in the structure of the PA elastic laminae were examined and analyzed with respect to changes in the PAwall tensile stress. The PA blood pressure increased rapidly within thefirst several hours of hypoxia and reached a stable level within 2 days, whereas the PA wall tensile stress increased initially due toelevated blood pressure and then decreased after 48 h due to vesselwall thickening and returned to the control level after 4 days. Inassociation with these changes, the elastic laminae, which appearedhomogeneous in normal control rats, changed into structures composed ofrandomly oriented filaments and edematous contents with an increase inthe volume during the early period of hypoxia and regained theirhomogeneous appearance and normal volume after 4 days. The changes inthe elastic laminae were correlated with changes in the tensile stress.These changes were associated with a transient decrease in thestiffness of the PAs. In hypoxic rats given nifedipine, no change wasfound in the blood pressure, the tensile stress, or the structure ofthe elastic laminae of the PAs despite continuous exposure to hypoxia.These results suggested that altered tensile stress in the PA wallplayed a critical role in the initiation and regulation of structuralchanges in the elastic laminae and that these changes might contribute to alterations in the mechanical properties of the PA in hypoxic hypertension.

     

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen

Summary The pharmacokinetics of ¹¹¹In-labeled 260F9, a murine monoclonal antibody directed against a breast-cancer-associated antigen, was determined in seven patients with advanced breast cancer. Six patients were administered 1 mg antibody containing 1 mCi ¹¹¹In. The seventh patient was administered 20 mg unlabeled antibody followed by 1 mg ¹¹¹In-labeled antibody all via a peripheral vein. Immunoprecipitation, HPLC and SDS-PAGE gels demonstrated the stability of radiolabel on the antibody. The serum clearance of the radiolabel closely fits (r ²>0.95) a two-compartment model for the first six patients. The apparent volume of distribution of the radiolabel approximated to the plasma volume (3 1) and its mean residence time was 23.7 h. The radiolabel had an average t _1/2 of 22.9±12.21 h at the 1-mg dose. At the 20-mg dose one-compartment elimination kinetics were observed with the radiolabel and antibody showing similar mean residence times (36–41 h) and a t _1/2 of 26–28 h. Whole-body imaging showed that the blood-pool:liver ratio of radioactivity increased fourfold (at 48 h postinfusion) at the higher dose and the percentage of the injected dose of radioactivity in the liver decreased from 25% to 8% (24 h postinfusion).In one patient 7–14 times more radioactivity was localized in a breast tumor than in fat (normal breast). Over the first 25 h an average (cumulative) 7.5% of the total dose was excreted in urine. A study of 260F9 in CDF-1 mice demonstrated that the radiolabel remained associated with the antibody in serum. The antibody, however, cleared 60-fold slower in mice than in patients and showed an increased mean residence time of 191 h. The disparity in the pharmacokinetics of the antibody seen in the mouse and in the clinic, points to the different behavior shown by murine monoclonal antibodies in humans. This points to the need for preliminary studies of antibodies in patients for preclinical evaluations of their effectiveness as drug-targeting agents.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

 We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. Accepted: 28 August 1996     

SITES OF GLYCOGEN SYNTHESIS IN RAT LIVER CELLS AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY AFTER ADMINISTRATION OF GLUCOSE-H3

Glycogen synthesis was investigated by giving tritium (H³)-labeled glucose with carrier to fasted rats in vivo or incubating liver slices from fasted rats in vitro using a glucose-H³-containing medium. After 15 min or 1 hr, pieces of liver were fixed and radioautographed for light and electron microscopy. In vivo and in vitro, radioautographic reactions appeared over "glycogen areas" and over zones transitional between these areas and ergastoplasm. Treatment of sections by alpha amylase removed all but about 5% of the radioactivity, so that about 95% of it consisted of glycogen (synthesized during the 15 min or 1 hr elapsing after administration of glucose-H³). Within glycogen areas and transitional zones, most silver grains were over or very close to glycogen granules and smooth (or partly smooth) vesicles. Presumably, much of the label was added onto growing glycogen granules, in accord with the biochemical view that glycogen may serve as substrate for further glycogen synthesis. The few silver grains located far from glycogen granules—15% at the 15 min interval in vivo—approximated smooth (or partly smooth) vesicles of endoplasmic reticulum. This observation raised the possibility that smooth membranes play a role in glucose uptake at an early stage in de novo formation of glycogen granules.     

Quantitative radioautographic study of intracellular localization of calmodulin antagonist,W-7, in Chinese-hamster ovary cells

Summary The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 M), 2 (33.4 M), 5 (83.5 M) and 10 Ci/ml (167 M) tritiated W-7. Some cells were preincubated in 10, 50 and 100 M unlabelled W-7 for 30 min and then labelled with 2 or 5 Ci/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 m-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

Mepiquat chloride seed treatment and germination temperature effects on cotton growth, nutrient partitioning, and water use efficiency

Cotton seed (Gossypium hirsutum L. cv. Stoneville 825), treated with 0, 0.2, 1.0, and 2.0 g active ingredient (a.i.) mepiquat chloride (MC) kg^–1, was evaluated for the effect of MC on early plant growth. Emergence rate and total emergence of MC-treated seed and control were similar regardless of germination temperature. However, the number of leaves and squares and the dry weight of leaves, stems, and roots for hydroponically grown cotton plants were significantly lower at lower germination temperatures (15°C for 3 day/30°C for 1 day and 15°C for 4 days) than at higher germination temperatures (30°C for 4 days and 30°C for 3 days/15°C for 1 day). All MC treatments significantly decreased the number of nodes, leaves, and squares, as well as dry weight of leaves, stems, and roots, as compared to control plants at 28 days after emergence. MC seed treatments also significantly reduced plant height and total leaf area compared to controls. Water-use efficiency (WUE) was significantly lower for the 1.0 g a.i. MC treatment than for control plants. In general, the highest rate of MC seed treatment resulted in greater concentrations of calcium, phosphorus, and nitrogen in plant leaves and stems and also in greater concentrations of magnesium, phosphorus, and nitrogen in roots than in controls.