Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzo- (Fig. la) was constructed containing a 6.4-kb genomic region including exons 7, 9, and 10 of the y2 subunit gene isolated from a 129SV mouse genomic library. A 1.2-kb genomic Pvu II-Nco I fragment including exon 8 (coding for amino acids 306-375 of the y2 polypeptide) was replaced with the phosphoglycerate kinase (PGK)-neo cassette (11), and a tk expression cassette (12) was added at the 3' end of the y2 sequence. Splicing from exon 7 to exon 9 would result in a stop of the translational reading frame and prohibit expression of sequences downstream of exon 7. Before electroporation into E14 ES cells (13), the plasmid was linearized at a polylinker site adjacent to the 5' end of the 7y2 genomic sequence. E14 ES cells were cultured on irradiated G418-resistant feeder cells obtained from CD1-M-TKneo2 mouse embryos [BRL, Fullinsdorf (Basel)] in GMEM (Glasgow modification of Eagle's medium; Flow Laboratories) containing 10% total calf serum and leukemia inhibitory factor (103 units/ml, Life Technologies). The cells were transfected and screened for homologous recombinants (14) by using PCR and the primers y2.19 (5'-CATCT CCATC GCTAA GAATG TTCGG derived from 7y2 sequences upstream of the targeting vector and Y2.20 (5'-ATGCT CCAGA CTGCC TTGGG AAAAG C-3') derived from PGK promoter sequences (11). Chimeric mice were generated (15) and mated to C57BL/6 females, and the offspring were genotyped by PtR amplification of tail DNAs. Reactions specific for the disrupted y2 allele [(0) Abbreviations: BZ, benzodiazepine; GABA, y-aminobutyric acid; DRG, dorsal root ganglion (ganglia); ES, embryonic stem; E, embryonic day; P, postnatal day.§To whom reprint requests should be addressed. 7749The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473 p-AKT, 13 of 21 (62%) Ser2448 p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.
The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA-and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses.
We compared a cryptococcal culture filtrate antigen referred to as CneF with chemically defined cryptococcal antigen fractions isolated by Cherniak and co-workers by using double immunodiffusion gels, polyacrylamide gel electrophoresis, immunoblots, and footpad reactivity of immunized mice. The three previously described components of cryptococcal culture filtrates are a high-molecular-weight glucuronoxylomannan (GXM), which is the major constituent, a galactoxylomannan (GalXM), and a mannoprotein (MP). In this study we demonstrated that CneF contained components which were serologically and electrophoretically similar to the three previously described cryptococcal culture filtrate fractions. The MP fraction elicited significantly stronger delayed-type hypersensitivity responses than did the GXM or GalXM fraction when used in mice immunized either with the CneF in complete Freund adjuvant or whole heat-killed Cryptococcus neoformans yeast cells. These findings were confirmed when the footpads of immunized mice were challenged with GalXM and MP preparations from a culture filtrate of a C. neoformans acapsular mutant that does not produce GXM. Thus, we concluded that the MP was the primary component recognized by the anticryptococcal cell-mediated immune response in mice. Cell-mediated immunity (CMI), which can be assessed by determining the level of delayed-type hypersensitivity (DTH), is an important host defense mechanism in cryptococcosis. The cryptococcal antigen preparations used for detection of DTH are heterogeneous mixtures of relatively high-molecular-weight (>50,000) polysaccharides and proteoglycans. One such preparation, which specifically detects CMI responses to Cryptococcus neoformans and has been extensively studied in the murine cryptococcosis model, is a culture filtrate antigen referred to as CneF. When injected subcutaneously into mice, CneF (200 ,ug of carbohydrate) in complete Freund adjuvant (CFA) induces an anticryptococcal DTH response (15). However, if given intravenously, CneF will induce antigen-specific immunological suppression (15). CneF can be separated electrophoretically in 3% polyacrylamide gels into two components which stain with periodic acid-Schiff reagent (PAS) (16). One component migrates only a short distance into the gel, is glycosidic, and does not elicit significant skin test reactions in C. neoformans-sensitized guinea pigs (16). The other component is a fast-migrating fraction which moves with the tracking dye. The rapidly moving component stains for both carbohydrate and protein and elicits positive skin test responses in sensitized guinea pigs (16). Recently, Cherniak and co-workers (4, 5, 19) have fractionated C. neoformans culture filtrates which were prepared in a manner similar to that used for the culture filtrates from which CneF was obtained. They characterized three major components, i.e., glucuronoxylomannan (GXM), a high-molecular-weight, serotype-specific polysaccharide; galactoxylomannan (GalXM), a polysaccharide with a molecular mass of approximately 27...
Dermatophytoses are known to cause considerable discomfort, cosmetic problems and financial loss that have been recognized as a significant health concern worldwide. Since currently available antifungal agents have limitations in their efficacy, new agents are being developed. This study was undertaken to optimize an in vivo model of experimental dermatophytosis for evaluation of the efficacy of antifungal compounds. Guinea pigs were infected with different inocula of T. mentagrophytes to establish dermatophytosis. The optimal conditions for dermatophytosis in guinea pigs were found to be an inoculum size of 1 x 10(7) fungal cells applied on abraded skin. After optimization, animals were treated with oral or topical formulations of terbinafine. The optimized guinea pig model was found to be highly reproducible, and useful in the primary screening and evaluation of the anti-dermatophytic efficacy of topical and oral formulations of antifungal agents.
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