Faculty Bibliography

Caspofungin acetate (CAS) is a member of a new class of clinically-approved echinocandin drugs to treat invasive aspergillosis. CAS inhibits the activity of beta-1,3-D-glucan synthase (GS), thus damaging the fungal cell wall. Although no clinical resistance of Aspergillus to CAS has been reported as yet, the development of in vitro reduced susceptibility is presumed to be inevitable. By contrast, echinocandin resistance in laboratory strains of Candida albicans and Saccharomyces cerevisiae has been well documented. To study the potential for clinical resistance in Aspergillus, two classes of Aspergillus fumigatus mutant strains were isolated that exhibited reduced susceptibility to CAS. In the first class, a site-directed mutation within the target gene (AfFKS1, encoding the putative catalytic subunit of GS) was introduced and shown to confer low-level (16-fold) reduced susceptibility. A second class of spontaneous mutants were sensitive to low levels of drug but displayed nearly normal growth above 0.5 microg/ml, suggesting induction of an unknown resistance mechanism. At higher levels of drug (> or = 16 microg/ml), the mutants displayed partially restored sensitivity. Preliminary studies indicate that neither target site mutations, nor changes in target gene expression are present in these strains, as has been documented for several yeasts. Instead, preliminary results indicate that the molecular mechanism(s) underlying reduced susceptibility of CAS in the A. fumigatus strains is novel, possibly due to remodeling of the cell wall components.

The aldose reductase pathway has been demonstrated to be a key component of myocardial ischemia reperfusion injury. Previously, we demonstrated that increased lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, is an important change that drives the metabolic cascade mediating ischemic injury. This study investigated signaling mechanisms by which the aldose reductase pathway mediates myocardial ischemic injury. Specifically, the influence of the aldose reductase pathway flux on JAK-STAT signaling was examined in perfused hearts. Induction of global ischemia in rats resulted in JAK2 activation followed by STAT5 activation. Pharmacological inhibition of aldose reductase or sorbitol dehydrogenase blocked JAK2 and STAT5 activation and was associated with lower lactate/pyruvate ratio and lower protein kinase C activity. Niacin, known to lower cytosolic NADH/NAD+ ratio independent of the aldose reductase pathway inhibition, also blocked JAK2 and STAT5 activation. Inhibition of protein kinase C also blocked JAK2 and STAT5 activation. Transgenic mice overexpressing human aldose reductase exhibited increased JAK2 and STAT5 activation. Pharmacological inhibition of JAK2 reduced ischemic injury and improved functional recovery similar to that observed in aldose reductase pathway inhibited mice hearts. These data, for the first time, demonstrate JAK-STAT signaling by the aldose reductase pathway in ischemic hearts and is, in part, due to changes in cytosolic redox state

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

The control of peripheral lymphocyte numbers is a fundamental aspect of the immune system. Regulatory T cells are involved in the suppression of autoimmune, antitumor, allergic, and other inflammatory responses, as well as in facilitating graft acceptance. In this paper, we discuss whether the control of homeostatic proliferation is another facet of the immune system that is controlled by regulatory T cells. A review of the published data connecting regulatory T cells with the control of homeostatic proliferation indicates that several key questions remain open. One of these relates to the stage at which regulatory T cells could play a role (i.e., T-cell proliferation vs. survival)

Mammalian fertilization has been the subject of intensified research in recent times. Application of recombinant DNA, transgenic and gene targeting technology, in particular, to issues in mammalian fertilization has revolutionized the field. Here, we present some of the latest results coming from application of these and other technologies to four aspects of mammalian fertilization: 1. formation of the egg zona pellucida (ZP) during oocyte growth; 2. species-specific binding of sperm to the egg zona pellucida; 3. induction of the sperm acrosome reaction (AR) by the egg zona pellucida 4. binding of sperm to and fusion with egg plasma membrane. In virtually every instance, new information and new insights have come from relatively recent investigations.

Octopamine biosynthesis requires tyrosine decarboxylase to convert tyrosine into tyramine and tyramine beta-hydroxylase to convert tyramine into octopamine. We identified and characterized a Caenorhabditis elegans tyrosine decarboxylase gene, tdc-1, and a tyramine beta-hydroxylase gene, tbh-1. The TBH-1 protein is expressed in a subset of TDC-1-expressing cells, indicating that C. elegans has tyraminergic cells that are distinct from its octopaminergic cells. tdc-1 mutants have behavioral defects not shared by tbh-1 mutants. We show that tyramine plays a specific role in the inhibition of egg laying, the modulation of reversal behavior, and the suppression of head oscillations in response to anterior touch. We propose a model for the neural circuit that coordinates locomotion and head oscillations in response to anterior touch. Our findings establish tyramine as a neurotransmitter in C. elegans, and we suggest that tyramine is a genuine neurotransmitter in other invertebrates and possibly in vertebrates as well

Seven carboxylic acid haptens of 8-prenylnaringenin (8-PN) were synthesized, coupled to cationized bovine serum albumin, and employed to raise specific antisera in rabbits. Two linkers of different lengths (C3H6COOH and C6H12COOH) were coupled to the C7-OH group and separated into their respective enantiomers yielding the first four haptens. Racemic derivatives with C4'-OH coupled linkers C5H10COOH and C9H18COOH were synthesized carrying a methylated C7-OH. Another racemic C4'-OH hapten (CH2COOH) was prepared starting from naringenin. The haptens elicited variable antibody titers dependent on linker lengths, with short linkers giving the best results. Three antisera were characterized in detail: anti-C7-carboxy-propyloxy-2S-(-)-8-PN (anti-H-11), anti-C7-carboxy-propyloxy-2R-(+)-8-PN (anti-H-10), and anti-C4'-carboxy-methoxy-rac-8-PN (anti-H-25). anti-H-10 and anti-H-11 showed about 9% enantiomeric cross-reactivity, and anti-H-11 did not discriminate between isoxanthohumol (IX) and 8-PN (84% cross-reactivity). For anti-H-10, cross-reactivities in the range of 2-5% were found for xanthohumol, IX, and 6-prenylnaringenin. Respective numbers for anti-H-25 were 0.02, 0.1, and 0.2%. Tritiated 8-PN was synthesized yielding a 3H-tracer of high specific radioactivity (2.22 GBq/mg). A radioimmunoassay using anti-H-25 and 3H-8-PN was established and used for the quantitative determination of 8-PN in various beer brands and in the urine of six men after the consumption of three different brands of beer. Furthermore, the dose-dependent excretion of 8-PN was tested after the consumption of a higher volume of a single beer brand with and without spiking with 8-PN and a small oral dose of authentic 8-PN, respectively. Conflicting results led to a pilot test on the in vivo conversion (demethylation) of IX into 8-PN in two men. Conversion rates of 1.9 and 4.4% were estimated. Thus, the total 8-PN dose in beer brands spiced with natural hop or hop products seems to be the sum of the 8-PN amount in a consumed volume and the amount arising from the conversion of IX.

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.

The Hedgehog-GLI signaling pathway is important in animal development and tumorigenesis. Recent findings indicate that the growth and survival of human prostate cancer cells rely upon sustained signaling from the Hedgehog-GLI pathway. These findings have prompted a novel rational strategy for therapeutic treatment of prostate tumors, including metastatic tumors.