Effects of dietary fat and a vegetable-fruit mixture on the development of intestinal neoplasia in the ApcMin mouse

The variation in colorectal cancer (CRC) incidence worldwide strongly suggests a role for dietary influences. Based on epidemiological data, protective effects of vegetables and fruit intake on CRC are widely claimed, while other data indicate a possible increased CRC risk from (higher) dietary fat intake. Therefore, we have investigated single and interactive effects of dietary fat and a vegetable-fruit mixture (VFM) in the ApcMin mouse, a mouse model for multiple intestinal neoplasia. In this study, four different diets (A-D) were compared, which were either low in fat (20% energy diets A/B) or high in fat (40% energy diets C/D). In addition, 19.5% (wt/wt) of the carbohydrates in diets B and D were replaced by a freeze-dried VFM. The diets were balanced so that they only differed among each other in fat/carbohydrate content and the presence of specific plant-constituents. Because the initiation of intestinal tumors in ApcMin mice occurs relatively early in life, exposure to the diets was started in utero. Without the addition of VFM, mice maintained at a high-fat diet did not develop significantly higher numbers of small or large intestinal adenomas than mice maintained at a low-fat diet. VFM added to a low-fat diet significantly lowered multiplicity of small intestinal polyps (from 16.2 to 10.2/mouse, 15 animals/group), but not of colon tumors in male ApcMin mice only. Strikingly, addition of VFM to female mice maintained on a low-fat diet and to both sexes maintained on a high-fat diet significantly enhanced intestinal polyp multiplicity (from 16.5 to 26.7 polyps/mouse). In conclusion, our results indicate that neither a lower fat intake nor consumption of VFM included in a high-fat diet decreases the development of polyps in mice genetically predisposed to intestinal tumor development.     

Effects of soy isoflavones on estrogen and phytoestrogen metabolism in premenopausal women

A stress-responsive gene highly expressed in brain and reproductive organs (BRE) is down-regulated after UV irradiation, DNA damaging agents, or retinoic acid treatment. The human BRE gene encodes a mRNA of 1.9 kb, which gives rise to a protein of 383 amino acids with a molecular size of 44 kilodaltons. BRE is not homologous to any known gene and its function has not been defined. Here we report that BRE was identified multiple times in a yeast two-hybrid screen of a murine cerebellar cDNA library, using the juxtamembrane domain of the p55 tumor necrosis factor alpha (TNF) receptor. The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by co-immunoprecipitation of transfected mammalian cells. In the yeast two-hybrid assay, BRE specifically interacted with p55 TNF receptor but not with other TNF family members such as the Fas receptor, the p75 TNF receptor, and p75 neurotrophin receptor. Overexpression of BRE inhibited TNF-induced NFkappaB activation, indicating that the interaction of BRE protein with the cytoplasmic region of p55 TNF receptor may modulate signal transduction by TNF-alpha.     

The effect of multiple cryopreservation procedures and blastomere biopsy on the in-vitro development of mouse embryos

Preimplantation genetic diagnosis is currently used in clinical practice. In experienced hands the biopsy procedure alone does not affect the further in-vitro and in-vivo developmental potential of animal and human embryos. No data exist on the combination of cryopreservation of embryos at the pronuclear and/or 8-cell stage and/or biopsy at the 8-cell stage. Pronuclear stages of mouse F1 hybrids (C57Bl/jxCBA) were harvested and divided into several experimental groups. The developmental rates of zygotes, which were neither biopsied nor cryopreserved were used as data control. Others were only cryopreserved at the pronuclear stage (C-PN), or at the cleavage stage (C-CS), or both. Each of these groups was also combined with or without a biopsy. Only the hatched blastocyst rate (HBR), but not the 'simple' blastocyst rate, showed significant differences between groups. Neither C-PN (HBR = 60.42%), nor C-CS (63.16%), nor a combination of both (59.46%) had an impact on the hatched blastocyst rate when compared with that of the control group (67.46%). The biopsy procedure (55.93%) also proved not to be harmful for the embryos. The embryos, which were C-PN and C-CS, and subsequently biopsied, showed a significantly lower hatched blastocyst rate (39.62%) than that of the control, C-PN, C-CS, and C-PN/C-CS groups (P < 0.05). The combination of C-PN and cleavage-stage biopsy also lead to a lower hatched blastocyst rate (42.22%), compared with that of the control group (P < 0.05). It was concluded that couples must be advised that an effect on embryos which have undergone a combined cryopreservation and micromanipulation procedure cannot be ruled out. However, cryopreservation at the pronuclear or at the 8-cell stage alone, or in combination with a biopsy procedure, does not influence the further development of the embryo.     

Analysis of the Mom1 modifier of intestinal neoplasia in mice

Although the methodology for mapping genes controlling susceptibility to tumor development in mice is becoming well established, it remains a formidable challenge to move from linkage to locus. Positional cloning, now commonly used in the identification of loci affecting a qualitative phenotype, has yet to be successfully applied to quantitative trait loci. This study describes the application of candidate gene testing, a method complementary to positional cloning. The method has been applied to evaluate candidates for the quantitative trait locus, Mom1, which modifies the susceptibility of ApcMin/+ mice to spontaneous intestinal tumor development. The authors also discuss the further testing of one candidate, the phospholipase gene Pla2g2a, by transgenesis. Finally, studies on the mode of action of Mom1 are discussed in light of the evidence that Mom1 encodes this secretory phospholipase.     

Dietary soy isoflavones inhibit in-vivo constrictor responses of coronary arteries to collagen-induced platelet activation

In effusion cytology the distinction of reactive mesothelia from metastatic carcinoma cells may be a diagnostic challenge. Immunocytochemistry using antibodies suitable to detect epithelial cells must be considered carefully due to limited sensitivity and specificity of these antibodies. Efficient results in histological differential diagnosis of malignant mesothelioma versus lung-adenocarcinoma applying a novel antiserum against the calcium binding protein calretinin inspirated us to investigate the value of anti-calretinin antibody in effusion cytology combined with an epithelial marker. Cytoslides prepared by cytocentrifugation from 42 malignant and 65 reactive effusion specimens were immunostained using antibodies against calretinin and the epithelial marker Ber-EP4. Positive immunoreaction for calretinin in normal and reactive mesothelial cells was noted in 93% of the cases, whereas immunoreaction for calretinin was completely negative in the metastatic cells in 95% of the malignant effusions. Metastatic carcinoma cells were detected with anti-Ber-EP4 in 83% of malignant effusions. Non-specific positive reactions for Ber-EP4 in single mesothelial cells were observed in 16% of all cases and, moreover, frequently with macrophages or neutrophilic granulocytes. Our results demonstrate high sensitivity and specificity of anti-calretinin antibody for mesothelial cells in effusion specimens. They support its application to improve the diagnostic reliability of epithelial markers, especially because anti-calretinin antibody could be helpful in the assessment of false positive and false negative reactions of epithelial markers.     

The effect of intestinal permeability on pancreatic enzyme-induced enteropathy in the rat

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.     

The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes

OBJECTIVE: To assess the effect of propofol on fertilization and early embryo development in a mouse IVF model. DESIGN: Controlled study. SETTING: Mouse IVF. INTERVENTION(S): Mouse oocytes were exposed in vitro to propofol at a concentration of 0 (control), 50, 250, 500, 1,000, or 5,000 ng/mL for 30 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the blastocyst stage was monitored daily. The potential to activate parthenogenetically oocytes also was evaluated by looking for spontaneous extrusion of the second polar body or development to the two-cell stage. In a second step, a pure propofol solution was added to culture medium and used as a standard. MAIN OUTCOME MEASURE(S): Two-cell and blastocyst-stage embryo. RESULT(S): Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of propofol solution in the medium, (i.e., 3% to 41%) in comparison with the control group (76%). Exposure of unfertilized oocytes for 30 minutes to propofol results in a parthenogenetic activation of 33% to 60%, which was significantly higher than the control (10%). When oocytes were kept in propofol for 24 hours, a mean of 30% of activation was observed as compared with 0.5% for the control. CONCLUSION(S): We can conclude from these experiments that even a brief exposure of cumulus-enclosed oocytes to a low concentration of propofol is deleterious to subsequent cleavage. Exposure of unfertilized oocytes to propofol results in a high degree of parthenogenetic activation.     

The effect of light deprivation on the mouse lens

This work was undertaken to test the hypothesis that first exposure of the eye to light is responsible for the changes in lens protein expression patterns observed around the time of birth. The effect of light deprivation on lens properties was examined in Balb c mice which were bred, reared and maintained in complete darkness for up to 7 months. Data were collected on body weight, lens weight, lens protein contents and crystallin distributions. The data were compared with those obtained from age matched mice maintained in natural light/dark conditions. No significant differences were observed in body weight between animals maintained in the light and dark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, the lens represented 0.02% of body weight compared with 0.031% in the light reared animals (P < 0.001). Lens protein concentration, insoluble protein contents and crystallin synthesis patterns were indistinguishable for the two groups of animals. It is concluded that light stimulation of the eye is required for optimal lens growth but does not affect the production of specific crystallins.     

The effect of short- vs. long-term platelet-activating factor exposure on mouse preimplantation embryo development

Preimplantation mouse embryos (n = 1540) were cultured in the presence of platelet-activating factor (PAF) (10(-7)- 10(-14) mol/l) to the hatched blastocyst stage. A dose-dependent negative correlation (-0.75783) relationship between blastocysts and the concentration of PAF was statistically significantly different (p < 0.001). Long-term but not short-term PAF exposure is detrimental to preimplantation Swiss Webster mouse embryos. Short-term PAF (10(-9) mol/l) exposure was found significantly (p < 0.05) to reduce blastocoel diameter. The effect of PAF during preimplantation development may be genotype dependent and be affected by the culture conditions.     

The positive effect of pretreatment with serotonin and gangliosides on the development of early mouse embryos after cryopreservation

Blood pressure (BP) during siesta declines to levels similar to those of night time sleep. The objective of the study was to assess the effect of siesta on 24-h ambulatory BP (ABP) data. Two different approaches were employed for the definition of day and night periods: (1) actual patient reported day and night intervals (ACT) with siesta period analysed as a third time period; and (2) arbitrary day and night time intervals (ARB) with the presence of siesta being ignored. A total of 203 24-h ABP recordings were analysed, with a siesta during ABP monitoring reported in 154 of them. Mean siesta BP was very close to ACT night time BP. Among recordings with a siesta, ACT daytime BP was higher and night time BP lower than the corresponding ARB BPs (P < 0.001). The magnitude of night time BP drop was greater with ACT intervals, resulting in a lower percentage of non-dippers (P < 0.001). Among 49 recordings without a siesta, differences between ACT and ARB BPs were less pronounced for daytime but not for night time. Differences in the magnitude of nocturnal BP drop between ACT and ARB periods, although statistically significant, did not affect the prevalence of non-dippers. In conclusion, analysis of 24-h BP profiles by using ARB instead of ACT day and night intervals results in underestimation of the nocturnal BP drop and overestimation of the proportion of non-dippers. This bias is more pronounced in patients who take a siesta during ABP monitoring.     

Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis

Soy isoflavones exhibit a number of biological effects, suggesting that they may have a role in cancer prevention. Our objectives are to determine whether components of soy products or purified soy isoflavones can inhibit the progression of bladder cancer. We compared the in vitro effects of pure soy isoflavones and soy phytochemical concentrate on growth curves, cell cycle progression, and apoptosis in murine and human bladder cancer cell lines. Pure soy isoflavones (genistein, genistin, daidzein, and biochanin A) and soy phytochemical concentrate exhibit dose-dependent growth inhibition of murine (MB49 and MBT-2) and human (HT-1376, UM-UC-3, RT-4, J82, and TCCSUP) bladder cancer cell lines, although the degree of inhibition varies among lines. Soy isoflavones induce a G2-M cell cycle arrest in all human and murine lines evaluated by flow cytometry. In addition, some bladder cancer lines show DNA fragmentation consistent with apoptosis. We next evaluated the ability of genistein, soy phytochemical concentrate, and soy protein isolate, respectively, to inhibit the growth of transplantable murine bladder cancer in vivo. C57BL/6 mice were randomly assigned to treatment groups (n = 12/group): (a) AIN-76A diet; (b) AIN-76A diet plus genistein, i.p., 50 mg/kg body weight/day; (c) AIN-76 diet with soy phytochemical concentrate at 0.2% of the diet; (d) AIN-76 diet with soy phytochemical concentrate at 1.0% of the diet; and (e) AIN-76A diet with soy protein isolate, 20% by weight. Mice were inoculated s.c. with 5 x 10(4) syngeneic MB49 bladder carcinoma cells, and tumor growth was quantitated. Neither genistein nor soy products reduced body weight gain. Tumor volumes from mice treated with genistein, dietary soy phytochemical concentrate at 1%, or dietary soy protein isolate were reduced by 40% (P < 0.007), 48% (P < 0.001), or 37% (P < 0.01), respectively, compared with controls. We characterized the effects of treatment on several biomarkers in tumor tissue: proliferation index by proliferating cell nuclear antigen staining, apoptotic index by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining, and angiogenesis by microvessel quantitation. Soy products reduced angiogenesis, increased apoptosis, and slightly reduced proliferation while showing no histopathological effects on the normal bladder mucosa. Our data suggest that soy isoflavones can inhibit bladder tumor growth through a combination of direct effects on tumor cells and indirect effects on the tumor neovasculature. Soy products warrant further investigation in bladder cancer prevention and treatment programs or as antiangiogenic agents.     

The effect of the live weight of rats on the intestinal in vitro absorption of nickel using everted intestinal sacs

The influence of the live weight of the experimental animals on the Ni absorption was investigated in vitro with everted sacs from rats. Totally 75 male rats in the live weight range from 30 to 250 g were used. With increasing live weight the Ni uptake by the intestinal wall and the Ni transfer across the intestinal wall decreased significantly. Ni transfer was already significantly reduced by 45% when body weight increased from 30 to 60 g. For the animals with a live weight above 200 g Ni transfer reached only about 10% of the Ni transfer measured for the animals with 30 g live weight. The decline of the Ni uptake by the intestinal wall was only slightly in the live weight range from 30 to 150 g. Within the live weights higher than 190 g Ni uptake by the intestinal wall decreased significantly to about 25% compared to the animals with 30 g live weight.     

The effect of intestinal transit time on bacterial translocation

Increase in intraluminal bacterial count, disruption of the mucosal integrity, changes in intestinal immunity and transit time are the factors involved in bacterial translocation. The relationship between intestinal transit time, intra luminal bacterial count and translocation rate were investigated in 40 Wistar-albino rats. The study was conducted in 4 groups with 10 animals in each. Group I (controls): saline + laboratory chow, Group II: saline + oral total parenteral nutrition (TPN) solution, Group III: morphine sulfate (MS) + oral TPN solution, Group IV: neostigmine bromide (NB) + oral TPN solution. Intestinal transit time was measured by using Indium111-labeled diethylene triamine pentaacetic acid (DTPA). It was prolonged in the MS-treated group and shortened in the NB-treated group (p < 0.01). The frequency of bacterial translocation was 60% in the oral TPN solution group, 100% in the MS-treated group, 20% in the NB-treated group and 10% in controls. Bacterial counts in duodenum, jejunum, ileum and caecum were significantly increased (p < 0.001) in the MS-treated group and decreased (p < 0.05) in the NB-treated group in comparison with the control group. In conclusion, the prolongation of intestinal transit time increased the intraluminal bacterial count and augmented bacterial translocation. The decrease in intestinal transit time had a converse effect.     

Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type Apc allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.     

The effect of ethoxyquin on the intestinal transport of glucose and water in vitro

A prospective study was made on the duration of lactation in 406 mothers injected postpartum for contraception with medroxyprogesterone acetate (DMPA), 150 or 250-300 mg, IM, at 3- or 6-month intervals. Breast feedings up to 2 per day were recorded, and findings compared with those from 173 nontreated controls. Eighty percent of the DMPA group were nursing successfully at the sixth month and 42% at the 12th month postpartum. These figures are significant and compare favorably with the control group. No advantage was found in the administration of DMPA earlier than the second or third month after delivery. Amenorrhea persisted during lactation. No pathologic breast conditions were observed. The eventual mechanism of DMPA in galactopoiesis has yet to be clarified.     

The effect of L-DOPA on the motor activity of mouse fetuses

Most patients with acute myocardial infarction do not receive or are ineligible for thrombolytic therapy, and thus their prognosis is worse than that of the populations studied in the major, randomized, lytic therapy trials. We need to devise a cost-effective strategy with which to appropriately stratify these patients. Simple, easily ascertained clinical variables that are evident soon after hospital admission can identify higher-risk patients, who are likely to be older and less able to adequately complete an exercise test. In some patients, nuclear imaging tests are appropriate; low-dose dobutamine echocardiography and ambulatory ECG monitoring may also have a role. Greater use of routine cardiac catheterization (with assessment of ventricular function) might be the most appropriate way to stratify patients because it may overcome some of the limitations of noninvasive testing, will clearly define high-risk patients, and may facilitate early discharge from the hospital. Left ventricular function and the patency of the infarct-related artery will be determined, and patients with left main coronary disease, significant three-vessel coronary artery disease, and two-vessel coronary disease (especially with proximal left anterior descending coronary artery involvement) will be identified. An aggressive strategy of revascularization to improve survival in appropriate patients may be employed. Greater use of routine coronary arteriography after acute myocardial infarction would inevitably lower the threshold for inappropriate, potentially risky, and expensive further interventions. We need to focus our attention on the most appropriate strategies for the management of patients whose prognosis is worse than the prognosis of those who receive lytic therapy after acute myocardial infarction.