Effect of repeated applications of buprofezin and acephate on soil cellulases, amylase, and invertase.

The impact of repeated applications of buprofezin and acephate, at concentrations ranging from 0.25 to 1.0 kg ha(-1), on activities of cellulases, amylase, and invertase in unamended and nitrogen, phosphorous, and potassium (NPK) fertilizer-amended soil planted with cotton was studied. The nontarget effect of selected insecticides, when applied once, twice, or thrice on soil enzyme activities, was dose-dependent; the activities decreased with increasing concentrations of insecticides. However, there was a rapid decline in activities of enzymes after three repeated applications of insecticides in unamended or NPK-amended soil. Our data clearly suggest that insecticides must be applied judiciously in pest management in order to protect the enzymes largely implicated in soil fertility.

4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway.

PURPOSE: To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum, on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action. METHODS: Rat skeletal muscle cells (L6-GLUT4myc) were treated with 4-HIL, and the effect on glucose uptake was determined by measuring the incorporation of radio-labeled 2-deoxy-[(3)H]-D-glucose (2-DG) into the cell. Translocation of GLUT4myc to plasma membrane was measured by an antibody-coupled colorimetric assay. RESULTS: The prolonged exposure (16 h) of L6-GLUT4myc myotubes to 4-HIL caused a substantial increase in the 2-DG uptake and GLUT4 translocation to the cell surface, without changing the total amount of GLUT4 and GLUT1. Cycloheximide treatment reversed the effect of 4-HIL on GLUT4 translocation to the basal level suggesting the requirement of new protein synthesis. The 4-HIL-induced increase in GLUT4 translocation was completely abolished by wortmannin, and 4-HIL significantly increased the basal phosphorylation of AKT (Ser-473), but did not change the mRNA expression of AKT, IRS-1, GLUT4, and GSK3ß. CONCLUSION: Results suggest that 4-HIL stimulates glucose uptake in L6-GLUT4myc myotubes by enhancing translocation of GLUT4 to the cell surface in a PI-3-kinase/AKT-dependent mechanism.

Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells [1]. ARNO is a member of the family of guanine nucleotide exchange factors for ARFs which includes cytohesin-1 and GRP-1 [2] [3-5]. Members of this family contain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) in vitro [3,4]. Here, we show that recombinant ARNO has the binding characteristics of a PIP3 receptor and that this activity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is localised exclusively in the cytoplasm. Stimulation with insulin, however, causes a rapid (< 50 second) PH-domain-dependent translocation of GFP-ARNO to the plasma membrane. This translocation is blocked by the PI(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002, and by co-expression with a dominant-negative p85 mutant, suggesting that the translocation is a consequence of insulin stimulation of PI 3-kinase. Our data strongly suggest that ARNO binds PIP3 in vivo and that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.

Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo.

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

Development of HPTLC-UV absorption densitometry method for the analysis of alprazolam and sertraline in combination and its application in the evaluation of marketed preparations.

A new simple, sensitive, and reproducible high-performance thin-layer chromatography method for the estimation of alprazolam and sertraline in combination is developed using silica gel plates with fluorescent indicators. The system is equipped with an automated sample applicator, and the detection was performed at 254 nm by using UV absorption densitometry. The mobile phase consists of carbon tetrachloride, methanol, acetone, and ammonia in the ratio 12:3:5:0.1. The retention factor values for alprazolam and sertraline are found to be 0.52 and 0.70, respectively. The limit of detection of alprazolam and sertraline in the mixture of given proportion is observed to be 0.05 microg/mL and 2.5 microg/mL and the limit of quantitation is 0.2 microg/mL and 10 microg/mL, respectively. The method has shown good linearity in the range of 0.2 microg/mL to 0.65 pg/mL for alprazolam (R2 > 0.9953) and 10 pg/mL to 32.5 microg/mL for sertraline (R2 > 0.9942). The intra- and inter-assay (n=5) variations in the linear range are less than 4% for alprazolam and 6% for sertraline. Three pharmaceutical products containing this combination are analyzed to test the applicability of the new method. The percentage of alprazolam and sertraline in the tablets studied range from 97.7% to 102.82% and 96.5% to 99.9%, respectively.

Sequences associated with human iris pigmentation.

To determine whether and how common polymorphisms are associated with natural distributions of iris colors, we surveyed 851 individuals of mainly European descent at 335 SNP loci in 13 pigmentation genes and 419 other SNPs distributed throughout the genome and known or thought to be informative for certain elements of population structure. We identified numerous SNPs, haplotypes, and diplotypes (diploid pairs of haplotypes) within the OCA2, MYO5A, TYRP1, AIM, DCT, and TYR genes and the CYP1A2-15q22-ter, CYP1B1-2p21, CYP2C8-10q23, CYP2C9-10q24, and MAOA-Xp11.4 regions as significantly associated with iris colors. Half of the associated SNPs were located on chromosome 15, which corresponds with results that others have previously obtained from linkage analysis. We identified 5 additional genes (ASIP, MC1R, POMC, and SILV) and one additional region (GSTT2-22q11.23) with haplotype and/or diplotypes, but not individual SNP alleles associated with iris colors. For most of the genes, multilocus gene-wise genotype sequences were more strongly associated with iris colors than were haplotypes or SNP alleles. Diplotypes for these genes explain 15% of iris color variation. Apart from representing the first comprehensive candidate gene study for variable iris pigmentation and constituting a first step toward developing a classification model for the inference of iris color from DNA, our results suggest that cryptic population structure might serve as a leverage tool for complex trait gene mapping if genomes are screened with the appropriate ancestry informative markers.

Conserved upstream sequence elements in plant 5S ribosomal RNA-encoding genes.

As a basis for further comparative studies, nuclear 5S rRNA gene repeats from two plants of the Solanaceae family, tobacco (Nicotiana rustica) and tomato (Lycopersicon esculentum), were isolated and sequenced. The more abundant 5S rRNA gene repeat in tobacco is 430 bp long, while a second less common variant is 521 bp long. In contrast, the 5S rRNA gene repeat from tomato is only 355 bp long. The spacer sequences from these gene repeats, as well as from other published plant nuclear 5S rRNA genes, were compared for repeating or conserved sequence elements. The results indicate that often observed, but non-conserved, repeating sequence elements probably arise spontaneously by unequal crossover with no functional significance. However, three conserved sequence elements immediately upstream of the coding sequence; a C residue at -1, a G + C-rich element centered at -13, and an A + T-rich element centered at -26 resemble regulatory features which have been identified in other types of genes.

The physiology, pathophysiology and therapeutic potential of gap junctions in smooth muscle.

Phenotypic variability in smooth muscle cells accounts, in large part, for the incredible functional diversity required of the involuntary hollow organs of the body (i.e., respiratory passages, blood vessels, gastrointestinal tract, urogenital tract, etc.). In all instances coordination of smooth muscle cell responses, that is, contraction and relaxation, is critical to normal organ function. While numerous biological mechanisms exist for coordinating smooth muscle cell responses, intercellular communication through gap junctions represents a common denominator present in all organ systems. In this report, we review the evidence documenting the presence and functional significance of myocyte gap junctions to physiologically distinct organ systems, and furthermore, provide some examples of their putative roles in organ pathology. Finally, we advance the thesis that despite their ubiquity and heterogeneous expression, gap junctions are nonetheless potentially attractive therapeutic targets for the treatment of certain smooth muscle disorders. Their therapeutic efficacy will necessarily hinge on the existence of connexin isoform-selective junctional effects. The overall rationale for targeting the intercellular pathway is therefore analogous to strategies that target other ubiquitously expressed ion channels, such as calcium or potassium channels. Such strategies have proved efficacious for the treatment of a wide range of human smooth muscle disorders including hypertension, urinary incontinence and sexual function.

Functional coupling of rat metabotropic glutamate 1a receptors to phospholipase D in CHO cells: involvement of extracellular Ca2+, protein kinase C, tyrosine kinase and Rho-A.

We report here that metabotropic glutamate 1a (mGlu1a) receptors, stably expressed in CHO cells, stimulate phospholipase D (PLD) activity. Several mGlu receptor agonists were found to exert this effect, with a rank order of potency of: L-quisqualate>L-glutamate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]=(S)-3,5-dihydroxyphenylglycine [(S)-DHPG]. Both L-glutamate- and (1S,3R)-ACPD-stimulated PLD activity were attenuated by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine. mGlu1a receptor-stimulated PLD was inhibited either by the selective protein kinase C (PKC) inhibitor, GF109203X, or via PKC downregulation. MGlu1a receptor-PLD coupling required extracellular Ca2+ and was sensitive to La3+ and Zn2+, inhibitors of intracellular Ca2+ store-operated Ca2+ influx. mGlu1a receptor-PLD coupling was inhibited by the selective tyrosine kinase inhibitor, genistein. In addition, mGlu1a receptor-PLD coupling was also inhibited by cell transfection with the selective Rho (small GTP-binding protein) inhibitors: C3-exoenzyme and dominant negative mutant RhoA constructs. Brefeldin A, a selective ADP-ribosylation factor (ARF) inhibitor, and a dominant negative ARF6 mutant, failed to significantly influence mGlu1a receptor-stimulated PLD activity. We conclude that mGlu1a receptors activate PLD via a mechanism that is dependent on extracellular Ca2+, PKC, tyrosine kinase and RhoA but independent of ARF.

Effects of heptanol on electrical activity in the guinea-pig vas deferens.

1. The effects of the putative intercellular uncoupling agent I-heptanol on electrical activity in the guinea-pig vas deferens were studied by use of intracellular and extracellular recording techniques. 2. At concentrations of 0.5, 1 and 2 mM, heptanol rapidly, monotonically and reversibly attenuated intracellularly recorded excitatory junction potential (e.j.p.) amplitude without affecting its time course, while spontaneous excitatory junction potentials (s.e.j.ps) were left unaffected. 3. Heptanol did not affect either the extracellularly recorded evoked excitatory junction current (e.j.c.), or the nerve terminal impulse that preceded it. These observations indicate that heptanol does not affect nerve impulse conduction, neurotransmitter release, or the postjunctional receptors involved in the production of the e.j.p. 4. E.j.ps appear to be suppressed by heptanol due to its intercellular uncoupling effects. Therefore, functional intercellular coupling may be necessary for the generation of the e.j.p. in smooth muscle.

Effects of heptanol on the neurogenic and myogenic contractions of the guinea-pig vas deferens.

1. The effects of the putative gap junction uncoupler, 1-heptanol, on the neurogenic and myogenic contractile responses of guinea-pig vas deferens were studied in vitro. 2. Superfusion of 2.0 mM heptanol for 20-30 min produced the following reversible changes in the biphasic neurogenic contractile response (8 trials): (i) suppression of both phases; (ii) delayed development of both the first as well as the second phase, accompanied by complete temporal separation of the two phases; (iii) prominent oscillations of force during the second (noradrenergic) phase only. 3. To eliminate prejunctional effects of heptanol, myogenic contractions were evoked by field stimulation of the vas in the presence of suramin (200 microM) and prazosin (1 microM). Heptanol (2.0 mM) abolished these contractions reversibly. 4. These results show that (i) heptanol inhibits both excitatory junction potential (EJP)-dependent and non EJP-dependent contractions of the vas; (ii) a postjunctional site of action of heptanol, probably intercellular uncoupling of smooth muscle cells, contributes to the inhibition of contraction.

Resistance to fluconazole in Candida albicans from AIDS patients correlated with reduced intracellular accumulation of drug.

Mucosal candidosis is an almost inevitable consequence of AIDS. Resistance to fluconazole therapy associated with enhanced tolerance, detectable in microbiological estimation of sensitivity, occurs in up to 10% of cases with late-stage AIDS. We report here our biochemical analysis of the basis of resistance in a study of two susceptible and two resistant isolates. Resistance was not associated with a change in the target enzyme sterol 14 alpha-demethylase, as indicated by equivalent levels of fluconazole inhibition of activity in extracts from all four isolates, or by mutations in sterol delta desaturase as previously observed in Saccharomyces cerevisiae and Ustilago maydis. Reduced cellular content of fluconazole in the resistant isolates of between six to ten-fold was observed which could account for their resistant phenotype.

Evidence for cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium.

The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448-450 and 452-454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH dependent and inhibited by carbon monoxide. Km values for activity showed a difference between the cellular fractions with a Km of 89 microM for microsomal P-450 and 400 microM for cytosolic P-450. The Vmax values observed were 0.83 nmol min-1 (nmol microsomal P-450)-1 and 0.4 nmol min-1 (nmol cytosolic P-450)-1. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.

Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus.

The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 x 10(-5) M and 50% of the growth at 1.3 x 10(-5) M and 2.1 x 10(-5) M respectively. A close relationship between accumulation of 14 alpha-methyl sterols (eburicol, obtusifoliol and 14 alpha-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 +/- 6.3 nM and 0.13 +/- 0.04 microM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.

Analysis of synaptic quantal depolarizations in smooth muscle using the wavelet transform.

The time-frequency characteristics of synaptic potentials contain valuable information about the process of neurotransmission between nerves and their target organs. For example, at the synapse between autonomic nerves and smooth muscle, two central issues of neurophysiology, i.e., 1) the probability of neurotransmitter release and 2) the quantal behavior of transmission can be deduced from analysis of the rising phases of evoked excitatory junction potentials (eEJP's) recorded from smooth muscle. eEJP rising phases are marked by prominent inflexions, which reflect these features of neuronal activity. Since these inflexions contain time-varying frequency information, we have applied recent techniques of time-frequency analysis based upon wavelet transforms to eEJP's recorded from the guinea-pig vas deferens in vitro. We find that these techniques allow accurate and convenient characterization of neuronal release sites, and that their probability of release falls between 0.001-0.004. We have also analyzed eEJP's recorded in the presence of the chemical 1-heptanol, which reveals quantal depolarizations. These results have helped clarify the nature of the quantal depolarizations that underly eEJP's. The present method offers significant advantages over those previously employed for these tasks, and holds promise as a novel approach to the analysis of synaptic potentials.

Estimation of substituted phosphates such as dibutyl and monobutyl phosphates, based on their reaction with lanthanum-Xylenol Orange complex.

Development of a new method for the estimation of substituted phosphates such as dibutyl phosphate (DBP) and monobutyl phosphate (MBP) is described in this paper. The method is based on the decolouration of lanthanum-Xylenol Orange complex by DBP and MBP at pH 5.

Estimation of variance components based on diallel model.

The problem of estimation of variance components based on diallel model for unbalanced data has been addressed. The least squares approach to quadratic estimation has been adopted in obtaining the explicit solutions for the design and genetic components of variance.

Hydatid disease of spleen treated by cyst enucleation and splenic salvage.

A 4-year-old boy with unilocular hydatid cysts of spleen and liver was successfully treated by enucleation of both the cysts and salvage of the spleen. The conventional surgical treatment of choice for hydatid cyst of the spleen is splenectomy. The authors demonstrate that preservation of a spleen afflicted by hydatid disease is technically feasible and is recommended as the choice of treatment to obviate the well-recognized postsplenectomy complications especially in children.

Stimulation of ammonification and nitrification in soils by the insecticides monocrotophos and quinalphos.

The application (up to 5 kg ha-1) of monocrotophos or quinalphos to four types of agricultural soils significantly stimulated the mineralization of peptone-nitrogen and the oxidation of ammonium-nitrogen. In soils treated with 2.5 kg ha-1 of either insecticide, the rate of ammonification and nitrification was fairly rapid after 2 and 4 weeks of incubation. The enhancement of both transformations, mediated by microorganisms, was significantly more in the quinalphostreated soils. The results suggest that a balance between the effect of insecticides on insect pests and their impact on beneficial microbial activities in soil must be determined.