Integrins regulate epithelial cell differentiation by modulating Notch activity.

Coordinating exit from the cell cycle with differentiation is crucial for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the switch from cell cycle exit to differentiation. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and signals downstream of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. Here, we have identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks the mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), by regulation of the cyclin-dependent kinase inhibitor (CKI) dacapo. In addition, integrin-mutant PFCs show defective Notch signaling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Taken together, our findings reveal a direct involvement of integrin signaling on the spatial and temporal regulation of epithelial cell differentiation during development.

Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis.

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.

Leiomyomatosis peritonealis disseminata with malignant degeneration. A case report.

Leiomyomatosis peritonealis disseminata (LPD) is an uncommon condition characterized by multiple nodules of smooth muscle within the peritoneal cavity. It usually occurs during reproductive age, and is especially associated to exogenous and endogenous exposure to female gonadal steroids. A limited number of cases of malignant transformation have been reported in the literature. We report a case of leiomyomatosis peritoneais disseminata with sarcomatous degeneration in a 37-year-old nulligravid patient with no exposure to exogenous estrogen or progesterone, revealed by increased abdominal perimeter. The imaging techniques showed occupation of the entire peritoneal cavity by bulky solid masses. The patient underwent a total hysterectomy with bilateral salpingo-oophorectomy and tumoral mass resection. The histopathologic diagnosis was leiomiomatosis peritonealis disseminata with leiomyosarcomatous degeneration. The patient was given systemic chemotherapy with tumoral progression, and died 24 months after the initial diagnosis.

Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2-/- mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.

Parkinsonism among Gaucher disease carriers.

An association between Gaucher disease and Parkinson disease has been demonstrated by the concurrence of Gaucher disease and parkinsonism in rare patients and the identification of glucocerebrosidase mutations in probands with sporadic Parkinson disease. Using a different and complementary approach, we describe 10 unrelated families of subjects with Gaucher disease where obligate or confirmed carriers of glucocerebrosidase mutations developed parkinsonism. These observations indicate that mutant glucocerebrosidase, even in heterozygotes, may be a risk factor for the development of parkinsonism. Understanding the relationship between altered glucocerebrosidase and the development of parkinsonian manifestations will provide insights into the genetics, pathogenesis, and treatment of Parkinson disease.

Monitoring the CNS pathology in aspartylglucosaminuria mice.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal storage disorder caused by the deficiency of the aspartylglucosaminidase (AGA) enzyme. The hallmark of AGU is slowly progressing mental retardation but the progression of brain pathology has remained uncharacterized in humans. Here we describe the long-term follow-up of mice carrying a targeted AGU-mutation in both alleles. Immunohistochemistry, histology, electron microscopy, quantitative magnetic resonance imaging (MRI) and behavioral studies were carried out to evaluate the CNS affection of the disease during development. The lysosomal storage vacuoles of the AGA -/- mice were most evident in central brain regions where MRI also revealed signs of brain atrophy similar to that seen in the older human patients. By immunohistochemistry and MRI examinations, a subtle delay of myelination was observed in AGA -/- mice. The life span of the AGA -/- mice was not shortened. Similar to the slow clinical course observed in human patients, the AGA -/- mice have behavioral symptoms that emerge at older age. Thus, the AGU knock-out mice represent an accurate model for AGU, both histopathologically and phenotypically.

Demonstration of remarkable sequence divergence in variants of a complex satellite DNA by molecular cloning.

Repeat units of a complex G + C-rich satellite of the Bermuda land crab have been cloned by insertion into either the PstI or EcoRI site of pBR322 or the EcoRI site of pUC9. While most of the recombinants contained inserts of approx. 2.1 kb, the average size of repeat units seen in cellular satellite digests, several inserts were markedly different in size. Two domains that account for major sequence differences among the satellite variants and that may be 'hotspots' for sequence modification have been subcloned to permit characterization of their secondary and tertiary structures independent of the influence of the other unusual sequences present. One of these domains is striking in its content of simple repeats; one strand is highly biased in pyrimidines which may permit the formation of unusual secondary and/or tertiary conformations. The other subcloned domain is rich in Pu/Py; preliminary data indicate a transition from B----Z DNA in this region.

Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition.

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 h without milk in their stomachs. OT injection into the dams restores the milk injection in response to suckling. These results indicate an absolute requirement for oxytocin for successful milk injection, but not for mating, parturition and milk production, in mice.

Blockade of transmission in rat sympathetic ganglia by a toxin which co-purifies with alpha-bungarotoxin.

Bungarus multicinctus venom was fractionated into its toxin components using ion-exchange chromatography on CM-Sephadex. According to previous reports, rechromatography of fraction II on a CM cellulose column yields chemically homogenous alpha-bungarotoxin (II2) of molecular weight 9000. However, in our hands, using the identical purification procedure, two discrete proteins of molecular weight 9000 and 15,000 were obtained as demonstrated by SDS gel electrophoresis. Subsequent fractionation of this alpha-bungarotoxin fraction (II2) was achieved on Sephadex G-50. The 9000 weight component (labelled II-S2) was identical to alpha-bungarotoxin; at a concentration of 1 microgram/ml it blocked transmission at the neuromuscular junction but did not block nicotinic responses in rat sympathetic ganglia. Very different properties were exhibited by II-SI, the 15,000 molecular weight component; it inhibited ganglionic transmission but was ineffective at the neuromuscular junction at the same concentration (1 microgram/ml). BGT II-S1 was equipotent in blocking the ganglionic action potential in the presence or absence of eserine; thus, it is not acting as an acetylcholinesterase by increasing acetylcholine breakdown. In the presence of toxin, [3H]choline incorporation into ganglionic acetylcholine during preganglionic stimulation was not altered, suggesting that the toxin did not block transmission by a presynaptic mechanism. Thus, the site of action of the toxin appears to be postsynaptic although it did not affect depolarization of the ganglia induced by carbachol.

Deoxyglucose uptake and choline acetyltransferase activity in cerebral cortex following lesions of the nucleus basalis magnocellularis.

The uptake of [3H]2-deoxyglucose (2-DG) into various brain regions of rats with unilateral or bilateral lesions of the nucleus basalis magnocellularis (nBM) was measured. The activity of choline acetyltransferase (ChAT) in these brain regions was also determined. Lesions of the nBM caused a significant decrease in cortical ChAT activity but had no effect on 2-DG accumulation. Pentobarbital treatment reduced 2-DG accumulation in all brain areas examined and these reductions were not influenced by the nBM lesions. The results indicate that a decrease in the cholinergic innervation of the cortex does not influence cortical glucose utilization. It appears unlikely, therefore, that the reported decrease in cortical glucose utilization in Alzheimer's disease is related to degeneration of the nBM-cortical cholinergic projection.

Targeted reduction of oxytocin expression provides insights into its physiological roles.

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 hours without milk in their stomachs. OT injection into the dams or rescue with the rat OT gene restores the milk ejection in response to suckling. OT is also needed for post-partum alveolar proliferation. These results indicate an absolute requirement for oxytocin for successful milk ejection, but not for mating, parturition and milk production, in mice. Furthermore, homozygous mutant mice show reduced aggression in some tests.

Relationship between rates of synthesis and intracellular distribution of ribosomal proteins during oogenesis in the mouse.

Ribosomal proteins are synthesized continuously in nonequimolar amounts during oogenesis in the mouse (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol. 73, 103), even though ribosomal proteins are found in equimolar amounts in ribosomes. In this report, the distribution of newly synthesized ribosomal proteins between the cytoplasm and germinal vesicle (nucleus) of fully grown mouse oocytes has been examined. As compared to total newly synthesized protein, ribosomal proteins were found to be highly concentrated in the oocyte's germinal vesicle. Furthermore, an inverse relationship was found between rates of synthesis of individual ribosomal proteins and percentages of newly synthesized ribosomal proteins associated with germinal vesicles. As a result of this relationship, the amounts of newly synthesized ribosomal proteins associated with germinal vesicles approximated an equimolar situation. Even in the presence of actinomycin D, oocytes continued to synthesize ribosomal proteins which were found associated with germinal vesicles in amounts similar to those observed in the absence of the drug. These results suggest that, although synthesis of ribosomal proteins by mouse oocytes is not coordinately regulated, a post-translational mechanism exists for adjusting the stoichiometry of these proteins within the oocyte's germinal vesicle; this mechanism apparently is not dependent upon concomitant ribosomal-RNA synthesis.

Effects of 4-aminopyridine on the cat superior cervical ganglion.

These experiments tested the effect of 4-aminopyridine (4-AP) on acetylcholine (ACh) release, 45Ca++ accumulation and transmission in the perfused superior cervical ganglion of the cat. The 4-AP increased the amount of ACh released during preganglionic nerve stimulation, but it did not alter spontaneous ACh release. The 4-AP-induced increase of ACh release was compensated for by increased ACh synthesis because stimulation in the presence of the drug did not deplete tissue ACh content. When ACh release was suppressed by Mg++ or by low Ca++, 4-AP restored release to normal, but it did not do so when Ca++ was absent. This is interpreted as consistent with the idea that 4-AP increases Ca++ influx into nerve terminals and this was supported by measures of 45Ca++ accumulation by ganglia. Thus, preganglionic nerve stimulation increased 45Ca++ accumulation by ganglia and 4-AP increased this measure; Mg++ decreased the stimulation-induced change in 45Ca++ accumulation and 4-AP reversed this effect of Mg++. Depression of ganglionic transmission caused by Mg++ was readily antagonized by 4-AP, but the compound did not as readily augment transmission depressed by tubocurarine or by trimethaphan.

Gonadotropins and the timing of progesterone-induced meiotic maturation of Xenopus laevis oocytes.

Isolated oocytes from 30 unstimulated Xenopus laevis females required from 2.50 +/- 0.13 to 14.59 +/- 0.77 hr after progesterone exposure for the first 50% of each group to complete meiotic maturation. Injecting 8 females with an amount of hCG not causing ovulation (25 micrograms, 96 IU) lowered oocyte maturation times by 45-83%. An enzyme-linked immunosorbent assay (ELISA) of the blood of 18 unstimulated animals found a constituent which bound to anti-hCG in amounts (equivalent to 0-1.03 micrograms/ml hCG) that had a direct relationship to the rates of GVBD in oocytes. Preincubation of manually isolated follicles in 0.25-1.25 micrograms/ml hCG shortens oocyte maturation times by 18-50% in a direct, nonlinear fashion and this priming effect is reversed when hCG is withdrawn. The action of gonadotropins in facilitating germinal vesicle breakdown (GVBD) mimics the previously reported priming effect produced by preincubation of oocytes in subthreshold levels of progesterone. Evidence suggests that individual variation in the time course of progesterone-induced meiotic maturation of amphibian oocytes is the result of priming differences caused by the action on follicle cells of fluctuating blood levels of an LH-like hormone.

Fundulus heteroclitus gonadotropin(s). I. Homologous bioassay using oocyte maturation and steroid production by isolated ovarian follicles.

Isolated ovarian follicles from several species were cultured to develop an in vitro bioassay system for Fundulus heteroclitus gonadotropin. An extract of F. heteroclitus pituitaries, when tested in heterologous systems using follicles from Rana pipiens. Xenopus laevis, and Carassius auratus, was ineffective in provoking either germinal vesicle breakdown or steroid production. In a homologous system using F. heteroclitus follicles, F. heteroclitus pituitary extract was capable of inducing both germinal vesicle breakdown and steroid production in a dose-dependent fashion. Testosterone, estradiol-17 beta, and 17 alpha-hydroxy,20 beta-dihydroprogesterone were detected in both the culture media and the follicle extracts after F. heteroclitus pituitary extract stimulation. The steroidogenic responses resulting from the pituitary extract stimulation were dependent on the size and stage of follicular development. Only large vitellogenic follicles (1.2-1.4 mm diameter) were able to produce 17 alpha-hydroxy,20 beta-dihydroprogesterone and testosterone. Small vitellogenic follicles (less than 1.2 mm) were unresponsive to stimulation by F. heteroclitus pituitary extracts as scored by either germinal vesicle breakdown or production of 17 alpha-hydroxy, 20 beta-dihydroprogesterone and testosterone. However, estradiol-17 beta production was detected in follicles of a much wider size range: Follicles as small as 0.8 mm diameter were responsive to F. heteroclitus pituitary extract stimulation and produced a large quantity of estradiol-17 beta. There was a marked seasonal sensitivity of F. heteroclitus follicles to pituitary extract stimulation in vitro. Follicles obtained from fish outside of the breeding season (January) were less responsive to stimulation by pituitary extract or steroid. The same preparation of pituitary extract was capable of provoking germinal vesicle breakdown in follicles obtained in May. Pituitary extracts prepared during October through January were also less potent than those prepared during the breeding season (February through September). We conclude that F. heteroclitus gonadotropin(s) shows a noticeable species specificity and that F. heteroclitus follicles exhibit both a season- and a size-dependent responsiveness to gonadotropin(s). Hence, with a judicious use of the appropriate types of F. heteroclitus ovarian follicles, we have been able to demonstrate that in vitro oocyte maturation and steroid production are sensitive, homologous bioassays for F. heteroclitus gonadotropin(s).

Chromatographic and electrophoretic heterogeneity of the cytochromes P-450 solubilized from untreated rat liver.

When the hepatic microsomes prepared from one or two untreated male rats were incubated for 3 hr at room temperature in buffered Emulgen 911, cholate, glycerol, and EDTA, almost complete recovery of the cytochromes P-450 in solubilized form was obtained. The intact cytochromes P-450 in this preparation were retained by Sephadex G-200. They could be resolved by anion-exchange chromatography into four fractions with 70--80% recovery of cytochrome P-450 and little or no conversion to P-420. In the process, the p-450 hemoproteins were separated from cytochrome b5, NADPH-cytochrome c reductase, and hemoglobulin. Each of the four fractions of cytochrome P-450 could be further resolved by electrofocusing in polyacrylamide into numerous protein bands, more than eight of which stained for heme. Extracts from focused gels retained P-450 spectral character. Mixtures of the various fractions electrofocused additively. Added hematin focused only in the acidic region of the gels. Although these results indicate extensive heterogeneity in the cytochromes P-450, the complexities of electrofocusing in the presence of detergent warrants cautious interpretation. These methods, however, should provide a basis for subsequent chemical, physical, catalytic, and immunological investigations of the heterogeneity, and its significance.

Absolute rates of protein synthesis during meiotic maturation of mammalian oocytes in vitro.

Measurements of the rates of incorporation of [35S]methionine into protein and the specific activities of endogenous free methionine pools have been used to calculate the absolute rates of protein synthesis in mouse oocytes during spontaneous meiotic maturation in vitro. Fluorodinitro[3H]benzene was used to determine the specific activity of the oocyte's free methionine pool. It was found that the absolute rate of protein synthesis decreased from 43 to 31 pg/hr per oocyte during meiotic progression from dictyate to metaphase II (meiotic maturation), while the size of the intracellular free methionine pool decreased from 61 to 35 fmol per oocyte during the same period. Comparable measurements made on ovulated mouse oocytes that had undergone meiotic maturation in vivo strongly suggest that the decrease in the absolute rate of protein synthesis observed during meiotic maturation in vitro is physiologically significant. An alternative method that depends upon differential expansion of the oocyte's endogenous methionine pool was also used to determine absolute rates of protein synthesis. The results of these experiments are in excellent agreement with those obtained by using fluorodinitro[3H]benzene, indicating that the oocyte's free methionine pool is not compartmentalized.

Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA.

The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.