Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth.

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.

Role of the Abcg2 transporter in plasma levels and tissue accumulation of the anti-inflammatory tolfenamic acid in mice.

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter that is expressed in the apical membrane of cells from relevant tissues involved in drug pharmacokinetics such as liver, intestine, kidney, testis, brain and mammary gland, among others. Tolfenamic acid is an anti-inflammatory drug used as an analgesic and antipyretic in humans and animals. Recently, tolfenamic acid has been repurposed as an antitumoral drug and for use in chronic human diseases such as Alzheimer. The aim of this work was to study whether tolfenamic acid is an in vitro Abcg2 substrate, and to investigate the potential role of Abcg2 in plasma exposure, secretion into milk and tissue accumulation of this drug. Using in vitro transepithelial assays with cells transduced with Abcg2, we showed that tolfenamic acid is an in vitro substrate of Abcg2. The in vivo effect of this transporter was tested using wild-type and Abcg2-/- mice, showing that after oral and intravenous administration of tolfenamic acid, its area under the plasma concentration-time curve in Abcg2-/- mice was between 1.7 and 1.8-fold higher compared to wild-type mice. Abcg2-/- mice also showed higher liver and testis accumulation of tolfenamic acid after intravenous administration. In this study, we demonstrate that tolfenamic acid is transported in vitro by Abcg2 and that its plasma levels as well as its tissue distribution are affected by Abcg2, with potential pharmacological and toxicological consequences.

Sterol uptake analysis in Saccharomyces and non-Saccharomyces wine yeast species.

Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis.

MmpL3 Inhibition: A New Approach to Treat Nontuberculous Mycobacterial Infections.

Outside of Mycobacterium tuberculosis and Mycobacterium leprae, nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.

Flavonoids as Novel Efflux Pump Inhibitors and Antimicrobials Against Both Environmental and Pathogenic Intracellular Mycobacterial Species.

Therapeutic treatment options for opportunistic non-tuberculous mycobacterial (NTM) infection and/or serious mycobacterial infections such as tuberculosis (TB) and leprosy are limited due to the spread of antimicrobial resistance mechanism. Plant-derived natural compounds as prospective efflux pump inhibitors may present a promising adjunct to conventional chemotherapy by enhancing mycobacterial susceptibility to antibiotics. This study served to evaluate the antimicrobial and resistance-modifying profile of a range of plant-derived flavonoids against the mycobacterial model strains: M. smegmatis, M. aurum, and M. bovis BCG. The minimum inhibitory concentrations (MICs) of the compounds against the mycobacterial strains were determined using both agar dilution and broth dilution assays, while their efflux inhibitory activity was investigated via an ethidium bromide-based fluorometric assay. All compounds were screened for their synergistic effects with ethidium bromide (EtBr) and rifampicin (RIF) against M. smegmatis. Skullcapflavone II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone, 1) exerted potent antimicrobial activity against M. aurum and M. bovis BCG and considerably increased the susceptibility of M. smegmatis to EtBr and RIF. Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, 2) was determined to be the most potent efflux-inhibitor in M. aurum and M. smegmatis. However, a connection between strong modulatory and putative efflux activity of the compounds could not be observed. Nevertheless, the results highlight two polymethoxyflavones, skullcapflavone II and nobiletin, with potent antimycobacterial and antibiotic resistance modulating activities as valuable adjuvants in anti-mycobacterial therapies.

Rab32 restriction of intracellular bacterial pathogens.

Our immune system is engaged in a continuous battle against invading pathogens, many of which have evolved to survive in intracellular niches of mammalian hosts. A variety of cellular processes are involved in preventing bacterial invasion or in killing bacteria that successfully invade host cells. Recently, the Rab GTPase Rab32 emerged as critical regulator of a host defense pathway that can eliminate bacterial pathogens. Salmonella enterica is an intracellular bacterium and a major cause of infections and deaths in humans. Rab32 and its guanine nucleotide exchange factor BLOC-3 are essential to prevent the growth of the human-restricted Salmonella enterica serovar Typhi (S. Typhi) in mice, a non-susceptible host. The importance of the Rab32/BLOC-3 pathway has been recently confirmed by the finding that broad-host Salmonella enterica serovars deliver 2 bacterial effectors to neutralize this pathway and infect mice. Rab32 has also been shown to control infection by Listeria monocytogenes, another medically relevant intracellular pathogen. In addition, genetic evidence indicate a possible role of Rab32 in controlling leprosy, a disease caused by Mycobacterium leprae in humans, suggesting that a Rab32-dependent pathway can also act as a host defense pathway in humans. The Rab32 role in bacterial pathogen restriction is discussed here and compared to the function of this GTPase in other cellular processes.

The Debaryomyces hansenii carboxylate transporters Jen1 homologues are functional in Saccharomyces cerevisiae.

We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1Δ ady2Δ strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (Km 0.94 ± 0.17 mM; Vmax 0.43 ± 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (Km 0.27 ± 0.04 mM; Vmax 0.11 ± 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, Km 0.31 ± 0.06 mM; Vmax 0.83 ± 0.04 nmol s(-1) mg(-1)and Km 0.16 ± 0.02 mM; Vmax 0.19 ± 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii.

pH Dependent but not P-gp Dependent Bidirectional Transport Study of S-propranolol: The Importance of Passive Diffusion.

PURPOSE: Recent controversial publications, citing studies purporting to show that P-gp mediates the transport of propranolol, proposed that passive biological membrane transport is negligible. Based on the BDDCS, the extensively metabolized-highly permeable-highly soluble BDDCS class 1 drug, propranolol, shows a high passive permeability at concentrations unrestricted by solubility that can overwhelm any potential transporter effects. Here we reinvestigate the effects of passive diffusion and carrier-mediated transport on S-propranolol. METHODS: Bidirectional permeability and inhibition of efflux transport studies were carried out in MDCK, MDCK-MDR1 and Caco-2 cell lines at different concentrations. Transcellular permeability studies were conducted at different apical pHs in the rat jejunum Ussing chamber model and PAMPA system. RESULTS: S-propranolol exhibited efflux ratios lower than 1 in MDCK, MDCK-MDR1 and Caco-2 cells. No significant differences of Papp, B->A in the presence and absence of the efflux inhibitor GG918 were observed. However, an efflux ratio of 3.63 was found at apical pH 6.5 with significant decrease in Papp, A->B and increase in Papp, B->A compared to apical pH 7.4 in Caco-2 cell lines. The pH dependent permeability was confirmed in the Ussing chamber model. S-propranolol flux was unchanged during inhibition by verapamil and rifampin. Furthermore, pH dependent permeability was also observed in the PAMPA system. CONCLUSIONS: S-propranolol does not exhibit active transport as proposed previously. The "false" positive efflux ratio can be explained by the pH partition theory. As expected, passive diffusion, but not active transport, plays the primary role in the permeability of the BDDCS class 1 drug propranolol.

Characterization of new polyol/H+ symporters in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H(+) (and sugar/H(+)) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H(+) symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H(+) symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H(+) 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H(+) 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.

Identification of a glucose permease from Mycobacterium smegmatis mc2 155.

We report here the molecular identification of a glucose permease from Mycobacterium smegmatis,a model organism for our understanding of the life patterns of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae. A computer-based search of the available genome of M. smegmatis mc(2) 155 with the sequences of well-characterized glucose transporters revealed the gene msmeg4187 as a possible candidate. The deduced protein belongs to the major facilitator superfamily of proton symporters and facilitators and exhibits up to 53% of amino acid identity to other members of this family. Heterologous expression of msmeg4187 in an Escherichia coli glucose-negative mutant led to the restoration of growth on glucose. The determination of the biochemical features characterize MSMEG4187 (GlcP) as a high affinity (K(m) of 19 microM), glucose-specific permease. The results represent the first molecular characterization of a sugar permease in mycobacteria, and thus supply fundamental data for further in-depth analysis on the nutritional lifestyle of these bacteria.

Yarrowia lipolytica vesicle-mediated protein transport pathways.

BACKGROUND: Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. RESULTS: We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. CONCLUSION: These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae.

Mechanisms underlying the halotolerant way of Debaryomyces hansenii.

The yeast Debaryomyces hansenii is usually found in salty environments such as the sea and salted food. It is capable of accumulating sodium without being intoxicated even when potassium is present at low concentration in the environment. In addition, sodium improves growth and protects D. hansenii in the presence of additional stress factors such as high temperature and extreme pH. An array of advantageous factors, as compared with Saccharomyces cerevisiae, is putatively involved in the increased halotolerance of D. hansenii: glycerol, the main compatible solute, is kept inside the cell by an active glycerol-Na+ symporter; potassium uptake is not inhibited by sodium; sodium protein targets in D. hansenii seem to be more resistant. The whole genome of D. hansenii has been sequenced and is now available at http://cbi.labri.fr/Genolevures/ and, so far, no genes specifically responsible for the halotolerant behaviour of D. hansenii have been found.

Metal ion transport and regulation in Mycobacterium tuberculosis.

The regulation of metal ion concentrations is central to the physiology of the interaction between pathogenic bacteria and their hosts. Apart from the NRAMP orthologue, MntH, metal ion transporters in Mycobacterium tuberculosis have not been studied. Mn, the physiological substrate of MntH in other bacteria, may play an important role as a structural and redox-active cofactor in a wide range of metabolic processes. Fe, Cu and Zn play structural and catalytic roles in metalloenzymes involved in oxidative stress responses. Fe and Mg are required for growth in macrophages. Genomic analyses reveal 28 sequences encoding a broad repertoire of putative metal ion transporters (or transporter subunits), representing 24% of all transporters in this organism. These comprise 8 families of secondary active transporters and 3 families of primary active transporters, including 12,P, type ATPases. Potential metal ion specificities include K+, Na+, Cu2+, Cd2+, Zn2+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Fe2+/3+, Hg2+, AsO2- and AsO4(2-). 17 of these transporters are also encoded as complete open reading frames in Mycobacterium leprae, suggesting a role in intracellular survival. Iron transcriptionally regulates a diverse set of genes via the iron-dependent DNA-binding proteins, Fur and IdeR. Changes in Fe and Mg concentrations signal entry into the intracellular compartment and potentially trigger up-regulation of virulence determinants. The plethora of putative transport systems encoded by the M. tuberculosis genome contrasts strikingly with the paucity of experimental data on these systems. The detailed analysis of the temporal pattern of M. tuberculosis transporter gene expression during infection will provide important insights into the basic biology of intracellular parasitism and may help to shape novel therapeutic strategies.

Studies on the time course of the effects of the probiotic yeast Saccharomyces boulardii on electrolyte transport in pig jejunum.

Orally administered Saccharomyces boulardii (synonym Saccharomyces cerevisiae Hansen CBS 5926) has already been shown to affect relevant functions of the mucosa in pig jejunum such as lowering the secretory response to theophylline or stimulating sodium/glucose cotransport, but knowledge of time-dependent relationship is minimal. In this study we examined the effects of S. boulardii on sodium (Na+) and chloride (Cl-) transport in pig jejunum under nonstimulated (basal) and stimulated (secretory) conditions. For this purpose the conventional Ussing chamber method was used for measuring electrical parameters (short circuit currents, Isc; tissue conductances, G(T)) and electrolyte transport of isolated intact jejunal epithelia in the absence and presence of the secretagogue theophylline (10 mM, serosal side). Time profiles of the mucosa response were assessed by treating animals perorally with S. boulardii for 0 (control), 3, 8, and 16 days. Intestinal tissues were obtained from growing pigs in the weight range between 25 and 40 kg. All animals were fed twice daily and received 1.0-1.6 kg/day of a standard diet avoiding probiotics as food additives. After a 9- to 10-day adaptation period the diets for treated animals were supplemented with approximately 1.8 x 10(7) colony forming units (CFU)/g feed of the probiotic. Whereas basal tissue conductances were not affected by treatment duration, basal Isc values decreased significantly during 8 days of treatment, by 26%, indicating a lower electrogenic net ion transport, which, however, was reconstituted after 16 days. This effect could be explained by almost the same reduction of basal Jms of Na+ during 8 days of treatment, whereas respective flux rates in the opposite direction remained stable. Under basal conditions unidirectional and net flux rates of Cl- were not affected by S. boulardii. Induction of secretory conditions by theophylline revealed pronounced increases in net Cl- secretion but this effect was more than 60% lower after 8-day S. boulardii application, and this was reflected by a respectively lower Isc stimulation. Interestingly, this inhibitory effect on the secretory response could no longer be observed in the 16-day group. And this was reflected by a respectively lower Isc stimulation. A similar effect could be observed regarding net Na+ flux rates. Residual fluxes were affected neither by S. boulardii nor by theophylline, therefore, Isc values can be explained completely by respective Na+ and Cl- fluxes. In conclusion, S. boulardii has specific duration-dependent effects on the secretory response of the pig jejunal mucosa which developed during 8-day treatment but disappeared during further application. Thus, this study supports the concept that probiotics may exert beneficial effects in the gastrointestinal tract.

CD1a and langerin: acting as more than Langerhans cell markers.

Langerhans cells (LCs) represent a unique DC subset populating the outermost body surface, i.e., the epidermis. Although CD1a and langerin (CD207) are used as specific markers to distinguish LCs from other DC subsets, their immunological functions have remained mostly unknown. A new paper (see the related article beginning on page 701) demonstrates that LCs utilize these markers to induce cellular immune responses to Mycobacterium leprae: CD1a mediates the presentation of nonpeptide antigens to T cells, while langerin facilitates uptake of microbial fragments and perhaps their delivery to a specialized subcellular compartment.

Passive and iontophoretic transport enhancement of insulin through porcine epidermis by depilatories: permeability and fourier transform infrared spectroscopy studies.

The effect of thioglycolate-based depilatory lotions was studied on the in vitro passive and iontophoretic permeability of insulin through porcine epidermis and biophysical changes in the stratum corneum (SC) lipids and proteins. The porcine epidermis and Franz diffusion cells modified for iontophoresis were used for the in vitro transport studies. Cathodal iontophoresis was performed at 0.2 mA/cm2 current density. Resistance of the control- and depilatory-lotion-treated epidermis was determined according to Ohm's law. Biophysical changes were studied on porcine SC before (control) and after treatment with the depilatory lotions using Fourier transform infrared (FT-IR) spectroscopy. Asymmetric (approximately 2915 cm(-1)) and symmetric approximately 2848 cm(-1)) Carbon-Hydrogen (C-H) stretching absorbances were studied to estimate the extent of lipid extraction. Fourier self-deconvolution and second derivative procedures were applied to amide I band (1700-1600 cm(-1)) in order to estimate quantitatively the changes in the secondary structure of the SC protein. The passive permeability of insulin was significantly (P <.05) increased through depilatory-lotion-treated (ie, Better Off, Marzena, and Sally Hansen) epidermis in comparison to control. Iontophoresis significantly enhanced (P <.05) the permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis. Further, we were able to achieve the desired flux of insulin (5.25 U/cm2/d) through Better Off-treated epidermis using 0.2 mA/cm2 current density and 100 U/mL donor concentration of insulin. The SC treated with depilatory lotions showed a decrease in peak areas of C-H stretching absorbances in comparison with untreated SC. Depilatory lotion treatment also decreased (P <.05) the epidermal resistance in comparison with the control epidermis. The decrease in the alpha-helix conformation and the increase in the random and turn structures were observed in the SC proteins due to depilatory lotion treatment. The changes in the secondary structure of proteins and lipid extraction from the SC are suggested as the cause of the decrease in the epidermal resistance and the increase in the passive and iontophoretic permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis.

Hypotonic cell swelling induces translocation of the alpha isoform of cytosolic phospholipase A2 but not the gamma isoform in Ehrlich ascites tumor cells.

We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor cells. Both enzymes are almost uniformly distributed throughout the cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic cell swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich cells, was immediately and transiently increased on hypotonic cell swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic cell swelling is not accompanied by any detectable increase in intracellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.

The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system.

We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N. O., and Roseman, S. (1997) Proc. Natl. Acad. Sci., U. S. A. 94, 14367-14371). A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-[(3)H]diacetylthiochitobioside ([(3)H]Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Here and in the accompanying papers (Keyhani, N. O., Boudker, O., and Roseman, S. (2000) J. Biol. Chem. 275, 33091-33101; Keyhani, N. O., Bacia, K., and Roseman, S. (2000) J. Biol. Chem. 275, 33102-33109; Keyhani, N. O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S. (2000) J. Biol. Chem. 275, 33110-33115), we report that transport of [(3)H]Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester. The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide. The [(3)H]Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3. The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.

Genomic arrangement of a putative operon involved in maltose transport in the Mycobacterium tuberculosis complex and Mycobacterium leprae.

A Mycobacterium bovis gene coding for a putative MalE maltose binding protein was cloned and its full-length sequence determined. Database searches revealed 99.9% identity with IpqY, encoding a putative sugar uptake protein from Mycobacterium tuberculosis strain H37Rv. The deduced protein product showed high sequence similarity to MalE-like proteins from a variety of bacterial species, including Mycobacterium leprae. Analysis of flanking database sequences from M. tuberculosis and M. leprae revealed the presence of malF-, malG- and malK-like genes. Comparison of these mycobacterial sequences with other maltose operons has allowed us to deduce a unique genomic arrangement of the genes involved in the uptake of maltose in members of the Mycobacterium tuberculosis complex and M. leprae.