Involvement of the C-terminal tail of Arthrobacter ureafaciens sialidase isoenzyme M in cleavage of the internal sialic acid of ganglioside GM1

Arthrobacter ureafaciens sialidase comprises four isoenzymes, L, M1, M2 and S, of which L, M1, and M2, but not S, have the unique ability to cleave GM1 ganglioside, but the hydrolysis of GM3 and colominic acid by S occurs at a higher rate than that by L, M1 and M2. Since the N-terminal amino acid sequences of L, M1, M2 and S were shown to be identical on protein sequencing, they were suggested to have arisen from the same protein through truncation at different C-terminal sites. A DNA segment containing an open reading frame was cloned from a genomic library, and the structural gene was found to comprise 2,970 bp encoding a protein of 990 amino acids including a signal peptide at the N-terminus, a conserved FYRIP-region and four Asp boxes. The molecular weights of the isoenzymes determined by MALDI-TOFMS revealed that L, M1, M2 and S should comprise amino acids 39-773, 39-653, 39-655 and 39-528, respectively. In fact, recombinant enzymes M2 and S prepared in Escherichia coli exhibited identical substrate specificities toward gangliosides as those of the purified enzymes. Consequently, the C-terminal tail of isoenzyme M2 might be involved in the hydrolysis of the internal sialic acid of GM1.     

Action of Arthrobacter ureafaciens sialidase on sialoglycolipid substrates. Mode of action and highly specific recognition of the oligosaccharide moiety of ganglioside GM1.

A new bacterial sialidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) isolated from the culture filtrate of Arthrobacter ureafaciens was characterized in detail with respect to its action on sialoglycolipids. Strong electrolytes had a reversible inhibitory effect on the action of the enzyme on brain gangliosides in accordance with Debye-Hückel effect of ionic environment on ionic activity, and resulted in an acidic shift and a broadening of the pH optimum. Both ionic and non-ionic detergents markedly enhanced the enzymic activity on the gangliosides, and caused an acidic shift on the pH optimum of this enzyme. Sulfhydryl groups seemed to be involved in its active site. This enzyme had a highly specific action on sialidase-resistant ganglioside GM1, showing about 100-fold higher activity on GM1 than Clostridium perfringens sialidase, the only sialidase so far reported to cleave the lipid substrate in the presence of bile salts. In the absence of detergents, the activity of A. ureafaciens sialidase on GM1 was very low. Ganglioside GM1 in either the monomeric or micelar form was hydrolyzed to asialo-GM1 by A. ureafaciens sialidase most efficiently in the presence of sodium cholate of about three times the GM1 molar concentration. The presence of detergents increased both the Km and Vmax values for ganglioside GM1. The oligosaccharide prepared from GM1 by ozonolysis was cleaved well by this sialidase in the absence of detergents, and no detergent was found to affect the hydrolysis. The Km value for the sugar substrate was about two orders of magnitude greater than that for the corresponding lipid substrate. It is suggested that the hydrophobic ceramide moiety increases affinity of the lipid substrate to the enzyme, but inhibits hydrolysis of the substrate, possibly due to its hydrophobic interaction with hydrophobic portions of the enzyme molecule (resulting in lower Km and Vmax for lipid substrates). This inhibition may be released by detergent due to formation of mixed micelles of sialoglycolipid and detergent molecules. It is also indicated that recognition of the specific saccharide structure of GM1 by individual sialidases is essential for release of the resistant sialyl residue, and that A. ureafaciens sialidase seemed to have an isoenzymic or oligomeric structure.     

Selection of GM2, fucosyl GM1, globo H and polysialic acid as targets on small cell lung cancers for antibody mediated immunotherapy

Glycolipids GM2, GD2, GD3, fucosyl GM1, sialyl Lewis a (sLe^a) and globo H, and polysialic acid on embryonal NCAM, are cell-surface antigens expressed on small cell lung cancer (SCLC) biopsy specimens. They are all candidates for inclusion in a polyvalent, antibody-inducing vaccine or for adoptive therapy with monoclonal antibodies (mAbs) against SCLC. To identify the minimum optimal combination of target antigens on SCLC and to confirm that antibodies against this combination might be able to mediate complement activation and lysis in the majority of cases, we tested ten SCLC cell lines with fluorescence activated cell sorter (FACS) and complement dependent cytotoxicity (CDC) assays using mAbs against these seven target antigens individually or pooled in different combinations. We find that (1) none of these mAbs demonstrated strong FACS reactivity with more than 6 of the 10 cell lines, (2) no mAb had strong CDC reactivity with more than 4 of the cell lines, (3) when the mAbs were pooled, nine cell lines were strongly positive by FACS and nine cell lines were strongly positive by CDC, and (4) mAbs against GM2, FucGM1, globo H and polysialic acid was the minimum optimal combination for inducing FACS reactivity. The addition of mAbs against sLe^a, GD2 and GD3 had no additional impact by FACS and only minimal additional impact in CDC assays. H345, the only cell line that had less than 30% CDC with the four mAb pool was strongly positive by FACS. To understand the lack of correlation between FACS and CDC in the case of H345, the ten cell lines were screened for expression of complement resistance factors CD55 and CD59. Three cell lines were strongly positive for CD55 and eight were strongly positive for CD59. Overall, no correlation was seen between expression of either of these factors on the ten cell lines and sensitivity to CDC. In the case of H345 however, complement resistance of H345 is demonstrated to be mediated primarily by CD59, and in the presence of mAb against CD59, the four mAb MEM-43 pool induced strong (94%) CDC. CD59 inhibits membrane attack complex formation but not activation of earlier complement components. Consequently, all ten cell lines are good targets for complement activation by the four antibody pool and for elimination by effector mechanisms including complement mediated inflammation and opsonization. These findings support our plan to develop a tetravalent vaccine against SCLC targeting GM2, fucosyl GM1, globo H and polysialic acid.Supported by grants from the NIH (PO1CA33049), and the Lawrence and Selma Ruben Foundations.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with ¹³C-¹H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all ¹H and ¹³C chemical shifts is presented.     

Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K,L, M1, M2, and M2' intermediates of the photocycle

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.     

Inhibition of sialidases from viral,bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position

The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus,Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.This paper is dedicated to the memory of Professor Dr E. Zbiral.     

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.     

Paraoxonase 1 L55M, Q192R and paraoxonase 2 S311C alleles in atherothrombosis

Increased oxidative stress is known to play a role in the pathogenesis of atherosclerosis, and polymorphisms in genes encoding for enzymes involved in modulation of oxidant stress, such as paraoxonases (PONs), provide a potentially powerful approach to study the risk of disease susceptibility. Aim of our study is to investigate the possible association among PONs polymorphisms, clinical and metabolic factors, and atherothrombotic events in an Italian population. We evaluated in 105 subjects, with or without atherosclerotic risk factors, the presence of PON1 L55M, PON1 Q192R, and PON2 S311C genetic variants, as well as lipid profile, the concentration of aminothiols (blood reduced glutathione, plasma total glutathione, homocysteine, cysteine, cysteinyl glycine), and malondialdehyde as markers of lipid peroxidation. Clinical, biochemical, and genetic variables were correlated with a history of atherothrombosis. Previous atherothrombotic events were found in 42 patients (40 %): myocardial infarction in 24, stroke or transient ischemic attack in 18. By multiple logistic regression analysis, hypertension (OR = 5.538; 95 % CI 2.202–13.902, P < 0.001), HDL-cholesterol concentration (OR = 0.947; 95 % CI 0.910–0.985, P = 0.007), and the presence of C allele in PON2 gene (OR = 3.595; 95 % CI 1.247–10.361, P = 0.018) were independently associated with atherothrombotic events. Our study sheds light on the role of PON2 as a possible cofactor in determining the risk of events together with the well-known risk markers HDL-cholesterol and hypertension.     

Allometry,merism, and tooth shape of the upper deciduous M2 and permanent M1

The aims of this study were to investigate the effect of allometry on the shape of dm² and M¹ crown outlines and to examine whether the trajectory and magnitude of scaling are shared between species. The sample included 160 recent Homo sapiens, 28 Upper Paleolithic H. sapiens, 10 early H. sapiens, and 33 H. neanderthalensis (Neandertal) individuals. Of these, 97 were dm²/M¹ pairs from the same individuals. A two‐block partial least squares analysis of paired individuals revealed a significant correlation in crown shape between dm² and M¹. A principal component analysis confirmed that Neandertal and H. sapiens dm² and M¹ shapes differ significantly and that this difference is primarily related to hypocone size and projection. Allometry accounted for a small but significant proportion of the total morphological variance. We found the magnitude of the allometric effect to be significantly stronger in Neandertals than in H. sapiens. Procrustes distances were significantly different between the two tooth classes in Neandertals, but not among H. sapiens groups. Nevertheless, we could not reject the null hypothesis that the two species share the same allometric trajectory. Although size clearly contributes to the unique shape of the Neandertal dm² and M¹, the largest H. sapiens teeth do not exhibit the most Neandertal‐like morphology. Hence, additional factors must contribute to the differences in dm² and M¹ crown shape between these two species. We suggest an investigation of the role of timing and rate of development on the shapes of the dm² and M¹ may provide further answers. Am J Phys Anthropol 154:104–114, 2014. © 2014 Wiley Periodicals, Inc.     

SDS-PAGE、同工酶等电聚焦和RAPD标记鉴定两系杂交水稻F1种子纯度对比研究

目的：探讨利用种子贮藏蛋白SDS—PAGE电泳、酯酶同工酶超薄等电聚焦电泳和RAPD分子标记鉴定五个两系杂交水稻组合F_1种子纯度的可行性。方法和结果：利用SDS—PAGE电泳技术未能找到所试五个组合各自的父本特征蛋白质带;利用酯酶同工酶超薄等电聚焦电泳技术和RAPD分子标记可找到所试五个组合的父母本特征酶带和RAPD标记,但酯酶同工酶的多态性同时也受种子萌发时间的影响。酯酶同工酶超薄等电聚焦电泳和RAPD分子标记可用于所试五个两系杂交水稻组合F_1种子纯度的鉴定。     

Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Abstract

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200?mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35?°C and pH 7.0 within 18?h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55?°C), and NaCl concentration (5–20?g/L). Further, these strains decolorize up to 800?mg/L concentrations of methyl orange within 24?h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12?h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01?µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.     

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.     

应用HA、NA、M1和M2蛋白制备H5N1高致病性禽流感病毒样颗粒

目的:应用重组杆状病毒表达系统制备由HA、NA、M1和M2蛋白组成的H5N1高致病性禽流感病毒样颗粒,为研究H5N1高致病性禽流感疫苗奠定基础。方法:构建能共表达A/chicken/Jilin/2003(H5N1)禽流感病毒血凝素(HA)和神经氨酸酶(NA)、A/PR/8/34(H1N1)流感病毒基质蛋白(M1)和离子通道蛋白(M2)的2个二元重组杆状病毒,共同感染HighFive细胞,同时表达HA、NA、M1和M2蛋白,使这4种蛋白在感染的细胞内自主组装成病毒样颗粒。经差速离心和蔗糖密度梯度超速离心收获病毒样颗粒,通过Western印迹鉴定病毒样颗粒的组成,透射电镜观察病毒样颗粒形态,血凝试验测定病毒样颗粒的活性。结果:HA、NA、M1、M2蛋白在昆虫细胞中共表达,并组装成病毒样颗粒;电镜观察到病毒样颗粒的形态与流感病毒一致,直径约80 nm;血凝试验显示该病毒样颗粒具有凝集鸡红细胞的活性。结论:应用该方法可以制备流感病毒样颗粒,为H5N1流感疫苗研究提供了可行方案。     

Stereoselectivity of (R)- and (S)-hexahydro-difenidol binding to neuroblastoma M1, cardiac M2, pancreatic M3, and striatum M4 muscarinic receptors

(R)-Hexahydro-difenidol has a higher affinity for M1 receptors in NB-OK 1 cells, pancreas M3 and striatum M4 receptors (pKi 7.9 to 8.3) than for cardiac M2 receptors (pKi 7.0). (S)-Hexahydro-difenidol, by contrast, is nonselective (pKi 5.8 to 6.1). Our goal in the present study was to evaluate the importance of the hydrophobic phenyl, and cyclohexyl rings of hexahydro-difenidol for the stereoselectivity and receptor selectivity of hexahydro-difenidol binding to the four muscarinic receptors. Our results indicated that replacement of the phenyl ring of hexahydro-difenidol by a cyclohexyl group (----dicyclidol) and of the cyclohexyl ring by a phenyl moiety (----difenidol) induced a large (4- to 80-fold) decrease in binding affinity for all muscarinic receptors. Difenidol had a significant preference for M1, M3, and M4 over M2 receptors; dicyclidol, by contrast, had a greater affinity for M1 and M4 than for M2 and M3 receptors. The binding free energy decrease due to replacement of the phenyl and the cyclohexyl groups of (R)-hexahydro-difenidol by, respectively, a cyclohexyl and a phenyl moiety was almost additive in the case of M4 (striatum) binding sites. In the case of the cardiac M2, pancreatic M3, or NB-OK 1 M1 receptors the respective binding free energies were not completely additive. These results suggest that the four (R)-hexahydro-difenidol "binding moieties" (phenyl, cyclohexyl, hydroxy, and protonated amino group) cannot simultaneously form optimal interactions with the M1, M2, and M3 muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of rat liver cytochrome P450 isoenzymes CYP 1A and CYP 2B by different fungicides, nitrofurans, and quercetin

The genotoxic activity of environmental xenobiotics is manifested either in their direct interaction with cellular genetic material or in provoking secondary events, among which reactive oxygen species (ROS) production is a common phenomenon. Both pathways can be mediated by the activity of the cytochrome P450 monooxygenase system. We studied induction of the CYP 1A or CYP 2B monooxygenases in rat liver by the fungicides: thiram, captan, captafol, dodine and the drugs: nitrofurazone, furazolidone and the plant flavonoid: quercetin. A cytochrome P450 induction assay (CYPIA test) was used. S9 prepared from livers of rats treated with the test compounds were used to activate ethidium bromide (EtBr) (CYP 1A isoenzyme) or cyclophosphamide (CPA) (CYP 2B isoenzyme) in the Ames test.It was found that among the tested compounds, the most potent inducer of CYP 1A was furazolidone (3× 80 mg/kg). Less potent was thiram (1× 100 mg/kg), as well as quercetin (3× 80 mg/kg), and captafol (1× 30 mg/kg). On the other hand, thiram (1× 100 mg/kg), captafol (1× 30 mg/kg), and quercetin (3× 80 mg/kg) were most potent in the CYP 2B isoenzyme induction, while furazolidone (3× 80 mg/kg), and nitrofurazone (3× 80 mg/kg) appeared to be less potent in this respect. Captan and dodine (3× 80 mg/kg) did not affect the activity of any of the cytochrome P450 isoenzymes.     

The Emerging Role of VHS Domain‐Containing Tom1, Tom1L1 and Tom1L2 in Membrane Trafficking

The maintenance of cellular homeostasis and execution of regulatory mechanisms to dynamically govern various cellular processes require the correct delivery of proteins to their target subcellular compartments. It is estimated that over 30% of the proteins encoded by the human genome, projected to encode about 25 000 proteins and other macromolecules, are delivered to the secretory and endocytic pathways where movement of proteins between various compartments is primarily mediated by vesicles/carriers budding from one compartment for delivery to another. Sorting of cargo proteins into budding vesicles/carriers is mediated by adaptors that link the cargo proteins to the coat proteins. The adaptor function of VHS domain proteins, GGA proteins, STAM proteins and Hrs is well‐established and is evolutionarily conserved from yeast to humans. Recent studies suggest that Tom1, Tom1L1 and Tom1L2 subfamily of VHS domain proteins, which do not exist in yeast, are emerging as novel regulators for post‐Golgi trafficking and signaling.