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Biomedical subjects

M A Davis

Publications and source records attributed to M A Davis.

At least 19 recordsLinked to original sources
PubMed2006

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity.

We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 muM gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT-PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

Apoptosis↗
PubMed2006

Effects of elevated atmospheric carbon dioxide on biomass and carbon accumulation in a model regenerating longleaf pine community.

Plant species vary in response to atmospheric CO2 concentration due to differences in physiology, morphology, phenology, and symbiotic relationships. These differences make it very difficult to predict how plant communities will respond to elevated CO2. Such information is critical to furthering our understanding of community and ecosystem responses to global climate change. To determine how a simple plant community might respond to elevated CO2, a model regenerating longleaf pine community composed of five species was exposed to two CO2 regimes (ambient, 365 micromol mol(-1) and elevated, 720 micromol mol(-1)) for 3 yr. Total above- and belowground biomass was 70 and 49% greater, respectively, in CO2-enriched plots. Carbon (C) content followed a response pattern similar to biomass, resulting in a significant increase of 13.8 Mg C ha(-1) under elevated CO2. Responses of individual species, however, varied. Longleaf pine (Pinus palustris Mill.) was primarily responsible for the positive response to CO2 enrichment. Wiregrass (Aristida stricta Michx.), rattlebox (Crotalaria rotundifolia Walt. Ex Gmel.), and butterfly weed (Asclepias tuberosa L.) exhibited negative above- and belowground biomass responses to elevated CO2, while sand post oak (Quercus margaretta Ashe) did not differ significantly between CO2 treatments. As with pine, C content followed patterns similar to biomass. Elevated CO2 resulted in alterations in community structure. Longleaf pine comprised 88% of total biomass in CO2-enriched plots, but only 76% in ambient plots. In contrast, wiregrass, rattlebox, and butterfly weed comprised 19% in ambient CO2 plots, but only 8% under high CO2. Therefore, while longleaf pine may perform well in a high CO2 world, other members of this community may not compete as well, which could alter community function. Effects of elevated CO2 on plant communities are complex, dynamic, and difficult to predict, clearly demonstrating the need for more research in this important area of global change science.

Atmosphere↗
PubMed2006

Comparison of a waterless hand-hygiene preparation and soap-and-water hand washing to reduce coliforms on hands in animal exhibit settings.

Outbreaks of enteric disease associated with exposure to live animals on exhibit have occurred with increasing frequency in recent years. Possibly the most important pathogen causing such outbreaks is Escherichia coli O157:H7, because of the serious illness it can cause. Hand hygiene is consistently protective against disease among persons exposed to animals implicated in these outbreaks. Livestock barns have limited hand-washing facilities, therefore a waterless hand-sanitizing gel would be a potentially preventive measure readily available to visitors and animal exhibitors. This study compared the reduction of bacterial counts on hands of animal exhibitors when soap and water was used or when an ethanol-based hand gel was used after animal handling. Participants were youth and adults involved with showing livestock. The sanitation methods were similar in reducing the total bacteria and coliform counts on the hands of the participants (Wilcoxon rank sum test P values 0.12 and 0.69 respectively).

Animals↗
PubMed2004

TamA interacts with LeuB, the homologue of Saccharomyces cerevisiae Leu3p, to regulate gdhA expression in Aspergillus nidulans.

Previous studies have shown that expression of the gdhA gene, encoding NADP-linked glutamate dehydrogenase (NADP-GDH), in Aspergillus nidulans is regulated by the major nitrogen regulatory protein AreA and its co-activator TamA. We show here that loss of TamA function has a more severe effect on the levels of gdhA expression than loss of AreA function. Using TamA as the bait in a yeast two-hybrid screen, we have identified a second protein that interacts with TamA. Sequencing analysis and functional studies have shown that this protein, designated LeuB, is a transcriptional activator with similar function to the homologous Leu3p of Saccharomyces cerevisiae. Inactivation of leuB revealed that this gene is involved in the regulation of gdhA, and an areA; leuB double mutant was shown to have similar NADP-GDH levels to a tamA single mutant. The requirement for TamA function to promote gdhA expression is likely to be due to its dual interaction with AreA and LeuB.

Amino Acid Sequence↗
PubMed2003

Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates.

Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances.

Animals↗
PubMed2002

Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells.

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.

Androstadienes↗
PubMed2001

A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans.

NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, whereas in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Northern blot analysis and reverse transcription-polymerase chain reaction revealed the presence of two idpA transcripts and two transcription start points were identified by sequencing cDNA clones and by 5'-rapid amplification of cDNA ends. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targeting sequence as well as a C-terminal tripeptide (ARL) as a potential peroxisomal targeting signal. The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targeting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targeting sequences were confirmed by microscopic analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localization to mitochondria and peroxisomes was observed during growth on glucose whereas cytoplasmic and peroxisomal localization was found upon acetate or fatty acid induction. Therefore, we have established that by the use of two transcription start points a single gene is sufficient to specify localization of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans.

Amino Acid Sequence↗
PubMed2001

AoHapB, AoHapC and AoHapE, subunits of the Aspergillus oryzae CCAAT-binding complex, are functionally interchangeable with the corresponding subunits in Aspergillus nidulans.

Two genes, AohapB and AohapE, encoding subunits of the Aspergillus oryzae CCAAT-binding complex were cloned and sequenced. The polypeptides encoded by AohapB and AohapE were expressed in Escherichia coli and used to reconstitute a DNA-binding complex with recombinant AoHapC. The DNA-binding activity was observed only in the presence of all three subunits, indicating that AoHapB, AoHapE and AoHapC are essential for CCAAT-binding. Furthermore, introduction of the AohapB, AohapC and AohapE genes into the A. nidulans hapB delta, hapC delta and hapE delta strains, respectively, revealed that the A. oryzae Hap subunits are functionally interchangeable with the corresponding subunits in A. nidulans.

Amino Acid Sequence↗
PubMed2001

Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans.

The tam A gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/Dal81p/DurLp of Saccharomyces cerevisiae. It has been proposed that TamA functions as a co-activator of AreA, the major nitrogen regulatory protein in A. nidulans. Because AreA functions as a transcriptional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus. We found that a GFP-TamA fusion protein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution. As the predicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important regions. We have cloned the tamA gene of A. oryzae and compared its functional and sequence characteristics with those of A. nidulans tamA and S. cerevisiae UGA35/DAL81/DURL. The Aspergillus homologues are highly conserved and functionally interchangeable, whereas the S. cerevisiae gene does not complement a tamA mutant when expressed in A. nidulans. Uga35p/Dal81p/DurLp was also found to be unable to recruit AreA. The sequence changes in a number of tamA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA. Changes in most regions of TamA appeared to destroy its function, suggesting that the overall conformation of the protein may be critical for its activity.

Alleles↗
PubMed2001

The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene.

The ability to utilize formamide as a sole nitrogen source has been found in numerous fungi. We have cloned the fmdS gene encoding a formamidase from Aspergillus nidulans and found that it belongs to a highly conserved family of proteins separate from the major amidase families. The expression of fmdS is primarily regulated via AreA-mediated nitrogen metabolite repression and does not require the addition of exogenous inducer. Consistent with this, deletion analysis of the 5' region of fmdS has confirmed the presence of multiple AreA-binding sites containing a characteristic core GATA sequence. Under carbon starvation conditions the response to nitrogen starvation is eliminated, indicating that the lack of a carbon source may result in inactivation of AreA. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS with its transcript overlapping the fmdS coding region. Disruption of the 5' gene and analysis of the effects of overexpression of this gene on fmdS expression has shown that expression of this upstream gene interferes with fmdS transcription, resulting in a strong dependence on AreA activation for expression. Therefore the relative position of these two genes is essential for normal regulation of fmdS.

Amidohydrolases↗
PubMed2001

Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans.

Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnADelta mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

Amino Acid Sequence↗
PubMed2001

The role of apoptosis in 2',2'-difluoro-2'-deoxycytidine (gemcitabine)-mediated radiosensitization.

The nucleoside analogue Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is active against a wide variety of solid tumors and is a potent radiation sensitizer. Because apoptosis has been shown to be an important mechanism of cell death for many cancers, we wished to investigate the role of apoptosis in dFdCyd-mediated radiosensitization. We evaluated HT29 colon cancer cells, UMSCC-6 head and neck cancer cells, and A549 lung cancer cells, which differ substantially in the ability to undergo radiation-induced apoptosis. We hypothesized that if dFdCyd produced radiosensitization by potentiating preexisting death pathways, then only the apoptotic-prone HT29 cells would show a substantial increase in apoptosis when treated with the combination of dFdCyd and radiation and that UMSCC-6 cells and A549 cells would be radiosensitized through nonapoptotic mechanisms. We found that the radiosensitization of HT29 cells (enhancement ratio, 1.81 +/- 0.16) was accompanied by an increase in apoptosis and by caspase activation and that inhibition of this activation by the caspase inhibitor Z-Asp-Glu-Val-Asp-fluoromethylketone (DEVD) significantly decreased radiosensitization (to 1.36 +/- 0.24; P < 0.05). In contrast, UMSCC-6 cells and A549 cells were modestly radiosensitized (enhancement ratio, 1.47 +/- 0.24 and 1.31 +/- 0.04, respectively) via a nonapoptotic mechanism. These findings suggest that although apoptosis can contribute significantly to dFdCyd-mediated radiosensitization, the role of apoptosis in dFdCyd-mediated radiosensitization depends on the cell line rather than representing a general property of the drug.

Apoptosis↗
PubMed2000

Radiosensitization produced in vivo by once- vs. twice-weekly 2'2'-difluoro-2'-deoxycytidine (gemcitabine).

PURPOSE: Gemcitabine (2'2'-difluoro-2'-deoxycytidine, dFdCyd) is a potent radiosensitizer of rodent and human tumor cells. Our Phase I clinical trial using once-weekly dFdCyd as a radiosensitizer in the treatment of patients with Stage IV squamous cell head and neck cancer has produced a high rate of tumor response and significant normal mucosal toxicity. These findings raised the question of whether we are using dFdCyd in the optimal dose and schedule. In vitro studies suggest that twice-weekly dFdCyd has the potential to be more effective than once-weekly dFdCyd when administered in combination with radiation (RT) given 5 days per week. Therefore, we have used a mouse model to assess whether the therapeutic ratio of combined modality therapy may be improved by using a twice-weekly drug regimen. We asked two questions: 1) Does a once-weekly or twice-weekly dFdCyd regimen cause more normal tissue radiosensitization? 2) Does a once-weekly or twice-weekly dFdCyd + RT regimen produce a better therapeutic index? METHODS AND MATERIALS: To assess normal tissue toxicity, C3H mice underwent mouth (60)Co RT (27.5 Gy in 5 daily fractions) +/- dFdCyd delivered intraperitoneally (IP) either once or twice weekly 6 hours prior to irradiation. Acute lip reactions were quantified according to a standard scoring system, and weight loss was measured. We measured tumor control using squamous cell carcinoma (SCC) VII murine squamous cell flank tumors (50-125 mm(3)) treated with the same regimens used in the mouth irradiation model. RESULTS: We found that dFdCyd delivered 800 mg/kg once weekly or 150 mg/kg twice weekly caused similar (and maximal tolerable) weight loss; therefore these regimens were chosen to test which schedule produced more acute lip radiosensitization. Twice-weekly dFdCyd + RT was somewhat more toxic by weight loss (800 mg/kg once weekly: 11.9%; 150 mg/kg twice weekly: 17.7%; p = 0.09). To assess therapeutic index, we treated SCC VII flank tumors with RT combined with isotoxic drug/RT regimens (dFdCyd 800 mg/kg once weekly or 100 mg/kg twice weekly). Tumors treated with twice-weekly dFdCyd + RT were significantly smaller than tumors treated with once-weekly drug + RT at 28 days from the start of treatment (p < 0.03). CONCLUSIONS: These findings demonstrate that equitoxic once- versus twice-weekly dFdCyd regimens cause differing levels of oral mucosal radiosensitization. This would suggest that each radiation-dFdCyd schedule will require its own dFdCyd dose escalation trial (which cannot be determined by the maximum tolerated dose (MTD) for dFdCyd alone using that schedule). In addition, our findings suggest that for head and neck cancers twice-weekly dFdCyd may have a higher therapeutic index compared with once-weekly dFdCyd when combined with daily RT.

Animals↗
PubMed2000

Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from the spinal cord is mediated by nitric oxide but not by cyclic GMP.

Recent data support a role for nitric oxide (NO) in pain processing at the level of the spinal cord, possibly via regulation of neuropeptide release. The goal of this study was to determine whether capsaicin, which selectively activates primary afferent neurons and evokes neuropeptide release, acts in an NO-dependent manner. Our results indicate that capsaicin (1 microM)-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) is significantly reduced in the presence of the NO synthase inhibitor, L-NAME (10-400 nM; F(3,45)=68.38; P<0.001) and, the selective nNOS inhibitor, 3-bromo-7-nitroindazole (170-680 nM; F(5,48)=56.2; P<0. 01). D-NAME (200 nM) had no effect on capsaicin-evoked iCGRP release. Hemoglobin (an extracellular scavenger of NO; 3 mg/ml) significantly reduced the effect of capsaicin on the release of iCGRP (F(1,8)=9.12; P<0.05). The NOS substrate, L-arginine, effectively reversed the inhibitory effect of 3-bromo-7-nitroindazole on capsaicin-evoked iCGRP release. To determine whether the NO-mediated release was NMDA-driven, we superfused spinal cord slices with competitive and non-competitive NMDA antagonists in the presence and absence of capsaicin. MK-801 (0. 1-10 microM; F(4,33)=8.49; P<0.0001) and AP-5 (0.01-10 microM; F(4, 38)=3.34; P<0.05) reduced capsaicin-evoked iCGRP release. CNQX, an AMPA/kainate antagonist (10 nM-10 microM), significantly decreased capsaicin-evoked release of iCGRP (F(6,42)=8.76; P<0.01) in a dose-dependent fashion. Additionally, our results demonstrate that while capsaicin-evoked release is significantly reduced in the presence of LY-83583 (10 microM; F(2,18)=3.46; P<0.01; a cyclic GMP lowering agent), there is no effect of ODQ (a potent and selective inhibitor of guanylate cyclase). Moreover, the application of a cell permeable analog of cyclic GMP (8-bromo-cGMP; 0.01-1000 microM) is without effect on both basal and evoked iCGRP release. Finally, we observed no colocalization of immunoreactive neuronal NOS (nNOS) with CGRP in the dorsal horn. In summary, these data indicate that capsaicin evokes the release of iCGRP, in part, via the production of NO which enters the extracellular space prior to having an effect. Moreover, iCGRP and nNOS are produced in distinct populations of neurons within the dorsal horn. We conclude that capsaicin-evoked release involves the activation of the NMDA receptor but is also modified by the activation of AMPA or kainate receptors. Finally, these data suggest that while capsaicin-evoked iCGRP release is modified by NO, this release does not require the activation of guanylate cyclase and subsequent production of cyclic GMP.

Animals↗
PubMed2000

Knock down of spinal NMDA receptors reduces NMDA and formalin evoked behaviors in rat.

Chronic pain remains a major health problem afflicting an estimated 70% of patients with advanced cancer and inflammatory disorders, and up to 94% of patients with spinal cord injuries. Although progress has been made in the pharmacotherapy of chronic pain management, such as usage of adjuvant drugs and more effective methods of drug delivery, the mainstay of clinical pain management still depends on opiates. NMDA receptor activation, at the level of the spinal cord has been shown to play an important role in the facilitation of nociception (pain) in several animal models. Unfortunately, potent NMDA receptor antagonists, such as MK-801 and APV, have toxic properties and low safety margins that preclude their clinical use. We present evidence which indicates that the use of antisense oligonucleotides targeted to the NMDA-R1 receptor subunit (AS-NMDA-R1), but not sense, abolishes NMDA and formalin induced behaviors. Moreover, we demonstrate that spinal administration of AS-NMDA-R1 results in the abolition of staining for immunoreactive NMDA-R1 in the spinal cord. These data provide novel evidence supporting the feasibility of the use of gene therapy approaches in the management of neuropathic pain.

Animals↗