To identify proteins that may specifically characterize colonic polyps we have investigated the abundance of numerous proteins in epithelial cells from 15 normal colon specimens and 13 colonic polyps, using two-dimensional gel analysis to detect possible differences in expression. Silver-stained digitized images of the gels were analyzed with Melanie II 2.1 software. We consistently detected more than 700 protein spots on each gel, and found that the intensity of 59 of them was significantly altered in polyp specimens (Wilcoxon test assuming P< or =0.05). Immunostaining, microsequencing and mass spectrometry (matrix-assisted laser desorption/ionization - time of flight; MALDI-TOF) techniques were used to identify these proteins and selected others that did not show differential regulation. The expression of numatrin (nucleophosphine/B23), hsp 70, and hsp60 was increased in polyps; levels of fatty acid binding protein (L-FABP), 14-3-3 sigma, citokeratin 20, cytochrome c oxidase polypeptide Va, Rho GDP-dissociation inhibitor (Rho GDI), and beta- and gamma-actins were decreased. Although the levels of expression of a given protein often varied among polyp specimens, it generally held true that the direction of variation (up or down) remained constant across the panel. We concluded that proteins showing constant differential regulation across all or most of the polyp specimens represent the most characteristic regulatory pathways in colon polyps, while more sporadic variations reflect characteristics of individual polyps.

Adenomatous Polyps↗

PubMedDate not supplied

Preoperative chemoradiation for patients with locally advanced adenocarcinoma of the pancreas.

BACKGROUND: Improved resectability is a major theoretical benefit of preoperative chemoradiation for pancreatic cancer. Since 1994, patients at Duke University Medical Center with locally advanced pancreatic cancer have been treated with multimodality preoperative therapy. The purpose of this study was to review our experience with preoperative therapy for locally advanced pancreatic cancer and determine if an aggressive neoadjuvant regimen would not only downstage these tumors pathologically but also improve the odds of complete surgical resection. METHODS: The charts of 25 patients treated with neoadjuvant chemoradiation at Duke University Medical Center with biopsy-proven, locally advanced adenocarcinoma of the pancreas were reviewed. Tumors were defined as locally advanced based on radiographic or intraoperative evidence of disease that abuts the superior mesenteric artery or vein (n = 22) or involves lymph nodes that are within the proposed radiation field (n = 3). All 25 patients received external beam radiotherapy (median dose 4500 cGy) in daily fractions of 180 cGy over 5 weeks. All patients concurrently received 5-fluorouracil (FU), and many also received mitomycin C or cisplatin, or both. Patients were given a 3- to 4-week break before a restaging computed tomographic (CT) scan was performed. Three patients were not restaged: one died from metastatic disease; one was reclassified as having a neuroendocrine tumor; and one was lost to follow-up. RESULTS: On restaging after neoadjuvant therapy, 64% of patients had stable or decreased primary tumor size. Radiographically, two patients appeared potentially resectable, and seven others developed evidence of metastatic disease. Eight patients underwent exploration, but only five could be resected. Of the five patients resected, only one had negative margins and negative lymph nodes. This patient had significant pancreatitis on initial exploration. After neoadjuvant therapy, he had a complete response radiographically, and there was no residual cancer in his resection specimen. Pathologic examination of the other resection specimens suggested that despite significant tumor fibrosis, malignant cells persist even at the periphery of the lesions. CONCLUSION: Although neoadjuvant chemoradiation has many theoretical advantages in managing pancreatic malignancy, true pathologic downstaging of locally advanced lesions into tumors that can be removed with negative nodes and margins appears to be a rare event with currently used therapeutic regimens.

Adenocarcinoma↗

PubMedDate not supplied

Beta-amyloid accumulation in aged canine brain: a model of early plaque formation in Alzheimer's disease.

We characterized eight aged beagles (maintained from birth in a laboratory colony) and one black Labrador using Bielschowsky's, thioflavine S, and Congo red staining, and antibodies to the beta-amyloid peptide, dystrophic neurites, and other plaque components. All plaques within these canine brains were of the diffuse subtype and were neither thioflavine S- nor Congo red-positive. The majority of plaques in the entorhinal cortex contained numerous neurons within them while plaques in the dentate gyrus did not. beta-Amyloid immunoreactivity was also present within select neurons and neuronal processes and was detected as a diffuse linear zone corresponding to the terminal fields of the perforant path. There was no significant correlation between extent of beta-amyloid accumulation and neuron number in entorhinal cortex. Neither tau-1, PHF-1, nor SMI-31-immunostaining revealed dystrophic fibers, confirming the classification of these plaques as diffuse. Canine plaques did not appear to contain bFGF- or HS-positive immunostaining. This may explain why neuritic involvement was not detected within these canine plaques. It is possible that the beta-amyloid within the canine brain has a unique primary structure or may not be in an assembly state that adversely affects neurons.

Adolescent↗

PubMedDate not supplied

The pharmacokinetics in mice and dogs of nitroimidazole radiosensitizers and chemosensitizers more lipophilic than misonidazole.

We have examined the pharmacokinetic properties of nitroimidazole radiosensitizers and chemosensitizers more lipophilic than misonidazole (MISO). In dogs, 2 analogues showed comparable peak plasma concentrations with considerably shorter half-lives (t1/2) and reduced areas under curves (AUC). Benznidazole (Ro 07-1051) had a much longer t1/2, a higher AUC, and somewhat higher peak concentrations. In mice tumor/plasma, brain/plasma, and tumor/brain ratios were generally similar to MISO, as was penetration of brain and peripheral nerve by benznidazole in dogs. Selection of lipophilic analogues with appropriate pharmacokinetic properties may facilitate accommodation of the potentially different requirements for improved radiosensitization or chemosensitization.

Animals↗

PubMedDate not supplied

A semi-automated method for measuring the potential for protein covalent binding in drug discovery.

INTRODUCTION: Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins. METHODS: Here we present a semi-automated method in which a Brandel cell harvester is used to collect and wash proteins that have been incubated with radiolabeled drug. This method utilizes glass fiber filter paper to capture precipitated protein, rather than the more traditional exhaustive extraction/centrifugation approach. Using model compounds (including [14C]diclofenac, [3H]imipramine, [14C]naphthalene, and [14C]L-746530) we compare the covalent binding results obtained using this method to results generated using the traditional method and we performed cross-laboratory testing of assay reproducibility. RESULTS: It was found that results from new method correlated highly with the traditional method (R2=0.89). The cross-laboratory testing of the method showed an average interlaboratory coefficient of variation of only 18.4%. DISCUSSION: This method provides comparable results to the more traditional centrifugation-based method with considerable time and labor savings.

Animals↗

PubMedDate not supplied

Developmental toxicity evaluation of unleaded gasoline vapor in the rat.

To evaluate the potential of unleaded gasoline vapor for developmental toxicity, a sample was prepared by slowly heating API 94-02 (1990 industry average gasoline) and condensing the vapor. The composition of this vapor condensate, which comprises 10.4% by volume of the starting gasoline, is representative of real-world exposure to gasoline vapor encountered at service stations and other occupational settings and consists primarily of volatile short chain (C4-C6) aliphatic hydrocarbons (i.e. paraffins) with small amounts of cycloparaffins and aromatic hydrocarbons. A preliminary study in rats and mice resulted in no developmental toxicity in either species. However, a slight reduction in maternal body weight gain in rats led to the selection of rats for this guideline study. Groups of pregnant rats (n = 24/group) were exposed to unleaded gasoline vapor at concentrations of 0, 1000, 3000, or 9000 (75% lower explosive limit) ppm equivalent to 0, 2653, 7960, or 23,900 mg/m3, for 6 h/day on gestation days 6-19. All rats were sacrificed on gestation day 20. No maternal toxicity was observed. Developmentally, there were no differences between treated and control groups in malformations, total variations, resorptions, fetal body weight, or viability. The maternal and developmental NOAEL is 9000 ppm. Under conditions of this study, unleaded gasoline vapors did not produce evidence of developmental toxicity.

Administration, Inhalation↗

PubMedDate not supplied

Construction of linkage maps with DNA markers for human chromosomes.

DNA markers and sampling of three-generation families can be used to construct complete linkage maps of human chromosomes. This is important in mapping disease loci and in determining the genetic or environmental component of a disease.

Chromosome Mapping↗