Biosynthetic relationship between tetrahydroanthracene and anthraquinone in Aloe saponaria

The incorporation of Me¹⁴COONa into aloesaponol I, laccaic acid D methyl ester and aloesaponarin I was demonstrated. The biosynthetic relation between aloesaponol I and aloesaponarin I was established, but incorporation of aloesaponol I into laccaic acid D methyl ester, or vice versa was not demonstrated and this result was confirmed by an investigation using labelled laccaic acid D methyl (¹⁴CH₃) ester. It was possible to show that aloesaponol I and laccaic acid D methyl ester were biosynthesized in parallel in Aloe saponaria.     

Composition of the honeydew excreted by the lac insect, Kerria lacca (Homoptera: Coccoidea). I. Free amino acids

Honeydew of the lac insect, Kerria lacca is a clear watery fluid. By means of paper partition chromatography seventeen free amino acids and amides, viz., aspartic acid, asparagine, arginine, alpha and beta alanine, glycine, glutamic acid, histidine, lysine, leucine and or isoleucine, methionine, proline, serine, threonine, tyrosine, valine and possibly homoserine were identified in the honeydew of the mature female lac insect. The plant sap of Moghania macrophylla on which the insects were reared contained comparatively fewer amino acids.

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Zusammensetzung des von dem lackinsekt kerria lacca (Homoptera : Coccoidea) ausgeschiedenen honigtaues. I. Freie aminosäuren

Zusammenfassung Der Honigtau der begatteten weiblichen Lackschildlaus Kerria lacca (Kerr) wurde mit Hilfe zweidimensionaler Papierchromatographie auf Gehalt an freien Aminosäuren untersucht. Es wurden 17 freie Aminosäuren nachgewiesen: Asparaginsäure, Asparagin, Arginin, -und -Alanin, Glycin, Glutaminsäure, Histidin, Lysin, Leucin und (oder) Isoleucin, Methionin, Prolin, Serin, Threonin, Tyrosin, Valin und wahrscheinlich Homoserin. Außerdem erscheint ein nicht identifizierter Fleck (mit Rf-Wert 0,69 in Phenolwasser) etwas oberhalb des Methionins. Proteine und Peptide fehlten.Der Pflanzensaft von Moghania macrophylla (Willd.) O. Ktze., auf dem die Insekten gehalten wurden, enthielt vergleichsweise weniger Aminosäuren.

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Recently it has been found by one of us (R.K.V.) that the generic name Laccifer Oken, which is presently being used for the lac insect with sp. lacca, is invalid and should be substituted by Kerria Targ., 1884. This change has been approved of by the International Commission on Zoological Nomenclature.     

Antimicrobial Efficacy of Methylated Lac Dye,an Anthraquinone Derivative

A natural red dye which is produced by the tiny insects Kerria lacca while feeding on host trees is popularly known as lac dye. Lac dye is a mixture of at least five closely related pure compounds all being anthraquinone derivatives designated as laccaic acid A, B, C, D and E. Anthraquinones isolated from different natural sources and reported to have potent antimicrobial activity. The lac dye, which is also a mixture of anthraquinone derivatives, is expected to exhibit antifungal and antibacterial activity. Lac dye cannot be used as antibacterial and antifungal agent due to its low water solubility and high polarity. Therefore, it is modified into its methyl derivative to enhance its bio-efficacy. Methylated lac dye is characterized with the help of TLC, UV–Vis spectroscopy and FT-IR, NMR analysis. An in vitro spore germination assay was carried out to evaluate the antifungal efficacy of methylated lac dye against some phytopathogenic fungi which commonly caused a various foliar diseases in crop plants viz., Alternaria solani, Curvularia lunata, Erysiphe pisi, Helminthosporium oryzae and Verticillium sp. Among the tested fungi, Verticillum sp. showed highest sensitivity, which showed 100% inhibition at 750 and 1000 µg/ml as compared to control. However, E. pisi an obligate parasite also showed varied sensitivity but at 1000 µg/ml showed 100% spore germination as compared to control. Methylated lac dye also showed strong antibacterial properties against Ralstonia solanacearum at very low concentration (40 and 50 µg/ml). Hence, lac dye may serve as potent antifungal and antibacterial agent in plant disease management.     

Inhibition of Plasma Hyaluronan-Binding Protein Autoactivation by Laccaic Acid

Plasma hyaluronan-binding protein (PHBP) is a serine protease implicated in proteolysis under inflammatory conditions. We identified laccaic acid, a widely used food coloring from scale insects, as a potent inhibitor of the protease in terms of both autoactivation of the PHBP proenzyme (IC₅₀ = 0.35–0.55 μg/ml) and the catalytic activity of the active enzyme (IC₅₀ = 1.1 μg/ml).     

The role of Wolbachia and the environment on sex determination of the Indian lac insect,Kerria lacca (Coccoidea: Tachardiidae)

The Indian lac insect, Kerria lacca (Kerr) exhibits a highly variable sex ratio, apparently influenced by various environmental factors, in the absence of a chromosomal sex determination mechanism. The endosymbiont Wolbachia, well known as sex ratio distorter, was reported in this species but its role in the sex determination of the host has hitherto remained unexplored. Distinct variation in the presence of Wolbachia was observed in relation to the developmental stages and the sexes of K. lacca as well as the host plant species; adult males of W + whereas adult females are W-. The post-settlement environmental factors such as host plant, season and genetic makeup affect the sex ratio in this insect, probably mediated through Wolbachia dynamics. Denser settlement of the crawlers led to comparatively higher Wolbachia presence with an elevated sex ratio. Suppression of Wolbachia through antibiotic administration resulted in a nearly threefold increase in the sex ratio. Distinct transitions in the proportion of insects with heterochromatic genome (cytological males) have been observed during the first instar on different host plants, indicating that changes in the sex ratio occur during early development phase. Environmental factors appear to influence Wolbachia, which in turn brings about sex ratio changes, probably mediated through changes in juvenile hormone levels, as the male and female lac insects show distinct modes of metamorphosis.     

SIX NEW SPECIES OF THE GENUS MICROTERYS OF CHINA (HYMENOPTERA: ENCYRTIDAE) *

Abstract This paper deals with six new species of the genus Microterys, collected from Zhejiang, Fujian, Yunnan and Guangdong Provinces. They are M. metaceronemae Xu, sp. n. M. nuticaudatus Xu, sp. n. M. postmarginis Xu, sp. n. M. pseudonietneri Xu, sp. n. M. unicoloris Xu, sp. n. and M. zhaoi Xu, sp. n. The host of these parasitoids are Metaceronema japonica on Camellia oleosa Cerococcus muratae on Magnolia officinalis, Coccus hesperidum on citrus, Ceroplastes japonicus on citrus, Chloropulvinaria aurantii, Laccifer lacca Ceroplastes japonicus and Kerria lacca (Homoptera: Coccidoidea)     

The red insect dyes: carminic,kermesic and laccaic acids and their derivatives

Three groups of insect dyes are described: three cochineal dyes, the kermes dye and the lac dye. The major color components are carminic acid, kermesic acid and laccaic acids, respectively. These dyes are red anthraquinone derivatives. The chemical structures are described. All of these dyes have extensive histories that are related briefly; however, only American cochineal is of commercial importance today. Two manufactured derivatives of cochineal, carmine and acid-stable carmine (4-aminocarminic acid) are described in some detail including the chemical identity, toxicity, stability, and staining and non-staining applications.     

Utilization of phenoxyacetic acid, by strains using either the ortho or meta cleavage of catechol during phenol degradation, after conjugal transfer of tfdA, the gene encoding a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase

The degradation of recalcitrant pollutants in contaminated soils and waters could be facilitated by broadening the degradative capabilities of indigenous microbes by the conjugal transfer of catabolic genes. The feasibility of establishing bacterial populations that degrade phenoxyacetic acid by conjugal transfer of tfdA, the gene encoding 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase, to phenol-degrading strains of Pseudomonas and Ralstonia was examined. The mobilizable plasmid pKJS32 served as a vector for delivery of tfdA and the regulatory gene, tfdS. Transconjugant strains that degraded phenol by an ortho cleavage of catechol grew well on phenoxyacetic acid while those employing a meta cleavage could only grow on phenoxyacetic acid in the presence of benzoic acid or after a prolonged lag period and the appearance of mutants that had gained catechol 1,2-dioxygenase activities. Thus, an ortho cleavage of catechol was essential for degradation of phenoxyacetic acid, suggesting that a product of the ortho-cleavage pathway, probably cis,cis-muconic acid, is an inducer of tfdA gene expression. Establishment of phenoxyacetic-acid-degrading soil populations by conjugal transfer of tfdA would depend on the presence of phenol-degrading recipients employ- ing an ortho cleavage of catechol. Received: 7 August 1998 / Received revision: 29 October 1998 / Accepted 30 October 1998     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Cover Image,Volume 88, Issue 5

Human pathogenic and commensal bacteria have evolved the ability to scavenge host-derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known—here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N-acetyl-d -glucosamine, N-acetyl-d -galactosamine and N-acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N-acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate—a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.     

Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS

Summary Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.     

Effect of selected pesticides on larval mortality of the neuropteran predator,Chrysopa lacciperda Kimmins,of the lac insect,Kerria lacca (Kerr)

Laboratory bioassays were carried out to evaluate the bioefficacy of some pesticides against larval Chrysopa lacciperda Kimmins, a lac insect predator, to develop a suitable strategy for field management of this serious neuropteran pest of Indian lac insect, Kerria lacca (Kerr). Seven insecticides (lambdacyhalothrin, carbosulfan, spinosad, indoxacarb, fipronil, alphamethrin and ethofenprox) were identified based on field trials against the lac insect. They were evaluated for their bioefficacy against C. lacciperda by spraying the insect directly and by exposing the insect to a residual film. Direct spray of lambdacyhalothrin (0.005 and 0.008% a.i.), carbosulfan (0.02 and 0.03% a.i.), fipronil (0.005 and 0.01% a.i.), alphamethrin (0.005 and 0.01% a.i.), spinosad (0.02% a.i.), indoxacarb (0.02% a.i.), and ethofenprox (0.02% a.i.) exhibited 100% mortality of C. lacciperda within 24 h of treatment. Fipronil (0.005 and 0.01% a.i.) and indoxacarb (0.02% a.i.) were equally effective as 100% mortality was observed within 24 h of treatment with both modes of treatment. For most insecticides, direct spray was more effective compared to residual films. It is therefore, suggested that lambdacyhalothrin, carbosulfan, indoxacarb, spinosad, fipronil, alphamethrin and ethofenprox shall be incorporated in IPM programs for the effective management of this neuropteran predator of lac insect without adversely affecting the lac insect.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Lavandula stoechas L. subsp. stoechas,a New Herbal Source for Ursolic Acid: Quantitative Analysis,Purification and Bioactivity Studies

In this study, methanol, ethanol, methanol-dichloromethane (1 : 1, v/v), acetone, ethyl acetate, diethyl ether, and chloroform extracts of lavender (Lavandula stoechas L. subsp. stoechas) were prepared by maceration, and the ursolic acid contents in the extracts were determined quantitatively by HPLC analyses. The present results show that the methanol-dichloromethane (1 : 1, v/v) solvent system is the most efficient solvent system for the extraction of ursolic acid from the plant sample with the highest yield (2.22 g/100 g plant sample). In the present study, a new practical method for the isolation of ursolic acid from polar extracts was also demonstrated for the first time. The inhibition effects of the extracts and ursolic acid were also revealed on α-glycosidase, acetylcholinesterase, butyrylcholinesterase, and human carbonic anhydrase I and II enzymes by determining IC₅₀ values for the first time. The extracts and ursolic acid acted as potent antidiabetic agents by strongly inhibiting the α-glycosidase activity, whereas they were found to be very weak neuroprotective agents. In view of the present results, L. stoechas and its major metabolite, ursolic acid, can be recommended as a herbal source to control postprandial blood sugar levels and prevent diabetes by delaying the digestion of starch in food.     

Identification of linoleic acid,a main component of the n-hexane fraction from Dryopteris crassirhizoma,as an anti-Streptococcus mutans biofilm agent

Dryopteris crassirhizoma is a semi-evergreen plant. Previous studies have shown the potential of this plant as an agent for the control of cariogenic biofilms. In this study, the main antibacterial components of the plant were identified by correlating gas chromatography–mass spectrometry data with the antibacterial activity of chloroform and n-hexane fractions and then evaluating the activity of the most potent antibacterial component against Streptococcus mutans UA159 biofilms. The most potent antibacterial component was linoleic acid, a main component of the n-hexane fraction. Linoleic acid reduced viability in a dose dependent manner and reduced biofilm accumulation during initial and mature biofilm formation. Furthermore, when the biofilms were briefly treated with linoleic acid (10?min/treatment, a total of six times), the dry weight of the biofilms was significantly diminished. In addition, the anti-biofilm activity of the n-hexane fraction was similar to that of linoleic acid. These results suggest that the n-hexane fraction of D. crassirhizoma and linoleic acid may be useful for controlling cariogenic biofilms.     

Studies on the Bacterial Formation of a Peptide Antibiotic,Colistin

The biosynthetic pathway of α,γ-diaminobutyric acid, 6 moles of which are involved in the colistin molecule as a main component, was investigated. On the basis of the isotopic results using aspartic acid-U-¹⁴C as a precursor and also the finding of transaminase activity between α-ketog?utaric acid and α,γ-diaminobutyric acid, though in reverse reaction, α,γ-diaminobutyric acid was proved to be synthesized from aspartic acid via aspartyl-phosphate and aspartic β-semialdehyde. α,γ-Diaminobutyric acid did not inhibit asparto-kinase activity of this bacterium, the first enzyme involved in the process of α,γ-diamino-butyric acid synthesis from aspartic acid, while the end product amino acids such as lysine, threonine and methionine showed inhibition for aspartokinase activity.

On the other hand, α,γ-diaminobutyric acid might be rate-limiting factor in colistin formation, because of stimulatory effect of this diamino acid when added to the medium on colistin production. Furthermore, colistin production appeared to be related with the defect of TCA-cycle and further the resultant increase in activities of the key enzymes such as isopropylmalate synthetase, α-acetolactate synthetase and aspartokinase involved in the biosynthetic pathways of valine, leucine and isoleucine, respectively.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.