Protein tyrosine phosphatase 1B (PTP1B) contributes to leptin resistance by inhibiting intracellular leptin receptor signaling. Mice with whole body or neuron-specific deletion of PTP1B are hypersensitive to leptin and resistant to diet-induced obesity. Here we report a significant increase in PTP1B protein levels in the mediobasal hypothalamus (P = 0.003) and a concomitant reduction in leptin sensitivity following 28 days of high-fat (HF) feeding in rats. A significant increase in PTP1B mRNA levels was also observed in rats chronically infused with leptin (3 microg/day icv) for 14 days (P = 0.01) and in leptin-deficient ob/ob mice infused with leptin (5 microg/day sc for 14 days; P = 0.003). When saline-infused ob/ob mice were placed on a HF diet for 14 days, an increase in hypothalamic PTP1B mRNA expression was detected (P = 0.001) despite the absence of circulating leptin. In addition, although ob/ob mice were much more sensitive to leptin on a low-fat (LF) diet, a reduction in this sensitivity was still observed following exposure to a HF diet. Taken together, these data indicate that hypothalamic PTP1B is specifically increased during HF diet-induced leptin resistance. This increase in PTP1B is due in part to chronic hyperleptinemia, suggesting that hyperleptinemia is one mechanism contributing to the development of leptin resistance. However, these data also indicate that leptin is not required for the increase in hypothalamic PTP1B or the development of leptin resistance. Therefore, additional, leptin-independent mechanisms must exist that increase hypothalamic PTP1B and contribute to leptin resistance.

No abstract

SummaryThe manifestation of virulence traits in Cryptococcus neoformans is thought to rely on intracellular transport, a process not fully explored in this pathogenic fungus. Through interaction cloning, we identified a multi-modular protein, Cin1 (cryptococcal intersectin 1), whose domain structure is similar to that of the human endocytic protein ITSN1. Cin1 contains an N-terminal EH domain, a central coiled-coil region, a WH2 domain, two SH3 domains and a C-terminal RhoGEF (DH)-PH domain. Interestingly, alternative mRNA splicing resulted in two Cin1 isoforms, and Cin1 homologues are also restricted to basidiomycetous fungi. Disruption of the CIN1 gene had a pleiotropic effect on growth, normal cytokinesis, intracellular transports and the production of several virulence factors. Additionally, Cin1 interacts with cryptococcal Cdc42 and Wsp1 (a WASP homologue) proteins in vitro, suggesting a conserved role in the regulation of the actin cytoskeleton. However, deletion of RhoGEF or SH3 and RhoGEF domains did not result in any phenotypic changes, suggesting that functional redundancy exists in proteins containing similar domains or that the activities by other domains are necessary for Cin1 function. Our study presents the first evidence of a multi-modular protein whose function in intracellular transport underlies the growth, differentiation and virulence of a pathogenic microorganism.

Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14 C or 3 H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined. [U- H]glucose were shown to be C-1 and H-5, respectively. These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations. Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria. However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion. Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.The structure (Fig. 1) of the mycobacterial cell wall arabinan moiety of the arabinogalactan (henceforth referred to simply as arabinan) reveals that it is a very complex branched polysaccharide of arabinofuranosyl (Araf) resides (1). It provides a key structural connection between the peptidoglycan and the mycolic acids (9). Thus, the mycolic acids are attached in a specific fashion to a nonreducing end pentaarabinofuranosideThe reducing end of the arabinan is attached to a galactofuran (1) which is finally attached to the peptidoglycan via the actinomycete-specific linker disaccharide [34-Rha-(133)-GlcNAc-(13phosphate)] (10). This structural information suggests that arabinan is essential for cell wall integrity, and the antimycobacterial activity of ethambutol, an inhibitor of arabinan formation (3, 14), confirms this expectation.In parallel with what is known about the biosynthesis of other polysaccharides (12), it is expected that the arabinan is synthesized via arabinosyltransferases. These enzymes should utilize a 1-phosphorylated arabinose (either an arabinosyl sugar nucleotide or an arabinosyl polyprenyl phosphate) as the donor and the incomplete growing arabinan as the acceptor. Indeed, ␤-D-Araf-monophosphodecaprenol has been isolated and characterized from a variety of mycobacteria (18,19), and it has been demonstrated to function as an arabinosyl donor (7a, 21). It remains unknown if ␤-D-Araf-monophosphodecaprenol is the sole donor of arabinosyl residues or if there is an aqueous soluble arabinosyl nucleotide donor as well. In this regard, a preliminary report of a partially purifie...

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.

Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen Cryptococcus neoformans. Crg1 negatively regulates pheromone responses and mating through direct inhibition of G␣ subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving ⌬crg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated ⌬crg2-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that ⌬crg2 mutants formed enlarged capsules that mimic cells expressing a constitutively active GPA1(Q284L) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.Regulators of G-protein signaling (RGS) are a family of proteins that share the highly conserved RGS domain of about 125 amino acid residues and play an important role in cell communication among eukaryotic organisms. The RGS domain allows it to interact specifically with the cognate G␣ to promote signaling inactivation through acceleration of G␣-bound GTP to GDP hydrolysis (7,8,10,15,21,34). In humans, more than 20 RGS proteins have been reported, which are also known to have at least 20 G␣ subunits, 6 G␤ subunits, and 11 G␥ subunits (20,31,43). In the lower eukaryotes, such as the budding yeast Saccharomyces cerevisiae that contains two G␣ subunits, one G␤ subunit, and one G␥ subunit, four RGS and RGS-like proteins, including Sst2, Rgs2, Rax1, and Mdm1, have been described. Sst2 is an archetypical RGS protein that functions as a GTPase-activating protein (GAP) to inhibit G␣ Gpa1 and promote pheromone desensitization (3, 9), whereas Rgs2, identified through a screen for multicopy suppressors of Gpa2-mediated glucose-induced loss of heat resistance, was shown to act as a negative regulator of Gpa2 and glucoseinduced cyclic AMP (cAMP) signaling (38). Although Rax1 (return to axial) and Mdm1, a multidomain-containing protein similar to mammalian sorting nexin 13, contain the conserved RGS and RGS-like domains, there is no evidence demonstrating their direct involvement in G-protein signaling (13,27,28).The...

Abstract. The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.

No abstract