Chlorophyll interference with phytochrome measurement

Measurements of phytochrome by (A_{725–815 nm}) were completely suppressed at chlorophyll concentrations of the order of 20–40 g g^-1 f.wt. in vivo and 37 g cm^-3 in vitro, and the readings were reduced by 50% at only 12 g cm^-3 in vitro. At these concentrations of chlorophyll in aqueous methanol, the loss of phytochrome signal in vitro appeared to be due to failure of phytochrome photoconversion rather than to interference with A measuremebt by chlorophyll fluorescence in the 125/815 nm measuring beam.Abbreviations Chl chlorophyll - P phytochrome - P_r and P_fr phytochrome in red absorbing and far-red absorbing forms     

Cryptomonad biliprotein: Phycocyanin-645 from a Chroomonas species

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution.Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm.The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below.Phycocyanin-645 is heterogenous in charge as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment components differing in mobility were found. We propose the term multiple pigment forms for these five phycocyanin-645 modifications.Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (₁, ₂), having molecular weights of 9200 and 10400, respectively, and one heavy chain (), having a molecular weight of 15500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is _{1 1}-_{2 1}.Abbreviations PC-645 phycocyanin-645 - C-PC C-phycocyanin - SDS sodium dodecyl sulphate - pI isoelectric point - mol. wt. molecular weight     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Vergleichende fluoreszenzmikroskopische und elektronenmikroskopische Untersuchungen am zentralen Nervensystem von Planorbarius corneus L. (Basommatophora)

Zusammenfassung Durch Vergleich fluoreszenz- und elektronenmikroskopischer Untersuchungen am zentralen Nervensystem von Planorbarius corneus L. wird nachgewiesen, daß in den Schlundringganglien Neurosekretzellen vorkommen (Nachweis mit Pseudoisocyaninchlorid), die mit Nervenzellen nicht identisch sind, die durch ihren hohen Gehalt an biogenen Aminen auffallen (Nachweis durch die Methode von Falck und Owman, 1965). Es können daher im Schlundring von Planorbarius corneus peptiderge und aminerge Neurosekretzellen unterschieden werden. Die PSC-positiven Neurosekretzellen enthalten elektronendichte Elementargrana und die aminergen Neurone dense-core Vesikel. Der Nachweis biogener Amine in einigen Nervenzellen von Planorbarius corneus spicht für deren chemische Identität mit Transmittersubstanzen, ihre hohe Konzentration aber für eine Abgabe in die Körperflüssigkeit.

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Comparative fluorescence- and electron microscopic studies of the central nervous system of Planorbarius corneus L. (Basommatophora)

Summary The neurosecretory system of the snail Planorbarius corneus has been investigated by fluorescence and electron microscopy. With the fluorochrome Pseudoisocyanin the established neurosecretory system in the cerebral ganglia and single neurosecretory cells in the other ganglia show an intensive yellow fluorescence. Electron micrographs reveal the presence of electron dense granules (elementary granules) in the pericarya and the axons of neurones which have the same localisation in the ganglia as the pseudoisocyanin-positive cells. The fluorescence technique for biogenic amines produces yellow and green fluorescence within neurons and in the neuropil and nerves. The fluorescence obtained in determinable areas and neurones correlates well with the electron microscopic location of dense-core vesicles within the pericarya and axons of cells in even the same areas. It is discussed, that in this animal both types of cells are so-called neurosecretory cells, because the high content of elementary granules in the peptidergic neurosecretory cells and of dense-core vesicles in the aminergic neurosecretory cells is an indication for secretion of these products in neurohaemal areas (circulatory channels).

     

Potential-sensitive response mechanism of diS-C3-(5) in biological membranes

Summary The potential-sensitive response mechanism of 3,3-dipropylthiodicarbocyanine iodide (diS-C₃-(5)) was examined based on our previous model of diS-C₃-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (<10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986.J. Membrane Biol. 90:163–175). Transmembrane potentials () were induced in BBMV by K⁺ gradients and valinomycin. Steady-state diS-C₃-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with . The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C₃-(5) with BBMV at varying . The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing (–17 to 87 mV); the time constant for the faster exponential (30–35 msec) was independent of . There were single exponential kinetics (0.5–1.5 sec) for diS-C₃-(5) fluorescence response to a rapid (<2 msec) change in in the absence of a diS-C₃-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C₃-(5) response, where induced membrane potentials drive diS-C₃-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C₃-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C₃-(5) rotational characteristics or in the polarity of the diS-C₃-(5) environment with changes in . Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C₃-(5) fluorescence which accompany changes in at arbitrary dye/lipid ratios.     

The general occurrence of “plastid initials” and their development into plastids in cortical cells of woody plants in winter

Summary Electron microscopic studies revealed that plastid initials, presumed precursors of plastids, occur in cortical cells of the following plants studied in February and March: Betula ermanii Cham.; Prunus sargentii Rhed.; Pyrus communis L.; Ribes sinanense F. Maekawa; Salix matsudana Koidz. forma tortuosa Rhed.; and Sambucus sieboldiana var. miquelii Hara. Since plastid initials were found previously in Malus pumila Mill., Morus bombycis Koidz. and Populus euramericana cv. gelrica (Sagisaka 1991), plastid initials have been found in all woody plants examined to date. In P. euramericana cv. gelrica, at later stages of the development of the initials in March, the conglomerates of plastid initials became heterogeneous in terms of size, extent of thylakoid formation and ability to form starch granules. The formation of prolamellar structures was frequently observed in cells of Magnolia kobus var. borealis Sarg., which was sampled on April 19. These observations suggest the course of events in the development of the plastid initial and the continuity of the life of amyloplasts over a year in the life of woody plants.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Sequence of β<Subscript>2</Subscript>-microglobulin from rhesus macaque (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) includes an allelic variation in the 3′-untranslated region

The rhesus macaque (Macaca mulatta) has become a popular animal model for several human infectious diseases, such as HIV (modeled by SIV infection), hepatitis, and malaria. Investigation of T-cell responses in experimental infectious diseases in rhesus macaques has benefited from an expanding understanding of the diversity of macaque MHC class I heavy chains and the restriction of antigen presentation by macaque class I molecules. Here we add to this understanding with the first nucleotide sequences of M. mulatta ₂-microglobulin (₂m) mRNA, including a portion of the 3-untranslated region (3UTR). In pairwise comparison, the ₂m protein of M. mulatta differs from human and chimpanzee ₂m by nine amino-acid substitutions (92% identity), and from Macaca fascicularis by one amino-acid difference in the signal peptide region (99% identity). Allelic variations were identified at one site in the 3UTR. A structural analysis of human or chimpanzee ₂m and M. mulatta ₂m suggests that the differences cluster in three solvent-exposed clusters and do not involve contacts with the class I heavy chain. We predict that human and macaque ₂m should bind interchangeably with the class I heavy chains of the other species, and show that four M. mulatta class I alleles form cell surface complexes with human ₂m. Further, we predict that W6/32 (a monoclonal antibody that recognizes a combined epitope of some class I heavy chains and ₂m with a subtle species dependence) should bind similarly human or macaque class I molecules that are bound with ₂m of either species, supported by evidence of recognition of both heterologous and homologous complexes of macaque class I heavy chains. Our findings contribute to the growing understanding of rhesus macaque histocompatibility antigens and antigen presentation, and to the phylogeny of ₂m in primates.The nucleotide sequences data reported in this article have been submitted to GenBank under accession numbers AY349163 and AY445843     

On the localization,the fine structure,and the chemical composition of crystalline inclusions in theThesium humifusum haustoria

Summary TheThesium humifusum haustoria onMedicago marina roots, fixed in October–November, frequently contain in the cytoplasm of their cells inclusions consisting of sticks 0,3 m in thickness and 8 to 10 m in lenght, alone or associated in stacks of 3 or 6 units. These sticks consist of fibres, 10 nm in thickness, oriented in the same direction and separated from the others by a gap of 8 nm; these fibres seem to be composed of helically wound filaments and a less electron-dense matrix.The chemical composition of these inclusions was studied by enzymatic digestion in ultra thin sections; pronase digested the cytoplasmic paracrystals. This demonstrates that they are composed primarily of protein.The physiological significance of the inclusions is discussed: presumably the haustorium functions as a storage organe.     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Localization and function of an FMRFamide-like substance in the aorta of Helix aspersa

Summary With an antiserum (aFM) against the molluscan cardio-active FMRFamide (Phe-Met-Arg-Phe-NH₂) numerous immunoreactive axons were found in the outer, longitudinal, muscle layer of the anterior aorta of Helix aspersa. Immunoreactive axons were rare in the inner, circular, muscle layer. At the ultrastructural level four types of axons could be distinguished. The granules containing the immunoreactive substance (mean diameter ca. 100 nm) are present in type-2 axons. The effect of synthetic FMRF-amide was tested in vitro on preparations of ring- and tubule-shaped pieces of the anterior aorta. Physiological doses (3 × 10^-7 M) provoked contractions of the circular muscle fibres, but had no effect on the longitudinal muscle cells. Apparently in vivo the FMRF-like substance diffuses from the richly innervated longitudinal muscle layer to the circular muscle layer, where it exerts its effect. This conclusion is sustained by the observation that the contents of the aFM-immunoreactive granules in type-2 axons are released by exocytosis in a non-synaptic fashion.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

(T2AG3)n Telomeric Sequence Hybridization Indicating Centric Fusion Rearrangements in the Karyotype of the Rodent Oryzomys Subflavus

Chromosome preparations of 30 specimens of Oryzomys subflavus trapped in eight Brazilian localities were C-, and G-banded and analyzed by fluorescence in situ hybridization (FISH). Two karyotypes were found, 2n=50/FN=64, at three coastal localities of the Atlantic Forest domain, and 2n=58/FN=70 at two sites located in the Cerrado biome, Brazil Central. Two fluorescence in situ hybridization (FISH) patterns of the telomeric sequence (T₂AG₃)_n were observed: in both karyotypes the probes hybridized to the telomeres of all chromosomes and also a hybridization signal in the centromeric regions of two autosome pairs was seen in the 2n=50 karyotype. These results, together with the occurrence of other diploid numbers described in the literature, suggest that O. subflavus is a complex species, bearing fusion/fission rearrangements proper to the different biomes which it inhabits.     

Studies on the histochemical „Leucine aminopeptidase“ reaction

Summary Since it was recently observed that purified cathepsin B will hydrolyse the chromogenic substrate LNA the obvious possibility of demonstrating the activity of this enzyme in tissue sections was explored. Chemical and histochemical experiments suggested that all aminopeptidase activity could be excluded by choosing a sufficiently acid pH for incubation, whereby the selective demonstration of cathepsin B activity was possible. This conclusion was further supported by different attempts to influence the histochemical reaction by preincubation with suitable activating and/or inhibiting reagents. This more specific procedure was used on sections from liver, kidney and granulomas rich in active macrophages. The suggested cathepsin B activity was demonstrated in autophagic vacuoles, and in other lysosome-like enlarged vacuoles or granules, appearing as a consequence of cellular injury as well as in actively phagocytosing macrophages.The following Abbreviations have been used AM Pase(s) aminopeptidase(s) in general - NA naphthylamides in general. — Substrates - LA Leucine amide - GPA Gly-Phe-amide - LNA L-leucyl--naphthylamide - Arg-NA L-arginyl--naphthylamide - BANA Benzoyl-dl-arginine--naphthylamide     

Assignment of human β-galactosidase-A gene to 3p21.33 by fluorescence in situ hybridization

GM₁ gangliosidosis and Morquio syndrome type B (MPS IVB) are inherited lyosomal storage disorders associated with deficiency of -galactosidase-A (GAL_A) activity. A recombinant plasmid containing a biotinylated cDNA (2.4-kb insert) encoding human GAL_A was used to localize the enzyme locus by fluorescence in situ hybridization (FISH). The human GAL_A gene was assigned to 3p21.33 by FISH.     

Cytokinesis in the green alga Eremosphaera viridis: Plasmalemma formation from “open membranes”

Summary Cytokinesis in the unicellular chlorococcalean alga Eremosphaera viridis de Bary has been investigated by electron microscopy of thin sections. The new plasmalemmata of the daughter cells in this organism form centrifugally within a phycoplast. Unlike other cell division systems each new plasmalemma is formed, not by the fusion of vesicles, but rather by the fusion of open membranes which are characteristically heavily stained. Measurements of these open membranes reveal that they are 11 nm thick with a central 4,5 nm unstained portion. The possible origin of these open membranes as burst-open vesicles has been suggested from the presence of intensely straining vesicles in the vicinity of the cell equator. Calculations of vesicle and open membrane surface areas support this contention.     

Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.