Xcat2 RNA is a translationally sequestered germ plasm component in Xenopus.

In Xenopus, the inheritance of germ plasm by a small subset of blastomeres during early development is thought to direct these cells into the germ cell lineage. We show that Xcat2 RNA, related to Drosophila nanos, is a germ plasm component that is translationally repressed during oogenesis. Xcat2 protein was not detected in oocytes at times prior to, or after its RNA was localized in germ plasm, suggesting Xcat2 RNA is functionally sequestered soon after transcription. Indeed, Xcat2 RNA is found in a dense non-polysomal compartment in oocytes. Repression of translation was not relieved by substituting the Xcat2 3'UTR with that of beta-globin. Immunodetection of Xcat2 protein during blastula and gastrula stages coincides with the time of symmetric segregation of the germ plasm and a net increase in the number of primordial germ cells. Xcat2 is capable of binding RNA in vitro and we propose that it may function to translationally regulate other RNAs specific to primordial germ cells.

DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A.

DEADSouth was selected in a screen for localized RNAs in Xenopus oocytes. In situ hybridization analysis shows that DEADSouth localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with germ plasm during early embryogenesis. These results lend further support for the general concept that the role of the early RNA localization pathway in Xenopus is to localize germ cell components (reviewed in King, M.L., Zhou, Y., Bubunenko, M. , 1999. BioEssays 21, 546-557). Further analysis shows that DEADSouth is a germline specific RNA, found exclusively within the germ plasm of oocytes and PGCs, as well as in male germ cells. Sequence comparisons with DEADSouth show it to be a member of a small sub-family of the DEAD-box RNA-dependent helicases related to eIF4A.

Loss of Cdh1 and Trp53 in the uterus induces chronic inflammation with modification of tumor microenvironment.

Type II endometrial carcinomas (ECs) are estrogen independent, poorly differentiated tumors that behave in an aggressive manner. As TP53 mutation and CDH1 inactivation occur in 80% of human endometrial type II carcinomas, we hypothesized that mouse uteri lacking both Trp53 and Cdh1 would exhibit a phenotype indicative of neoplastic transformation. Mice with conditional ablation of Cdh1 and Trp53 (Cdh1(d/d)Trp53(d/d)) clearly demonstrate architectural features characteristic of type II ECs, including focal areas of papillary differentiation, protruding cytoplasm into the lumen (hobnailing) and severe nuclear atypia at 6 months of age. Further, Cdh1(d/d)Trp53(d/d) tumors in 12-month-old mice were highly aggressive, and metastasized to nearby and distant organs within the peritoneal cavity, such as abdominal lymph nodes, mesentery and peri-intestinal adipose tissues, demonstrating that tumorigenesis in this model proceeds through the universally recognized morphological intermediates associated with type II endometrial neoplasia. We also observed abundant cell proliferation and complex angiogenesis in the uteri of Cdh1(d/d)Trp53(d/d) mice. Our microarray analysis found that most of the genes differentially regulated in the uteri of Cdh1(d/d)Trp53(d/d) mice were involved in inflammatory responses. CD163 and Arg1, markers for tumor-associated macrophages, were also detected and increased in the uteri of Cdh1(d/d)Trp53(d/d) mice, suggesting that an inflammatory tumor microenvironment with immune cell recruitment is augmenting tumor development in Cdh1(d/d)Trp53(d/d) uteri. Further, inflammatory mediators secreted from CDH1-negative, TP53 mutant endometrial cancer cells induced normal macrophages to express inflammatory-related genes through activation of nuclear factor-κB signaling. These results indicate that absence of CDH1 and TP53 in endometrial cells initiates chronic inflammation, promotes tumor microenvironment development following the recruitment of macrophages and promotes aggressive ECs.

WNT7A/ß-catenin signaling induces FGF1 and influences sensitivity to niclosamide in ovarian cancer.

We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/ß-catenin signaling directly regulates FGF1 expression via TCF binding elements in the FGF1-1C promoter locus. In vitro gene manipulation studies revealed that FGF1 is sufficient to drive the tumor-promoting effects of WNT7A. In vivo xenograft studies confirmed that the stable overexpression of WNT7A or FGF1 induced a significant increase in tumor incidence, whereas FGF1 knockdown in WNT7A overexpressing cells caused a significant reduction in tumor size. Niclosamide most efficiently abrogated WNT7A/ß-catenin signaling in our model, inhibited ß-catenin transcriptional activity and cell viability, and increased cell death. Furthermore, niclosamide decreased cell migration following an increase in E-cadherin subsequent to decreased levels of SLUG. The effects of niclosamide on cell functions were more potent in WNT7A-overexpressing cells. Oral niclosamide inhibited tumor growth and progression in an intraperitoneal xenograft mouse model representative of human ovarian cancer. Collectively, these results indicate that FGF1 is a direct downstream target of WNT7A/ß-catenin signaling and this pathway has potential as a therapeutic target in ovarian cancer. Moreover, niclosamide is a promising inhibitor of this pathway and may have clinical relevance.

Isolation and characterization of xnov, a Xenopus laevis ortholog of the chicken nov gene.

We have isolated an ortholog (xnov) of the chicken nov gene (for nephroblastoma overexpressed; encoding a putative avian proto-oncogene) from Xenopus laevis (Xl) by screening an Xl ovary cDNA library and genomic library using the entire coding region of human CTGF (encoding connective tissue growth factor) as a probe and by 5'RACE (rapid amplification of cDNA ends). xnov has the same genomic organization as chicken nov, mouse fisp12 and cyr61, but has a unique promoter sequence. The Xl open reading frame (ORF) encodes a 343-amino-acid (aa) polypeptide of 37.9 kDa. Xnov shows 62.9, 60.5, 52.2, 52.1, 47.6 and 45.8% identity with the chicken Nov, human NovH, human CTGF, mouse Fisp12, chicken Cef10 and mouse Cyr61 proteins, respectively. Xnov contains four aa domains which characterize the CTGF family. RT-PCR (reverse transcription-polymerase chain reaction) analysis shows that the xnov mRNA is very low in abundance and appears to be present throughout early Xl development. Our results also indicate that xnov and nov are not orthologs of human CTGF.

Adjuvant radiation therapy for rectal carcinoma: predictors of outcome.

PURPOSE: To review predictors of outcome, including sequencing of modalities and pretreatment findings for adjuvantly treated rectal cancer. METHODS AND MATERIALS: From 1975 through 1990, 307 patients with adenocarcinoma of the rectum underwent adjuvant radiation therapy. In 251 cases the radiation therapy was administered preoperatively, either 40-50 Gy (median dose 45 Gy) followed in 6-7 weeks by surgery (210 cases), or 20 Gy in five fractions immediately prior to surgery (41 cases). In 56 cases, patients were referred postoperatively for radiation (median dose 50 Gy). Adjuvant chemotherapy was never given concurrently with the preoperative radiation (RT), although 43 of the cases (including 14 of the preoperative RT cases) received postoperative chemotherapy. RESULTS: Multivariate analysis (Cox model) indicated that significant predictors of better overall freedom from disease were preoperative rather than postoperative RT (p < 0.001), low surgical stage (p < 0.0001), specialist surgeon (p = 0.007), low or moderate histologic grade (p = 0.026), and proximal lesion (p = 0.033). The significant predictors for better local control included use of preoperative RT (p < 0.001), low or moderate grade (p = 0.001), and low surgical stage (p = 0.015). The 5-year local control and freedom from disease for the preoperative RT patients were 90% +/- 2% and 73% +/- 3%, respectively. The selected cases that received the short course of 20 Gy preoperatively did well. Although 24 out of 41 patients proved to have Astler Coller B2 or C disease, local control at last follow-up was 39 out of 41 (95%). A second multivariate analysis of pretreatment factors was performed on the preoperative RT cases. The significant factors for both local control and overall freedom from disease were noncircumferential vs. circumferential tumor, proximal vs. distal lesion, and background of the surgeon. Additional negative factors on univariate analysis (although not achieving independent significance on multivariate analysis) included the finding of near-obstructing lesions and elevated carcinoembryonic antigen (CEA). Grade > or = 3 sequelae occurred in 8% of cases (including 3% bowel obstruction). The only significant factor for complications was background of the surgeon (4% for colorectal specialists vs. 12% for nonspecialists, p = 0.015). CONCLUSIONS: Significant factors for better tumor control included preoperative as opposed to postoperative RT and the experience of the surgeon. In selected cases, excellent results can be obtained with a short course of preoperative radiation. Concurrent chemotherapy need not be given routinely with preoperative radiation. Subgroups of preoperative RT cases at risk for distant metastases (who might benefit from postoperative chemotherapy), and at high risk for local failure (for whom concurrent preoperative chemotherapy and radiation might be considered), are identified.

Exertional hypotension in spinal cord injury.

STUDY OBJECTIVE: To assess blood pressure (BP) response to continuous maximal arm ergometry in patients with spinal cord injury (SCI). DESIGN: Cross-sectional analysis of data collected for a prospective study of functional electrical stimulation in patients with SCI. SETTING: Short-term rehabilitation hospital. PARTICIPANTS: Twenty individuals with SCI; 4 cervical (C6 to C8), 10 high thoracic (T1 to T6), and 6 low thoracic (T7 to T12). MEASUREMENTS AND RESULTS: Each subject performed continuous maximal arm ergometry with expired gas analysis. Blood pressure was measured using a technician-assisted protocol. The BP at maximal exercise was compared with the highest submaximal BP reached during the test (delta BP = final BP minus highest submaximal BP). All 20 subjects had a negative delta BP (mean +/- SD; -22.8 +/- 12.1 mm Hg) for mean BP and 19 of 20 had a negative delta BP (-25.8 +/- 14.4 mm Hg) for systolic BP. The delta BP was not significantly related to maximum exercise parameters, resting BP, or level of lesion. Four able-bodied subjects and six wheelchair-bound individuals without SCI showed no exertional hypotension. Repeated testing on the four able-bodied subjects showed excellent reproducibility for mean BP (coefficient of variation [CV] = 3.6 percent; r = 0.98; p < 0.01) and systolic BP (CV = 2.2 percent; r = 0.99; p < 0.01) using this protocol. CONCLUSIONS: These data describe, for the first time to our knowledge, that exertional hypotension is present in all individuals with SCI during continuous arm ergometry. Further studies are needed to clarify the mechanisms responsible for this phenomenon and to evaluate the long-term consequences for individuals with SCI.

The hemodynamic effects of isotonic exercise using hand-held weights in patients with heart failure.

BACKGROUND: Controversy surrounds the use of resistance exercise in patients with heart failure because of concerns that increases in rate-pressure product and systemic vascular resistance might lead to increased afterload and decreased cardiac output. METHODS: Following pharmacologic left ventricular unloading therapy using a pulmonary artery catheter, 34 patients with advanced heart failure performed isotonic weightlifting exercise at 50%, 65%, and 80% of the calculated one repetition maximum. Measurements were made of hemodynamics, ST segment, rate-pressure product, serum norepinephrine, rating of perceived exertion, and dysrhythmias following each exercise set. RESULTS: Repeated analysis of variance showed significant increases in systolic blood pressure (p = 0.0005), diastolic blood pressure (p = 0.01), rate-pressure product (p = 0.005); serum norepinephrine (p = 0.004), and rating of perceived exertion (p = 0.0005). However, systemic vascular resistance and cardiac output did not change significantly (p>0.05). Pulmonary capillary wedge pressures, the incidence of dysrhythmias, and ST segments did not significantly differ from baseline. No patients experienced angina or dyspnea during the study. CONCLUSIONS: Isotonic exercise using hand-held weights was well tolerated hemodynamically and clinically, and no patients experienced adverse outcomes during exercise.

Evaluation of supercritical fluid extraction in the pharmaceutical industry.

Various steroids precipitate from supercritical CO(2) as very small particles but still retain their crystallinity. This suggests that SCFE in this industry could be utilized as a size reduction method as an alternative to milling. The application of SCFE as a separation technique is demonstrated by the simultaneous extraction and reaction of mevinolin, a metabolite of the fungus Aspergillus terreus.

Extent of choledochoscopic utilization in common bile duct exploration.

Various reports have demonstrated that the intraoperative utilization of the choledochoscope has significantly reduced the incidence of unsuspected retained common duct stones from approximately 10 percent to 0 to 2 percent. Our series revealed a 4 percent incidence of retained stones before choledochoscopy was utilized. This incidence was reduced to 1.1 percent after it was employed. Since the availability of the choledochoscope is unknown, all hospitals in Alabama with 60 or more beds (total of 86) were surveyed to determine the rate of choledochoscopic utilization. Although this instrument was noted to be readily available in many of those institutions with greater than 150 beds (47 percent), only 25 (29 percent) of all the hospitals surveyed had the choledochoscope. Furthermore, only 17 (20 percent) of all hospitals used the instrument routinely for common duct exploration. Although the rate of retained common bile duct stones in Alabama is unknown, it is probably similar to the 10 percent average reported. Consequently, it is believed that wider acceptance of the choledochoscope will reduce the incidence and associated morbidity of retained common bile duct stones.

Predictors of isotonic exercise in patients with heart failure.

PURPOSE: Study aims were to determine the predictors of isotonic resistance exercise performance in patients with advanced heart failure and to compare the preexercise values of patients who experienced a negative physiologic response to resistance exercise with those who had minimal or no response. METHODS: A correlational design was used. After pharmacologic left ventricular unloading therapy using a pulmonary artery catheter, 34 patients with advanced heart failure performed graduated isotonic weight-lifting exercises. Measurements were made of hemodynamics and rating of perceived exertion after each test. RESULTS: The following variables, measured at baseline, were significantly correlated with the amount of weight patients were able to lift: rating of perceived exertion (RPE; r = -0.42, P = 0.014); diastolic blood pressure (DBP; r = 0.49, P = 0.03); systolic blood pressure (SBP; r = 0.40, P = 0.017); pulmonary capillary wedge pressure (PCWP; r = 0.39, P = 0.026); and right atrial pressure (RAP; r = 0.35, P = 0.041). Multiple regression analysis, using a stepwise procedure, showed that 47% of the variance in exercise performance was explained by DBP, RPE, and PCWP. There were no significant differences in baseline hemodynamics, ejection fraction, or age between the group of patients who had a negative hemodynamic response at peak exercise and the group of patients who had minimal or no response. CONCLUSIONS: Resting PCWP, DBP, and RPE can provide important information to help clinicians predict isotonic resistance exercise performance in patients with advanced heart failure. However, those patients who have a negative response to this type of exercise cannot be distinguished at baseline by clinical characteristics or age.

Determination of fenoverine, a modulator of smooth muscle motility, in capsules and in human plasma: application to dosage form stability and a pilot study in humans.

Fenoverine is a novel, potent, musculotropic, spasmolytic agent that affects primarily the gastrointestinal tract, bile duct, and female genital organs. A simple, specific, and accurate HPLC method was developed for the determination of fenoverine in capsules and plasma. This method has been successfully applied to stability studies of fenoverine capsules and to a pilot study in a normal, healthy volunteer following oral administration of fenoverine. For the determination of fenoverine in capsules, a Nucleosil 5-micron CN column, with acetonitrile:0.1 M ammonium acetate (60:40) as mobile phase and detection at 254 nm, was employed. The mean correlation coefficient of the calibration curve (n = 6) for the assay was 0.9999 over a concentration range of 24.6 to 147.6 micrograms/mL of fenoverine standard solutions. Fenoverine did not decompose significantly at 4, 45, 55, and 65 degrees C for 3 months. The mean correlation coefficients of within-day and between-day calibration curves were 0.9995 and 0.9999, respectively, over a range of 10 to 1000 ng/mL of fenoverine in plasma. The limit of detection was 10 ng in plasma.

Single-arc volumetric-modulated arc therapy can provide dose distributions equivalent to fixed-beam intensity-modulated radiation therapy for prostatic irradiation with seminal vesicle and/or lymph node involvement.

OBJECTIVES: Volumetric-modulated arc therapy (VMAT) is becoming an increasingly utilised modality for treating a variety of anatomical sites. However, the efficacy of single-arc VMAT to treat prostate cancer suspicious for extraprostatic extension was heretofore unknown. In this work, we report our institutional experience with single-arc VMAT and fixed-beam intensity-modulated radiation therapy (IMRT) for prostate cancer patients treated for seminal vesicle and/or lymph node involvement. METHODS: Single-arc VMAT and 7- or 9-field IMRT treatment plans were compared for 10 prostate cancer patients treated for seminal vesicle involvement and/or lymph node involvement. All treatment plans were constructed using the Philips Pinnacle treatment planning system (v.9.0, Fitchburg, WI) and delivered on an Elekta Infinity radiotherapy accelerator (Crawley, UK). Resulting plans were compared using metrics that characterised dosimetry and delivery efficiency. RESULTS: No statistically significant differences in target coverage, target homogeneity or normal tissue doses were noted between the plans (p>0.05). For prostate patients treated for seminal vesicle involvement, VMAT plans were delivered in 1.4±0.1 min (vs 9.5±2.4 min for fixed-beam IMRT) (p<0.01) and required approximately 20% fewer monitor units (p=0.01). For prostate patients treated for lymph node involvement, VMAT plans were delivered in 1.4±0.1 min (vs 11.7±1.3 min for fixed-beam IMRT) (p<0.01) and required approximately 45% fewer monitor units (p<0.01). CONCLUSION: Single-arc VMAT plans were dosimetrically equivalent to fixed-beam IMRT plans with significantly improved delivery efficiency.

Role of cyclin inhibitor protein p21 in the inhibition of HCT116 human colon cancer cell proliferation by American ginseng (Panax quinquefolius) and its constituents.

American ginseng and its ginsenoside constituents have been shown to exert anti-cancer effects although the mechanism of action remains unclear. The present study determined the effects of water-extracted ginseng (GE) or its ginsenoside (GF) and polysaccharide (PS) fractions on the proliferation of human colon cancer cells and examined the role of p21 in mediating these effects using wild-type and p21-/- HCT116 human colon carcinoma cells. Proliferation was inhibited by GE, GF, and PS in wild-type and p21-/- cells, and the p21-/- cells were more sensitive to these treatments. Wild type cells treated with GE were arrested in the G0/G1 phase of the cell cycle and the expression of p53 and p21 proteins was increased while phospho-MEK levels decreased. In contrast, cells deficient in p21 displayed reduced cell viability, elevated number of dead cells, and increased expression of Bax and cleaved caspase-3 proteins. Both polysaccharides and ginsenosides appear to be responsible for the anti-proliferative and proapoptotic effects of GE. This study suggests that p21 functions to arrest HCT116 wild-type cells treated with GE, while p21-deficient cells undergo cell death in a ginseng constituent-dependent manner.

Potential liability in utilization review: the risk grows.

The use of cost containment measures in utilization review is a continuing topic of concern to physicians and payors. Focusing on a recent California case that dealt with this issue, this article discusses the potential liability of physicians and payors when such measures are used.

Germ plasm and molecular determinants of germ cell fate.

One mechanism for the specification of cell types during embryonic development is the cytoplasmic localization of determinants in the egg into certain blastomeres. Primordial germ cell (PGC) development in many organisms is characterized by the inheritance of germ plasm, a cytologically distinct assembly of mitochondria and electron-dense germinal granules. This chapter reviews the structure of germ plasm and the experimental evidence for its importance in PGC specification in Caenorhabditis elegans, Drosophila, and Xenopus. It then compares and contrasts recent data on the identification of germ plasm components in these organisms. Many components are potentially RNA-binding proteins, implicating the regulation of RNA metabolism, transport, and translation as critical processes in PGC development. Germ plasm components also mediate transcriptional repression, regulate migration, and control mitotic divisions in PGCs. The chapter concludes with a discussion on the general roles of germ plasm components and how they might act to specify PGC fate.