Bio efficacy of some piperaceae plant extracts against Plutella xylostella L. (Lepidoptera: Plutellidae).

The extracts of Piper sarmentosum Roxb., Piper retrofractum Vahl, Piper interruptum Opiz and Piper nigrum plants belonging to the family Piperaceae were evaluated for their efficacy against diamondback moth, Plutello xylostella L. third instars under laboratory conditions. Comparative toxicity of various extracts prepared in hexane, dichloromethane, ethyl acetate and ethanol following sequential extraction procedure were applied topically to the larvae. The hexane extract of Piper retrofractum was most active (LD = 237 ppm). The toxicity was dose dependant and correlated to duration of exposure. The hexane extract of P. nigrum was least active with an LD50 of 18,435 ppm. The mode of action of these extracts and effect on other developmental parameters is in progress.

Effect of thymol on reproductive biology of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).

The objective of this study was to determine the possibilities for use of thymol, a common compound in essential oils of Thymus and Origanum plants, as an alternative biopesticidal compound to control Helicoverpa armigera. Thymol was applied to Helicoverpa armigera larvae topically and also administered orally at sublethal levels to determine the effects on their reproduction and development. Thymol did affect egg development and oviposition was reduced by 9.42 and 38.86% under two different applications, respectively. Growth inhibition was also observed and 6.7% of insects were dead as larvae, 42.4% as pupae and 6.6% as pupal-adult intermediates. The present studies clearly indicate that thymol used at sublethal levels can alternatively control bollworms by altering reproductive mechanisms.

Bioefficacy of Alpinia galanga (Zingiberaceae) rhizome extracts, (E)-p-acetoxycinnamyl alcohol, and (E)-p-coumaryl alcohol ethyl ether against Bactrocera dorsalis (Diptera: Tephritidae) and the impact on detoxification enzyme activities.

The application of insecticides to control oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae), is a principal component of the current management of these fruit flies. However, we evaluated four extracts of Alpinia galanga Wild Linn (Zingiberaceae) rhizomes against adult flies and found hexane and ethanol extracts to be most effective (LC50 = 4,866 and 6,337 ppm, respectively, after 24 h). This suggested that both nonpolar and polar compounds could be active in the candidate plant. Accordingly, the hexane extract was further processed to isolate nonpolar active compounds from this plant source. Two compounds, (E)-p-acetoxycinnamyl alcohol and (E)-p-coumaryl alcohol ethyl ether, were identified as active ingredients and found to be more active than total hexane extract (LC50 = 3,654 and 4,044 ppm, respectively, after 24 h). The data suggested that the compounds were not synergistic but may have some additive effect in a mixture. The activity of the hexane extract against detoxification enzymes, carboxylesterase (CE) and glutathione transferase (GST) also was determined in vitro. CE was inhibited by 70%, whereas GST was not significantly inhibited. Insect CEs mediate insecticide resistance via their induction; therefore, inhibition of these enzymes by plant allelochemicals could be a useful alternative approach for the management of the pest in the field.

Retinoic acid synthesis for the developing telencephalon.

The small lipid retinoic acid is known to promote neuronal differentiation in vitro and to act as a teratogen in the embryonic brain, but very little is known about the natural role of endogenously synthesized retinoic acid in forebrain development. Retinoic acid is synthesized mainly by three retinaldehyde dehydrogenases. We show here where the retinaldehyde dehydrogenases for the developing telencephalon are expressed and how their expression patterns change over developmental time. Retinoic acid diffusing from the retinaldehyde dehydrogenase sites is likely to influence the early telencephalon before the beginning of neurogenesis, as well as differentiation and radial migration of neurons into the cerebral cortex. Because of its diffusible character, retinoic acid represents a unique tool for the coordination of growth processes over an intermediate distance range in the developing telencephalon.

A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges.

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.

[Determination of substrate specificity of fucosyltransferase from rat brain using synthetic acceptors]. / Opredelenie substratnoi spetsifichnosti fukoziltransferazy iz mozga krys s pomoshch'iu sinteticheskikh aktseptorov.

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Gal beta 1-4GlcNAc beta 1-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated whereas Fuc alpha 1-2Gal beta 1-4GlcNAc displayed the same substrate properties as Gal beta 1-4GlcNAc. FT from cerebellum and forebrain was shown to have the specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from forebrain, the specificity profiles being similar.

Differential labelling of sphingolipids by [3H]serine and ([3H]methyl)-methionine in fish leukocytes.

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([3H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [3H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.

N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity.

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme. Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells. Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role. However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.

Evidence for a role of gammadelta T cells in demyelinating diseases as determined by activation states and responses to lipid antigens.

In this report we review current information on the phenotypic and functional properties of gammadelta T cells in demyelinating disorders. The results support the conclusion that although gammadelta T cells show evidence of activation in patients with either multiple sclerosis (MS) or Guillain Barrè syndrome (GBS), differences exist in the phenotypic and functional properties of these cells between the two diseases. In particular, our data indicate that in patients with MS the Vdelta2 subset is activated and that these cells can be induced to secrete high levels of proinflammatory cytokines. In contrast, in patients with GBS, the Vdelta1 subset is expanded and can be induced to secrete cytokines more associated with a humoral response.

Growth inhibitory and antifeedant activity of extracts from Melia dubia to Spodoptera litura and Helicoverpa armigera larvae.

The growth inhibitory activity and deterrency of Melia dubia (Meliaceae) extracts to Spodoptera litura and Helicoverpa armigera were investigated. Artificial diet bioassays using neonate larvae of both S. litura and H. armigera indicated that dichloroethane (DCE) and methanol (Me) extracts of M. dubia inhibited growth in a dose dependent manner. DCE and Me-5II fractions also resulted in 50% deterrency at concentrations of 22.5 and 16.8 micrograms/cm2 respectively against S. litura larvae in a leaf disc-choice test. The DCE-5 fraction was found to be more toxic to larvae (LC50 of 0.65%) than the Me-5II (LC50 of 0.8%), 72 hr after topical application. Both fractions lack contact toxicity, but the deterrent effect persisted for at least 60 hr under laboratory conditions. Although salannin was isolated from the DCE fraction to show antifeedant activity, the physico-chemical characteristics of the active fractions DCE-5 and Me-5II were not identical with either salannin or azadirachtin.

Phytosphingosine biosynthesis differs from sphingosine in fish leukocytes and involves a transfer of methyl groups from [3H-methyl]methionine precursor.

We have studied the incorporation of radioactivity from either [3-3H]serine as the direct or [3H-methyl]methionine as the indirect precursor into sphingoid bases of free ceramides in lymphocytes from fish. Radioactivity from serine was incorporated mostly in the sphingosine moiety of ceramides. In contrast, the radioactivity from methionine was exclusively incorporated into phytosphingosine base (i.e., 4-hydroxy-sphinganine) and the incorporation increased by about twofold in the presence of folic acid or niacinamide. Identity of the long-chain bases, phytosphingosine and sphingosine, was established chemically by thin-layer chromatography, chemical degradation, and gas-liquid chromatography.

alpha1,3 Fucosyltransferase, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, and Le(x) glycoconjugates in developing rat brain.

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Le(x)) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15-P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10-P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an alpha1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5-10) with different types of acceptors, and the differential expression of Le(x) containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, alpha-L-fucosidase, beta-D-galactosidase and alpha-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

The rat alpha1, 3-fucosyltransferase (rFucT-IV) gene encodes both long and short forms of the enzyme which share the same intracellular location.

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to play a role in cell-cell interactions. A recently cloned rat FucT (rFucT-IV) is expressed in the testes, and thus is a candidate for encoding the cell-surface FucT activity. This study maps the 5'-ends of several rFuc-T-IV mRNAs, and these results suggest that initiation of transcription may occur both upstream of the first ATG, as well as between the first two closely spaced, in-frame ATGs. Thus, in certain tissues, notably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic properties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be expressed both on the cell surface and in the Golgi. Plasmids that encode FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro using cell-free rabbit reticulocyte lysate, or after transfection in tissue culture. The results from these studies demonstrate that rFucT-IV is a glycosylated, transmembrane protein with a short cytoplasmic tail, and that either of the two ATGs in the 5' region of the rFucT-IV gene are capable of acting as functional initiators of translation in vitro, to produce enzymatically active glycoproteins. However, no difference in the intracellular localization between the transferase containing a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like staining in several different cell types, with no indication of surface expression. Thus, the additional amino-terminal 33 amino acids of the long form of rFucT-IV do not appear to influence its intracellular location in the cell types investigated.

A two-dimensional thin-layer chromatography procedure for simultaneous separation of ceramide and diacylglycerol species.

We have developed a novel, simple, and rapid, two-dimensional thin-layer chromatography method to separate 1,2-, 1,3-diacylglycerols and ceramides containing alpha-hydroxy and normal fatty acids from other neutral lipids on one 10 x 10 cm precoated silica gel plate. The three solvent systems used in succession leave the phospholipids at the origin and separate neutral lipids of interest into component species. We have applied this method to incorporation of 9,10-[3H]myristic acid into lipids of gills from sea bass and obtained results that are similar and comparable to those obtained by described methods.

Auditory discrimination reversal learning and assessment of behavioral teratogenesis in rats.

Qualitative auditory discrimination procedures were used to evaluate discrimination acquisition and reversal learning in rats. Twelve adult rats prenatally exposed to ethanol (ETOH) and 12 unexposed isocaloric controls (CON) were given training with a positively reinforced successive discrimination procedure. Most ETOH subjects were impaired relative to CON subjects on accuracy during early training sessions and the number of sessions required to meet an 80% accuracy criterion. Some ETOH subjects were also impaired on the rate of learning over a series of repeated discrimination reversals. Individual differences in reversal learning rates varied more widely with ETOH subjects than with CON subjects. Our results indicate that the auditory discrimination procedures may find application in assessments of behavioral teratogenesis.

[Glycosyltransferase inhibitors and study of UDP-galactose-globoside galactosyltransferase]. / Ingibitory glikoziltransferaz i izuchenie UDP-galaktoza-galobozidgalaktoziltransferazy.

There is now a large body of evidence indicating that glycoconjugates are involved in a wide variety of processes that influence cell growth, differentiation, cell sociological behavior and response to environmental conditions. The synthesis and expression of this class of compounds appears to be regulated to a large extent by the activities of the glycosyltransferases which are responsible for their biosynthesis. The level of glycosyltransferase activities in cells could be regulated at many levels. Thus, factors that influence transcription and splicing of the transferase genes, stability of the mRNAs, translation, and post-translation processes are all probably involved. Post-translation processes that affect the level of enzyme activity could include proteolytic degradation, glycosylation, phosphorylation, sulfation, acylation, and the presence of activator and inhibitory factors. Although the presence of a least seven different glycosyltransferase inhibitors have been reported, this type of regulation has received relatively little attention. It appears from the data cited in this review that in many instances, cellular inhibitors of glycosyltransferase activities may play a key role in the regulation of transferase activities and the expression of glycoconjugates. This type of post-translational regulation of glycosyltransferase activities deserves increased attention.

Developmentally regulated Gb3 galactosyltransferase and alpha-galactosidase determine Shiga toxin receptors in intestine.

The receptor for Shiga toxin on rabbit intestinal microvillus membranes (MVMs) has been identified as a developmentally regulated glycolipid, globotriaosylceramide [galactose alpha 1-4 galactose beta 1-4 glucose beta 1-1 ceramide (Gb3)]. MVM Gb3 levels increase markedly in the third week of life, concomitant with fluid secretory responses to the toxin. To study mechanisms controlling developmental regulation of MVM Gb3, we measured the specific synthetic Gb3 galactosyltransferase and degradative alpha-galactosidase activities and subcellular distribution of Gb3 at various ages. Quantitative high-performance liquid chromatography demonstrated a similar developmental pattern in both microsomal and MVM Gb3, indicating that late expression of MVM Gb3 is not due to delayed migration of Gb3 from the microsomal to the MVM. The specific Gb3 galactosyltransferase activity increased with age, with a sharp increase seen at 18 days of age, whereas alpha-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age. The nature of the signals for developmental regulation of Gb3 levels is unknown, as are the physiological consequences of altered MVM glycolipid composition other than mediating response to Shiga toxin.

RH-5849, a nonsteroidal ecdysone agonist, does not mimic makisterone-A in Dysdercus koenigii.

The response of final instar nymphs of Dysdercus koenigii to topical application of the non-steroidal ecdysone agonist, RH-5849, was dose dependent. The candidate compound produced mortality even at moderate doses, but precocious adult development was not observed. Similar results were obtained after oral administration or injection. Conversely, injections of makisterone-A (the principal moulting hormone of Dysdercus) into 5th instar nymphs resulted in precocious adult development within 4 days. We conclude that RH-5849 does not mimic makisterone-A, as is the case with ecdysone, and that toxicity is mediated instead through non-endocrine targets in this insect species.

Rapid isolation of monosialogangliosides from bovine brain gangliosides by selective-overload chromatography.

A procedure for rapid isolation of monosialogangliosides from purified bovine brain gangliosides has been developed. It utilizes the selective difference in association between monosialogangliosides and polysialogangliosides for the ion-exchange resin Q-Sepharose. When the ion-exchange column is overloaded with a bovine brain ganglioside mixture in the proper ganglioside to column bed-volume ratio, the polysialogangliosides are selectively retained by the column while the monosialogangliosides emerge with the void volume without the use of salt for elution. With the critical ganglioside to bed-volume ratio (1 g:8.32 ml), and an appropriate column bed-height to column radius ratio of 6.9, monosialogangliosides are reproducibly obtained in high purity with greater than 90% yield. The method has been used at both the analytical and preparative scale. We call this separation technique selective-overload chromatography.

Developmental expression of HNK-1-reactive antigens in rat cerebral cortex and molecular heterogeneity of sulfoglucuronylneolactotetraosylceramide in CNS versus PNS.

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.