Changes in the innervation of the mouse internal anal sphincter during aging.

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

A simple and accurate method for determination of microsatellite total allele content differences between DNA pools.

DNA pooling is a potential tool for the efficient analysis of the large numbers of samples and DNA markers that are necessary for genome-wide association studies. A simple accurate method for measuring total allele differences in comparisons between two pools containing large numbers of DNA samples is presented. This method compares relative peak height differences between electrophoretograms for each allele of a microsatellite. The method was evaluated by the analysis of 11 microsatellite markers and DNA pooled sample sizes of 50, 100, and 200 individual DNA samples from the same number of different subjects. Pools were created from previously individually genotyped subjects and constructed so that the pool comparisons would provide real total allele differences varying from 0% to 55%. Calculated pool differences were then compared with the real total allele differences determined by individual genotyping results. Together over 200 comparisons demonstrated a correlation coefficient of 0.96, which compared favorably with other previous methods of analysis. This method could provide a rapid screen for total allele differences of greater than 10%, a threshold that should be applicable to detecting low relative risk genes in common diseases. Therefore, these studies suggest that DNA pooling could be a useful tool in association studies for the determination of candidate regions for a range of complex genetic diseases.

Outpatient combination chemoimmunotherapy for patients with metastatic melanoma. Results of a phase I/II trial.

BACKGROUND: Few studies have examined the feasibility, safety, and efficacy of an outpatient biochemotherapy regimen of low dose, subcutaneously administered interleukin-2 (IL-2) for patients with metastatic (Stage IV) melanoma. METHODS: Nineteen patients were treated with intravenous cisplatin and dacarbazine (DTIC), oral tamoxifen, and subcutaneous IL-2 and interferon-alpha-2b (IFN). Eligibility requirements included bidimensionally measurable metastatic melanoma, a Karnofsky performance score of 60 or higher, absence of significant cardiac or pulmonary dysfunction, no prior DTIC or cisplatin chemotherapy, and no evidence of central nervous system involvement. Patients were given a minimum of 2 6-week cycles. Treatment was continued in the absence of progressive disease, and patients were monitored for response at two-cycle intervals. RESULTS: Of the 19 patients, 1 (5%) achieved a complete response; 6 (32%) a partial response; 3 (16%) stable disease; and 9 (47%) progressive disease, for an overall response proportion of 37% (95% confidence interval, 16-61%). The median survival of the treated cohort was 10.6 months. The mean time to disease progression for patients with stable disease or better was 8.4 months, with a mean response duration of 5.1 months. The most common toxicities noted were constitutional symptoms, weight loss, nausea, neutropenia, and fatigue. The 19 patients received a total of 59 cycles of treatment, and IL-2, IFN, or both were held in 14 of these cycles secondary to Grade 3 or 4 toxicities. In addition, six patients required dose reduction of IL-2 and/or IFN. CONCLUSIONS: Chemoimmunotherapy consisting of cisplatin, DTIC, and tamoxifen combined with subcutaneous IL-2 and IFN can be safely administered in an outpatient setting. The described regimen yields moderate activity in metastatic melanoma, and efforts to improve its efficacy merit further examination.

Flavonoid transport by mammalian endothelial cells.

Despite the ever-growing body of literature reporting the effects of flavonoids on animals at both the cellular and systemic levels, one of the most basic questions-"Are the effects of flavonoids on animal cells initiated through their interaction with extracellular targets or intracellular targets?"-has yet to be addressed. Because many effects of flavonoids on cells can be detected within minutes of flavonoid application and because flavonoids diffuse across lipid membranes slowly or not at all, intracellular mechanisms would necessitate a flavonoid transport system for rapid flavonoid uptake. The specific aims of this investigation were (1) to determine if endothelial cells contain a mechanism that mediates rapid flavonoid uptake and (2) to provide evidence for or against the hypothesis that rapid flavonoid effects on endothelial cell synthesis of prostacyclin and endothelin are initiated through the interaction of flavonoids with intracellular targets. Data show that bovine and human aortic endothelial cells possess a transport system that mediates rapid uptake of the flavonoid morin and suggest that the flavonoid uptake system utilizes a variety of oxygenated phenolic compounds as substrates. Further investigation into flavonoid transport should expedite future investigation into the mechanisms of flavonoid actions, because it may allow research to focus on the cellular locations where flavonoids are concentrated. Although endothelial cells contain a mechanism for the rapid uptake of morin, data reported herein suggest that morin initiates its rapid effects on endothelial cell synthesis of prostacyclin and endothelin through an interaction with extracellular targets.

Unaltered meiotic chromosome segregation in Drosophila melanogaster raised on a 5% quercetin diet.

Flavonoid plant pigments are an integral part of the human diet. Although potentially negative mitotic effects of flavonoids have been observed in model organisms, investigation into meiotic effects of flavonoids has been neglected. As flavonoids affect cell signalling and DNA replication, and because the flavonoid content of the human food supply is being increased, determining the effects of flavonoids on meiotic fidelity is important. Here, the effect of the human food supply's most prevalent flavonoid, quercetin, on the level of meiotic recombination and the amount of X and 4th chromosome non-disjunction in Drosophila melanogaster females was determined. This model organism was chosen since Drosophila melanogaster and Homo sapiens share a remarkable number of commonalities in the meiotic processes of oogenesis and because genetic techniques allow a detailed analysis of meiotic processes in Drosophila. No significant effect on either non-disjunction levels or the percentage distribution of exchange bivalents was observed. A significant effect was observed on the number of offspring; F1 and F2 generations of flies raised on a quercetin diet produced over 10% more progeny than flies raised on a control diet. In this investigation, high quercetin consumption by Drosophila melanogaster females did not pose a threat to meiotic fidelity.

Spontaneous X chromosome MI and MII nondisjunction events in Drosophila melanogaster oocytes have different recombinational histories.

Recent studies of human oocytes have demonstrated an enrichment for distal exchanges among meiosis I (MI) nondisjunction events and for proximal exchanges among meiosis II (MII) events. Our characterization of 103 cases of spontaneous X chromosome nondisjunction in Drosophila oocytes strongly parallels these observations. The recombinational histories of MI (97/103) and MII (6/103) nondisjunctional ova were strikingly different. MI nondisjunction occurred primarily in oocytes with non-exchange X chromosomes; of the new nondisjoining exchange bivalents, most carried distal crossovers. Thus, spontaneous MI nondisjunction reflects the failure of the achiasmate segregation systems. MII nondisjunction occurred only in oocytes with proximal exchanges. We propose several models to explain how very proximal exchanges might impair proper segregation.