Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria

Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci II (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R_F value similar to Carlsen's peptide (adipokinetic hormone II).These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta, and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in ligated locusts.It is concluded that NCC II controls the release of adipokinetic hormones during flight and that two adipokinetic hormones are released during flight. One of these hormones adipokinetic hormone II also acts as a hyperglycaemic hormone illustrating that a hyperglycaemic hormone is released, during flight.     

The composition of suberin from the corks of Quercus suber L. and Betula pendula roth

The suberin constituents of Quercus suber and Betula pendula have been isolated after alkaline hydrolysis of the corks and over 80% by weight identified using thin-layer chromatography, preparative thin-layer chromatography, gas-liquid chromatography and combined gas chromatography — mass spectrometry. Long-chain aliphatic acids ranging from C₁₆–C₂₆ comprise about 90% of both suberin fractions; monobasic, α,ω-dibasic, ω-hydroxymonobasic, dihydroxymonobasic, dihydroxydibasic and trihydroxymonobasic acid classes are present. The principal suberin acids of Q. suber are 18-hydroxyoctadecenoic (12%), 22-hydroxydocosanoic (25%), 9,10-dihydroxyoctadecane-1,18-dioic (15%) and 9,10,18-trihydroxyoctadecanoic (8%), and those of B. pendula 9,10,18-trihydroxyoctadecanoic (43%) and 22-hydroxydocosanoic (16%).     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide,but not to isonicotinic acid: Differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (M_r, 137) to isonicotinamide (M_r, 122), not to isonicotinic acid (M_r, 123), in the presence or absence of H₂O₂. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1:0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.     

A simple and rapid thin-layer chromatography method to follow the kinetics of product formation by 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase and 7,8-dihydropteroate synthetase.

A rapid, simple, and inexpensive thin-layer chromatography method was developed to follow and to quantify enzymatic reactions of the title enzymes without need of a microbiological test system. The method was used to measure the kinetics of product formation by the folate-synthesizing enzymes 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase (I) and 7,8-dihydropteroate synthetase (II) of Escherichia coli. Thus test conditions could be optimized for the reaction of H₂PtCH₂OH to H₂PtCH₂OPP by I and of H₂PtCH₂OH to H₂-pteroic acid as well as its derivatives by I plus II. Furthermore this tle system allows direct measurements of I and/or II activity in fractions obtained by column chromatography (gel filtration and ion exchange, respectively).     

Female sex pheromone components of the cotton bollworm, Heliothis armigera

Detailed examination of abdominal tip extracts from adult female Heliothis armigera revealed the presence of two components which elicit electroantennographic responses from the male moth. These olfactory stimulants have been fully identified as (Z)-11-hexadecenal (I) and (Z)-11-hexadecen-1-ol(II), and detected in airborne volatiles from a ‘calling’ female moth. A third olfactory stimulant was detected only in female tip extracts from some moths of Malawi origin, and this was tentatively identified as (Z)-9-hexadecenal (III). No other olfactory stimulants could be found, although hexadecenal (IV) and 1-hexadecan-1-ol (V) were detected by gas chromatography. In field tests in Malawi, (Z)-11-hexadecenal (I) attracted a few male H. armigera moths to traps but was very much less attractive than the virgin female moth. The attractiveness of (I) was not consistently affected by addition of alcohol (II), aldehydes (III) and (IV), or (E)-11-hexadecenal. Significant numbers of male Earias biplaga moths were found to be attracted to (Z)-11-hexadecenal (I).     

Les aglycones flavoniques d'Anthyllis vulneraria

Anthyllis vulneraria (Leguminosae, subfamily Lotoideae) has been investigated for flavonoids by means of polyamide column chromatography and TLC. The following flavonols have been characterized: quercetin, kaempferol and isorhamnetin, previously reported in this genus, and rhamnocitrin (I), rhamnetin (II), 3,7,4′-trihydroxy-flavone (III), fisetin (IV) and geraldol (V). This last compound has only been isolated once before as a natural product.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Bound TPN as the determinant of polymorphism in methemoglobin reductase

The pyridine nucleotide-dependent, dye-linked methemoglobin reductase from human erythrocytes can be resolved chromatographically or electrophoretically into two principal forms (I and II). Form (II), which migrates more rapidly than (I) toward the positive electrode during electrophoresis at pH 8.3, contains 1 mole of non-covalently bound TPN per mole of protein (MW = 22,000) as judged by spectral characteristics (absorbance maxima at 327 and 340 nm when the protein is treated with cyanide and hydrosulfite, respectively) and by analysis (thin-layer chromatography and coenzymatic activity with isocitrate dehydrogenase) of the nucleotide released by heat denaturation. TPN can also be displaced by treatment of (II) with p-chloromercuriphenyl-sulfonate. Incubation of (I) with TPN at neutral pH results in the formation of (II).     

Isolation and characterization of a glycolipid from Dictyostelium discoideum

A glycolipid was isolated from a lipid extract of the cellular slime mold Dictyostelium discoideum and characterized. From the results of analyses by thin-layer chromatography and infrared spectrometry, it was identified as a steryl glycoside. The steryl glycoside was further analyzed by gas-liquid chromatography/mass spectrometry as a trimethylsilyl ether derivative, and its quantitative and qualitative changes during the development of D. discoideum were examined. Δ²²-Stigmastenyl-d-glucoside was the major constituent of the steryl glycoside and comprised more than 90% of the total steryl glycoside fraction in cells at all stages of development. The content of the steryl glycoside was higher in vegetative-stage cells, late aggregation-stage cells, and 1-day sorocarps than in cells of other stages. The glycolipid fraction was often contaminated by a lipid which was also isolated and identified as a ceramide containing 2-hydroxy fatty acids and 4D-hydroxysphinganine.     

Defensive agents of the American water beetles Agabus seriatus and Graphoderus liberus

Electrical shocking and rapid temperature reduction induced the dytiscid beetles Agabus seriatus and Graphoderus liberus to excrete pygidial and prothoracic defensive substances. When minnows (Pimephales promelas) were introduced into aqueous solutions of the defensive excretions from either beetle, they were rapidly inactivated and killed. The active agent was extractable in chloroform and constituted the major lipid component of the prothoracic defensive excretions. By means of thin-layer and gas-liquid chromatography, and ultraviolet and mass spectrometry, the biologically active material from the prothoracic defensive glands of both beetles was identified as a single steroid 11-deoxycorticosterone (DOC).The sensitivity of P. promelas to low levels of DOC made possible the development of a quantitative bioassay for this steroid in the defensive excretions. Quantitation by u.v. absorption at 240 nm and by the Pimephales bioassay showed A. seriatus contained ca. 40 μg DOC per beetle and G. liberus contained ca. 20 μg DOC per beetle.The major lipid component of the pygidial excretions of both beetles, p-hydroxybenzaldehyde, exhibited no toxic effect against test fish.     

Neobetanin: isolation and identification from Beta vulgaris

A new natural plant constituent has been isolated from the root of red beet (Beta vulgaris subsp. vulgaris var. conditiva) and identified as the orange-coloured water-soluble pigment 5-O-β-D-glucopyranosylneobetanidin (neobetanin). Its structure was elucidated and characterized by ¹H and ¹³CNMR spectroscopy, FAB mass spectrometry, absorption spectroscopy, enzymatic determination of the glucose moiety, high-performance liquid chromatography and thin-layer electrophoresis. ¹H nuclear Overhauser enhancement spectra unambiguously identified the position of attachment of the sugar residue. Neobetanin was also found to occur as a minor constituent in petals of Opuntia ficusindica, Portulaca grandiflora, Zygocactus truncatus and in fruits of Phytolacca bogotensis and Rhipsalis warmingiana.     

Properties of three sets of isoenzymes of alcohol dehydrogenase isolated from barley (Hordeum vulgare)

Three sets of isoenzymes of alcohol dehydrogenase were separated from root and shoot tissue of Hordeum vulgare by DEAE-cellulose chromatography. Set I showed only one band of ADH activity after polyacrylamide gel electrophoresis; Set II—two and Set III—three, making a total of six discernable bands. Only one set (I) was detected in the dry seed and one set (III) in the M9 (Adh-1-null) mutant available in tissue culture. The sets were found to have identical molecular weights (90 000), were all located in the cytoplasm but showed small differences in pH optima and substrate specificity. The affinity for ethanol (K_m value, mM) varied between Set I (27.5), Set II (7.2) and Set III (3.5), whilst the affinity for NADH varied five-fold between the three sets. A dimeric quaternary structure was inferred from the random reassociation of enzyme subunits after dissociation in high ionic strength buffer.     

Selenium-containing tRNAs from Clostridium sticklandii. Cochromatography of seleno-tRNA I with l-valyl-tRNA

It has been previously shown that Clostridium sticklandii specifically synthesized three readily separable ⁷⁵Se-labeled tRNAs, designated seleno-tRNAs I, II and III, and the partially purified seleno-tRNA II cochromatographed with l-prolyl-tRNA on DEAE-Sephadex A-50 (Chen, C.S. and Stadtman, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1403–1407). In the present study a highly purified ⁷⁵Se-labeled tRNA I was obtained by chromatography on benzoylated DEAE-cellulose, DEAE-Sephadex A-50 and Sepharose 4B. The ⁷⁵Se-labeled tRNA I cochromatographed with an l-valine-accepting species on DEAE-Sephadex A-50 and Sepharose 4B. Addition of a 285-fold molar excess of unlabeled l-valine to the l-valine acceptor activity assay mixture markedly decreased the amount of l-[¹⁴C]valine bound to seleno-tRNA I.     

Reduction of the Gibberellin Content of Pharbitis Seeds by CCC and After-Effects in the Progeny

Plants of Pharbitis nil were treated with the growth retardant (2-chloroethyl) trimethylammonium chloride (CCC) shortly before and after anthesis. Fresh and dry weight of immature seeds were not affected by the CCC treatment.

The level of gibberellin-like activity in Pharbitis seeds as compared to control seeds was strongly reduced by CCC application. The progenies of the treated plants also had a much reduced GA content in the seedling stage. These results are interpreted to indicate that CCC blocks gibberellin biosynthesis in higher plants, as it does in the fungus Fusarium.

CCC applied via the roots accumulated in the immature seeds and was carried over to the following generation. Consequently, growth of CCC progenies was dwarfed and flower formation inhibited. Both phenomena were overcome by application of gibberellin A₃.

Three gibberellin-like substances (called fractions I, II, and III) were present in Pharbitis seeds and could be separated by thin-layer chromatography. All 3 fractions were also present in seeds treated with CCC. Fractions II and III were present in much higher quantities than fraction I. Both fractions II and III promoted growth of d5 corn but only fraction II was active in dwarf peas grown under red light.

     

Ingenol esters from the pro-inflammatory fraction of Euphorbia kamerunica

A series of unstable mono- and di-esters of the tetracyclic diterpene ingenol were isolated from the pro-inflammatory ether-soluble fraction of the latex of Euphorbia kamerunica. The esters were isolated by a neutral process involving column and thin-layer chromatography. The monoesters were identified by spectroscopic methods and hydrolysis reactions as ingenol-3-decanoate, ingenol-3-dodecanoate, ingenol-5-hexadienoate and ingenol-5-octenoate and the diesters as 20-acetyl-ingenol-3-octenoate and 20-acetyl-ingenol-3-angelate.     

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH₃Hg(I) and Hg(II), and took up significantly more CH₃Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH₃Hg(I) and Hg(II) across the bacterial membrane.