Effect of variety and location on growth and yield components of Roselle,HIbiscus sabdariffa L. and its infestation with the spiny bollworm Earias insulana (BOISD.)

Three roselle, Hibiscus sabdariffa L. varieties (Sudani, Masri and White) were cultivated at three different locations to recognize the transportation ability of roselle cultivation from the narrow old valley land to broad new land in Egypt. Qena as origin in situ old land, El-Kanater as ex situ old land and Nubaria as ex situ new land were the considered locations. Six growth quantitative characters and bolls infestation by spiny bollworm, Earias insulana were evaluated. Growth characters of roselle plants were affected significantly by either variety or location. Qena region was more suitable for roselle plant growth as judged with plant height, number of branches, number of fruits and sepals dry weight, followed by Nubaria followed by El-Kanater. Whereas, plants grown at Nubaria produced more fresh sepals weight than Qena or El-Kanater grown plants. As for Sudani, Nubaria exhibited the tallest plants, with the highest number of fruits and the heaviest fresh sepals as compared with the corresponding plants in Qena or El-Kanater. Values of broad sense heritability were highest for all characters in Qena. While the number of fruits per plant had the highest heritability in all locations. Dry sepals yield had highly significant correlation with all studied characters except percentage of water loss in Qena and Nubaria. Path coefficient analysis confirmed that fresh sepals yield had the highest direct and indirect effects on dried sepals yield. Chemical constituents responsible to sepal quality tended to produce significant variations due to the changes in varieties or locations. The highest levels of anthocyanins and sugars were achieved by Sudani variety, but the highest levels of free amino acids and total soluble solids were recorded for Masri variety. Moreover, Nubaria region was the most favourable for the accumulation of more anthocyanins in the sepals of all varieties followed by Qena. Plants grown at Qena produced sepals with the highest levels of sugars, free amino acids, organic acids and total soluble solids, followed by Nubaria followed by El-Kanater plants. Infestation with spiny bollworm Earias insulana was increased from Sudani up to Masri up to White varieties. Plants grown at Nubaria had the lowest number of attacks by bolls in all varieties, followed by those at El-Kanater followed by Qena plants. Spiny bollworm infestation was positively correlated with the number of branches and dry sepals weight, but negatively correlated with sepal moisture loss and anthocyanin contents. These findings clearly indicated that the Nubaria region was considered as a promising reclaimed area suitable for roselle cultivation, especially for Sudani, the most economic variety.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.

Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.     

Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore

The structure of the retinal chromophore about the C = N and C14-C15 bonds in bacteriorhodopsin's M412 intermediate has been determined by analyzing resonance Raman spectra of 2H and 13C isotopic derivatives. Normal mode calculations on 13-cis-retinal Schiff bases demonstrate that the C15-D rock and N-CLys stretch are strongly coupled for C = N-syn chromophores and weakly coupled for C = N-anti chromophores. When the Schiff base geometry is anti, the C15-D rock appears as a localized resonance Raman active mode at approximately 980 cm-1, which is moderately sensitive to 13C substitution at positions 14 and 15 (approximately 7 cm-1) and insensitive to 13C substitution at the epsilon position of lysine. When the Schiff base geometry is syn, in-phase and out-of-phase combinations of the C15-D rock and N-CLys stretch are predicted at approximately 1060 and approximately 910 cm-1, respectively. The in-phase mode is more sensitive to 13C substitution at positions 14 and 15 (approximately 15 cm-1) and at the epsilon position of lysine (approximately 4 cm-1). Calculations and comparison with experimental data on dark-adapted bacteriorhodopsin indicate that the in-phase mode at approximately 1060 cm-1 carries the majority of the resonance Raman intensity. M412 exhibits a C15-D rock at 968 cm-1 that shifts 8 cm-1 when 13C is added at positions 14 and 15 and is insensitive to 13C substitution at the epsilon-position of lysine. This demonstrates that M412 contains a C = N-anti Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Resonance Raman microscopy of rod and cone photoreceptors

We have constructed a Raman microscope that has enabled us to obtain resonance Raman vibrational spectra from single photoreceptor cells. The laser beam which excites the Raman scattering is focused on the outer segment of the photoreceptor through the epiillumination system of a light microscope. Raman scattering from the visual pigment in the photoreceptor is collected by the objective and then dispersed onto a multichannel detector. High-quality spectra are recorded easily from individual outer segments that are 5 x 50 micrometer in size, and we have obtained spectra from cells as small as 1 x 10 micrometer. We have used the Raman microscope to study photostationary steady-state mixtures in pigments from toad (Bufo marinus) and goldfish (Carassius auratus) photoreceptors; these photoreceptors were frozen in glycerol glasses at 77 degrees K. Comparison of our toad red rod spectra with previously published spectra of bovine rod pigments demonstrates that the conformation of the chromophore in the first photointermediate, bathorhodopsin, is sensitive to variations in protein structure. We have also studied the first photointermediate in the goldfish rod photostationary steady-state. This bathoporphyropsin has a much lower ethylenic stretching frequency (1,507 cm-1) than that observed in the toad and bovine bathoproducts (approximately 1,535 cm-1). Preliminary results of our work on goldfish cone pigments are also reported. These are the first vibrational studies on the vertebrate photoreceptors responsible for color vision.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Raman microscope and quantum yield studies on the primary photochemistry of A2-visual pigments.

The 77-K resonance Raman vibrational spectrum of intact goldfish rod photoreceptors containing 3,4-dehydro (A2) retinal is dominated by scattering from the 9-cis component of the steady state at all excitation wavelengths. Intact goldfish photoreceptors were regenerated with an A1-retinal chromophore to determine whether this behavior is caused by the protein or the chromophore. The resulting Raman spectrum was typical of an A1-pigment exhibiting significant scattering from all three components of the steady state: rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, regeneration of bovine opsin with A2-retinal produces a characteristic "A2-Raman spectrum" that is dominated by scattering from the 9-cis pigment. We conclude that the differences between the Raman spectra of the A1-and A2-pigments are caused by some intrinsic difference in the photochemical properties of the retinal chromophores. To quantitate these observations, the 77-K adsorption spectra and the photochemical quantum yields (phi) of the native A2-goldfish and the regenerated A2-bovine pigments were measured. In the goldfish A2-pigment, the value of phi 4 (9-cis----trans) is 0.05; phi 3 (trans----9-cis) is 0.10; and phi 2 (trans----11-cis) is 0.35. By contrast, in the bovine A1-pigment, these quantum yields are 0.10, 0.053, and 0.50, respectively. The reduced value of phi 4 and the increased value of phi 3 in the goldfish pigment confirms that the 9-cis isomer is photochemically more stable in A2-pigments.     

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.