Human MUC4 mucin induces ultra-structural changes and tumorigenicity in pancreatic cancer cells.

MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4alpha, and a membrane-bound growth factor-like subunit, MUC4beta. MUC4 mRNA contains unique 5' and 3' coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7-19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.

Endoscopic neuroanatomy through the sphenoid sinus.

OBJECT: The aim of the study was to perform endoscopic anatomical studies of skull base structures through the sphenoid sinus in order to better understand endoscopic approaches to skull base lesions. METHODS: Anatomical studies were performed on six cadavers using sinus endoscopes. The sphenoid sinus was entered via a sublabial nasoseptal approach. Stepwise removal of bone surrounding the sphenoid sinus was done to expose the suprasellar area, the retrosphenoid area and the cavernous sinus. The suprasellar dissection exposed the anterior communicating artery (ACom) complex, optic apparatus, pituitary stalk, lamina terminalis, third ventricle and pituitary gland. The retrosphenoid dissection exposed the mamillary bodies, basilar artery and its branches, the mid-brain, the pons, and the third, fifth, sixth, seventh and eighth cranial nerves. The cavernous sinus dissection exposed the carotid artery, and the third, fourth, fifth and the sixth cranial nerves. CONCLUSION: Through the sphenoid sinus, most of the structures in the suprasellar area, cavernous sinus and retrosphenoid area can be reached.

Behavioral and physiological sex differences observed in an animal model of fulminant hepatic encephalopathy in the rat.

Hepatic encephalopathy is characterized by a number of neuropsychiatric and motor disturbances observed in patients with liver dysfunction. The purpose of this study is to fully characterize behavioral and physiological sex differences in an animal model of fulminant hepatic encephalopathy (FHE). Male and female rats were administered thioacetamide (600 mg/kg) via i.p. (intraperitoneal) injection at Hours 0 and 24 and allowed to progress into the four stages of FHE. Male rats reached all four stages of FHE significantly earlier than female rats (p < 0.05). The performance of the male rats deteriorated more quickly (p < 0.05) than that of the females in all of the sensory and motor behavioral tests. Sex differences were observed in the liver enzymes of the FHE rats. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase were significantly greater (p < 0.05) in male rats in all four stages of FHE. Significant increases were also observed in the levels of direct and total bilirubin (p < 0.05). Neuronal damage was observed in the CA1 and CA2 regions of the hippocampus. In the CA1 region, male rats displayed greater pathological changes in Stages III and IV (p < 0.05) than female rats. The damage in the CA2 region was only observed in Stage IV male rats. Our data indicate that observable behavioral and physiological sex differences occur in thioacetamide-induced FHE in the rat.

Timing of breast cancer surgery during the luteal menstrual phase may improve prognosis.

A meta-analysis has been performed of available retrospective reports concerning the 5-15 year disease-free survival of 5,353 premenopausal breast cancer patients operated on either during the follicular or luteal phases of the menstrual cycle. Patients with surgery performed during the luteal phase (d 14-23+) had an overall mean 5% benefit compared to those operated on the follicular phase determined by date of onset of their last menstrual period p = 0.02 by Wilcoxon 2-tailed test. When nodal invasion was reported, node-negative patients had a 5 +/- 2% SEM benefit. Patients with positive nodes had a 34 +/- 3% SEM increase in survival (p = .05), including both estrogen and progesterone-receptor negative as well as positive neoplasms. In 3 of 4 reports from major cancer treatment centers, each containing 249-1175 cases, risk of recurrent cancer and/or death increased 5 to 6-fold after 10 years for women receiving surgery during d 7-14 of their cycle, compared to those resected during d 21-36. Improvement in prognosis was greatest for patients with the highest risk of recurrence due to node-invasive disease and receptor dysfunction. Several cell-mediated immunologic factors inimical to metastasis are maximal in the luteal phase of the menstrual cycle, including natural killer cell activity. A new drug which augments natural killer cell activity may extend any beneficial survival results to post-menopausal breast cancer patients in the future. We conclude that accurate menstrual histories should be included in the medical record from now on for all premenopausal women receiving any surgical procedure upon the breast, preferably using an objective method of determining the date of last ovulation. Prospective randomized clinical trials are necessary to determine the full extent of survival benefits of late luteal surgical timing.

Timing of breast cancer surgery during the luteal menstrual phase may improve prognosis.

A meta-analysis has been performed of available retrospective reports concerning the 5-15 year disease-free survival of 5,353 premenopausal breast cancer patients operated on either during the follicular or luteal phases of the menstrual cycle. Patients with surgery performed during the luteal phase (d14-23+) had an overall mean 5% benefit compared to those operated on the follicular phase determined by date of onset of their last menstrual period p=0.02 by Wilcoxon 2-tailed test. When nodal invasion was reported, node-negative patients had a 5 + 2% SEM benefit. Patients with positive nodes had a 34 + 3% SEM increase in survival (p = .05), including both estrogen and progesterone-receptor negative as well as positive neoplasms. In 3 of 4 reports from major cancer treatment centers, each containing 249-1175 cases, risk of recurrent cancer and/or death increased 5 to 6-fold after 10 years for women receiving surgery during d7-14 of their cycle, compared to those resected during d21-36. Improvement in prognosis was greatest for patients with the highest risk of recurrence due to node-invasive disease and receptor dysfunction. Several cell-mediated immunologic factors inimical to metastasis are maximal in the luteal phase of the menstrual cycle, including natural killer cell activity. A new drug which augments natural killer cell activity may extend any beneficial survival results to post-menopausal breast cancer patients in the future. We conclude that accurate menstrual histories should be included in the medical record from now on for all premenopausal women receiving any surgical procedure upon the breast, preferably using an objective method of determining the date of last ovulation. Prospective randomized clinical trials are necessary to determine the full extent of survival benefits of late luteal surgical timing.

Growth hormone-releasing hormone depletion in the female rat: similarities to aging.

The age-related decline in growth hormone (GH) secretion has been largely attributed to age-related degeneration of hypothalamic growth hormone-releasing hormone (GHRH)-producing neurons. GH decline has recently been linked to age-related bone changes in humans. Bone loss and decreased bone strength are common in aging rats and humans, but density of remaining mineral tissue is known to be increased. The effect of induced hypothalamic GHRH deficiency on bone was assessed, and similarities between bone changes encountered and those taking place in aging were identified. Female rats received monosodium glutamate (MSG) following birth, and they were euthanized at 19 weeks of age. Femurs from MSG-treated rats had greater mineral density (p < .05), greater mineral/matrix ratio (p < .01), lower mineral apposition rate (p < .005), and lower bone formation rate (p < .05). These results suggest that hypothalamic GHRH decline plays a substantial role in the development of bone pathology similar to that observed in aging individuals.

17 beta-Estradiol inhibits the production of dopamine by the tuberoinfundibular dopaminergic neurons of the male rat.

Previous work in our laboratory suggests that the indirect stimulation of prolactin gene transcription observed in male rats between 3 and 6 h following estrogen injection results from an inhibition of dopamine release from the tuberoinfundibular dopaminergic neurons. To evaluate further the role of dopamine in the regulation of prolactin gene transcription by estrogens, we have examined the acute effects of 17 beta-estradiol (E2) on dihydroxyphenylalanine accumulation in the arcuate median eminence region and the amount of dopamine associated with the arcuate median eminence region. Dihydroxyphenylalanine accumulation in the arcuate median eminence region was unaffected when examined 2 h following a single injection of E2 (10 micrograms), and was reduced by 60% when examined 4 h following E2 treatment. When examined 4 h after E2 injection, dopamine content in the arcuate median eminence region was significantly increased. These data suggest that estrogens exert acute inhibitory effects on the tuberoinfundibular dopaminergic neurons of the male rat, and further support the assertion that the indirect stimulatory actions of estrogens on prolactin gene transcription result from inhibition of hypothalamic dopamine production.

A quantitative evaluation of magnetic resonance image signal changes of the brain in chronic hepatic encephalopathy.

Hyperintensity in the basal ganglia of patients with serious liver disease is a common finding on T1-weighted magnetic resonance images. In this study, we used optical densitometry to quantitatively evaluate the hyperintense magnetic resonance image signal changes in the various regions of the brain of patients with chronic hepatic encephalopathy. The incidence and morphological distribution of the magnetic resonance signal changes were evaluated from T1-weighted magnetic resonance images of the brain from seven non-alcoholic patients and six healthy controls. Significant differences (p < 0.05) between the patient group and controls were found in the limbic system (hippocampus, temporal lobe, cingulate gyrus, and fornix), extrapyramidal system and associated myelinated pathways (lentiform nucleus, tectum, tegmentum, cerebral peduncles, internal capsule and the corpus callosum). No measurable differences were observed in the frontal, parietal, and occipital cortex, or the dorsomedial thalamus. The presence of the high signal intensity changes on T1-weighted magnetic resonance image suggests that characteristics alterations occur in functional regions of the brain in chronic hepatic encephalopathy.

Distribution of zinc metallothionein I mRNA in rat brain using in situ hybridization.

Metallothionein (MT) isoforms I and II were first identified and characterized in our laboratories in several regions of brain, in hippocampal neurons in primary culture, and in retinoblastoma and neuroblastoma cell lines. In this study, by having employed the MT-I cDNA as a probe, we sought to gain additional insight about the function of MT by discerning the regional distribution of its mRNA. Northern blot analyses of brain mRNA revealed that the administration of zinc enhanced dramatically MT-I mRNA (570 bp). The in situ hybridization study revealed that MT-I mRNA was located in several areas of brain, with the highest concentrations found in the cerebellum, hippocampus, and ventricles. The results of these studies are interpreted to suggest that zinc enhances the synthesis of MT mRNA and MT in turn may participate in zinc associated functions in neurons.

Is glutamate-induced reduction in growth hormone-releasing hormone a neuroendocrine model of aging in the rat?

Decreases in serum growth hormone (GH) associated with aging may be a result of age-related degenerative changes in neurons of the hypothalamus resulting in a decrease of growth hormone-releasing hormone (GHRH). This study tests the utility of glutamate-induced hypothalamic neuronal degeneration in the rat as a neuroendocrine model of aging. Sprague-Dawley female rats received three 4-mg/g monosodium glutamate (MSG) subcutaneous injections during the first 5 days following birth. Animals were anesthetized at 60 days of age and challenged with GHRH. Blood samples were assayed for GH. Serum GH levels following GHRH challenge in the MSG-treated group rose more slowly and to a lower peak than in controls (P < 0.05). There was no difference in total GH release over the 1-hr interval following challenge. MSG-treatment of animals resulted in lower gross body (P < 0.05) and kidney (P < 0.05) weights with no difference in ovary or adrenal weights. There were also no differences in pituitary GH or total protein content between the groups. Analysis of femurs in the MSG animals revealed both a lower bone strength (P < 0.05) and maximum mid-shaft diameter (P < 0.05), but no difference in length, mineral/matrix ratio, or tissue density. Our data suggest that the degree of neuroendocrine disruption resulting from neonatal MSG administration mimics in part systemic changes commonly observed in aged animals. Hence, definite similarities exist between MSG treatment and the documented aging-related changes in hypothalamic GHRH content and GH regulation in the rat.

Partitioning of vasoactive intestinal polypeptide into lipid bilayers.

Incubation of radiolabeled vasoactive intestinal polypeptide (VIP) with preformed lipid vesicles composed of phosphatidylcholine, phosphatidylglycerol and cholesterol resulted in reversible and saturable association of the peptide with the lipid bilayer. The pH-optimum for the reaction was in the physiological range. The vesicle-associated peptide displayed enhanced stability to proteolytic digestion, it was efficiently released into the supernatant by detergent-solubilization of the vesicles but remained vesicle-associated during treatment with agents that disrupt ionic interactions. Peptide binding by electrically neutral vesicles was lower than that by negative vesicles. Electron spin resonance studies with 5-doxylstearic acid or 16-doxylstearic acid labeled vesicles suggested that VIP decreased the fluidity close to the surface of the bilayer and increased the fluidity in its hydrophobic core. These observations suggest that VIP can bind and penetrate lipid bilayers.

Development of the anal fin appendicular support in the western mosquitofish, Gambusia affinis affinis (Baird and Girard, 1854): a reinvestigation and reinterpretation.

Development of the sexually dimorphic anal fin appendicular support of an internal fertilizing bony fish Gambusia affinis affinis was investigated by staining whole-mounted embryos, immature, and adult female and male G. a. affinis with alizarin red S and alcian blue. The tissue was examined histologically to assess development of the amphicelous centrum and to verify specificity of the stains. Our data confirm earlier claims about the development of the male and female characteristics in this species, and we provide for the first time direct embryonic evidence suggesting that development of the sexually dimorphic anal fin appendicular support is biphasic: (1) anteriorization of the most anterior caudal segments, and (2) growth and elongation of hemal arches of vertebrae 14-16. The first process involves a sequential homeotic transformation of hemal arches of vertebrae 11-13 through resorption of mineralized connective tissue, thus forming parapophyses that bear pleural ribs. This process begins in undifferentiated embryos and proceeds similarly in postnatal males and females. During the same period, the second process, likely induced by male gonadal hormones, causes the addition of mineralized connective tissue at the hemal arches of vertebrae 14-16. This second process, which occurs only in males, elongates the hemal arches of vertebrae 14-16 anteriorly. This elongation apparently translocates the anal fin appendicular support (including parts of the hemal spine of the hemal arch of vertebra 13) to the level of vertebra 11. It appears that the developmental programs of both female and male G. a. affinis create an area of 6 vertebrae which are markedly different from any vertebrae anterior to 11 and posterior to 16. We propose to term the area including these vertebrae and the associated anal fin, the genital area. We also propose that the first process, homeotic transformation of caudal into precaudal segments, is regulated by differential expression of control genes, such as homeobox genes, whereas the second process is regulated by gene expression under the control of male gonadal hormones. Conflicting data in the literature can be resolved with this model. Appropriate tests of the model are proposed.

Increased catechol estrogen metabolism as a risk factor for nonfamilial breast cancer.

The metabolism of estrone (E1) or estradiol-17 beta (E2) to catechols seldom has been investigated in biochemical studies related to the risk of development of human breast cancer, as a result of the extreme lability and reactivity of these hormones. A method of indirect calculation was developed in which estimated catechol estrogen excretion (ECE) from urinary excretion of E1, E2, and estriol (E3) was used, based on the obligate reciprocal relation between 16 alpha-hydroxylase activity (r3) and estrogen 2/4 hydroxylase function (r2). This relationship is expressed by r2 x r3 = K, the estrogen oxidative constant. From published data relating chiefly to 2-OH estrone excretion, K = 12.4 +/- 0.8 (standard error of the mean). Urinary E1 + E2 excretion rates reflect nonprotein-bound plasma ovarian estrogen concentrations available for cell metabolism, which influence the value of K. The equation: r2 = [E1 + E2] K/[E3 + 16 alpha OH E1] = ECE gives a median correlation coefficient between actual catechol estrogen excretion and ECE in micrograms/24 hours of +0.88 (range, 0.61 to 0.97). When tested against the best product isolation analysis of catechol estrogen excretion, ECE was 95% accurate. Using this method a metaanalysis was conducted of published fractional estrogen excretion collected from 2846 healthy women worldwide aged 15 to 59 years, with a risk of breast cancer varying fivefold. Overall ECE was 78% to 97% higher in high-risk women of all ages and menstrual cycle phases (P less than 0.001, by Wilcoxon test). With increasing cancer risk (as estimated by the authors), ECE rose linearly exponentially with a slope of 0.149 (follicular phase) and 0.136 (luteal phase). The correlation coefficient (R2) between the two variables was 0.77 and 0.57, respectively (P less than 0.05). These data derived from calculations of ECE in healthy women confirmed recent analytic results of a twofold increase in the ratio of 2-OH E1/4-OH E1 in healthy Finnish women compared with recent Japanese migrants to Hawaii. In Finnish women with breast cancer, this ratio increased further (almost twofold). Metaanalysis supported the conclusion that increased rates of oxidation of estradiol 17-beta to 2-OH catechols supply the principal proximal human mammary carcinogens active after menarche.

A method for estimating catechol estrogen metabolism from excretion of noncatechol estrogens.

The relationship of catechol estrogen metabolism to disease has seldom been investigated because of analytic difficulties. Estradiol (E2) and estrone (E1) are oxidized simultaneously at either ring A or ring D, and the rate of catechol estrogen formation (r2) is reciprocally related to the rate of 16 alpha-hydroxylation (r3). The rate of ovarian estrogen production (X10) can be summarized as to metabolic outcome: X10 = r10 + r2 + r3 + r(u), where r10 is the loss of E1 and E2 in urine, and ru is the fecal and urinary loss of unknown oxidative products. Assuming a constant r(u) between subjects: constancy of the X10 concentration between subjects during similar menstrual cycle phases. In the absence of xenobiotics, r2 x r3 are reciprocally interrelated: r2 x r3 = K (an oxidation constant whose limiting factor is the biologically available estrogen at the cell surface). To the extent that r10 approximates estrogens available for cellular metabolism, the rate of catechol estrogen metabolism may be determined from (Formula; see text) From published data K = 12.4 +/- 0.8 of the standard error of the mean. Pearson correlation coefficients between actual and estimated catechol estrogen excretion in groups of subjects ranged from 0.61 to 0.97 (median, 0.88). This method has been useful for clinical investigation of the relationship of catechol estrogen metabolism to disease until better methods to measure catechol estrogen directly are available.

Quantification of proteins in discrete brain regions of androgen-insensitive testicular feminized Tfm mice.

The effect of the testicular feminization mutation (Tfm) on the concentration of specific proteins in the medial preoptic area (MPO), ventromedial hypothalamus (VMH) and parietal cortex (CX) was examined. Adult Tfm and Swiss-Webster male mice were decapitated, the brains were removed and sectioned. Proteins from the three microdissected areas were separated by two-dimensional gel electrophoresis. Gels were stained with silver and then analyzed by quantitative computerized scanning densitometry. Of the 195 proteins quantified, the Tfm mutation significantly influenced the concentration of 16 proteins measured from gels of MPO tissue, 21 from VMH gels and 11 from CX. Of these, three proteins were affected in all brain regions; and three additional proteins were shown to vary in both MPO and VMH. One protein higher in the MPO and VMH of Tfm mice was identified as the glial fibrillary acidic protein. It is suggested that the proteins influenced by the Tfm mutation are regulated by steroids, most likely androgens. Thus, these proteins may prove to be important in hormone-regulated physiological functions.

Inhibition of radiogenic mammary carcinoma in rats by estriol or tamoxifen.

Mammary carcinomas have been induced by 3.5 Gy whole-body gamma radiation administered at age 40 to 50 days to virgin female Sprague-Dawley rats. In 142 irradiated controls carcinoma incidence averaged 7.8% in survivors observed less than 300 days and 38.3% of those surviving longer (P less than 0.001 by t test). Mammary cancer promotion was inhibited by two methods: estriol (E3) 638 micrograms/month (2.2 microns/mo) subcutaneously for natural life span begun 2 weeks after exposure reduced cancer incidence from 76% in controls to 48% after 331 to 449 mean days observation until neoplasia was palpable (P less than 0.02 by chi-square analysis). Uterine weights were similar in control and treated groups, and were 15% to 18% greater than uteri of nonirradiated controls from other simultaneous experiments. Six monthly 638-micrograms doses of 17 alpha ethinyl estriol (EE3) reduced tumors from 88% in controls to 64% (P less than 0.05 by chi-square analysis) and delayed cancer onset (P less than 0.01-0.04 by life table analysis). Ethinyl estradiol (EE2) after 6 months' treatment similarly delayed mammary tumor development reducing incidence to 75% (NS), with a six-fold increase in nonmammary epithelial malignant tumors. Estriol administration begun between 3 days before to 5 days after radiation did not alter mammary cancer incidence in six experiments. Monthly implantation of 2.5 mg tamoxifen (4.44 microns/mo) started 2 weeks after radiation reduced mammary cancer incidence from 83% to 14% after 307 to 314 days' observation (P less than 0.001 by chi-square analysis). Treated rats had atrophic ovaries and uteri consistent with blockade of endogenous estradiol activity. Short-term parenteral E3 or EE3 therapy using 10 to 30 micrograms/kg/day (35-100 microns/kg/day) rapidly differentiated virgin rat mammary glands without impairment of subsequent estrus cycles and offers an alternative to castration or life-long antiestrogen therapy for reduction of risk of radiogenic mammary carcinoma.

Neuroendocrine responses to exogenous estrogen: no differences between heterosexual and homosexual men.

Plasma LH concentrations were determined in 55 men before and for four days following injection of 10 mg, 20 mg, or 30 mg Premarin or a placebo injection of vehicle. Testosterone (T) and dihydrotestosterone (DHT) concentrations were also determined in plasma samples taken just prior to Premarin or placebo injection and in samples taken three days later. All subjects provided self-descriptions of their sexual orientation and, based upon these descriptions, were classified as heterosexual (N = 39) or homosexual (N = 16). Premarin injection resulted in a reliable reduction in plasma LH 24 hr later. In subsequent samples, LH values rose and in many cases exceeded baseline levels, most reliably in those subjects receiving the 10 mg dose. Contrary to some previous reports, we observed no significant differences between heterosexual and homosexual subjects in the likelihood of their exhibiting elevated LH concentrations following exogenous estrogens. T, but not DHT, concentrations were suppressed after Premarin injection in both groups of subjects. Other than Premarin dosage, we could not identify any variable which predicted the likelihood of elevated LH values.

Abolition of vagino-cervical stimulation-induced analgesia by capsaicin administered to neonatal, but not adult rats.

We have previously reported that vagino-cervical mechanical stimulation (VS or probing) produces analgesia in rats. Neonatal treatment with capsaicin (CAP) has been shown to reduce the concentrations of several neuropeptides in dorsal root ganglia, spinal cord, and autonomic ganglia, via a neurotoxic effect. In the present study, we report that CAP administered in the neonatal period abolishes the analgesic effect of probing in adulthood. In addition, we report that the ability of VS to potentiate the lordosis response to manual stimulation of the flanks is abolished by neonatal CAP treatment. By contrast, rats treated as adults with CAP show the typical VS-produced effects of analgesia and potentiation of the lordosis response. Our results suggest that neonatal, but not adult, CAP treatment depletes a neuropeptide(s) that mediates the analgesia and lordosis-inducing effects of VS.