Showing papers by "University of Milan published in 1990"

Two G protein oncogenes in human endocrine tumors

Abstract: Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.

Dietary Factors and Risk of Breast Cancer: Combined Analysis of 12 Case—Control Studies

Abstract: We conducted a combined analysis of the original data to evaluate the consistency of 12 case-control studies of diet and breast cancer. Our analysis shows a consistent, statistically significant, positive association between breast cancer risk and saturated fat intake in postmenopausal women (relative risk for highest vs. lowest quintile, 1.46; P less than .0001). A consistent protective effect for a number of markers of fruit and vegetable intake was demonstrated; vitamin C intake had the most consistent and statistically significant inverse association with breast cancer risk (relative risk for highest vs. lowest quintile, 0.69; P less than .0001). If these dietary associations represent causality, the attributable risk (i.e., the percentage of breast cancers that might be prevented by dietary modification) in the North American population is estimated to be 24% for postmenopausal women and 16% for premenopausal women.

Autoantibodies to GABA-ergic neurons and pancreatic beta cells in stiff-man syndrome.

Abstract: Stiff-man syndrome is a rare disorder of the central nervous system of unknown pathogenesis. We have previously reported the presence of autoantibodies against glutamic acid decarboxylase (GAD) in a patient with stiff-man syndrome, epilepsy, and insulin-dependent diabetes mellitus. GAD is an enzyme selectively concentrated in neurons secreting the neurotransmitter gamma-aminobutyric acid (GABA) and in pancreatic beta cells. We subsequently observed autoantibodies to GABA-ergic neurons in 20 of 33 patients with stiff-man syndrome. GAD was the principal autoantigen. In the group of patients positive for autoantibodies against GABA-ergic neurons, there was a striking association with organ-specific autoimmune diseases, primarily insulin-dependent diabetes mellitus. These findings support the hypothesis that stiff-man syndrome is an autoimmune disease and suggest that GAD is the primary autoantigen involved in stiff-man syndrome and the associated insulin-dependent diabetes mellitus. Our findings als...

Islet isolation assessment in man and large animals.

Abstract: Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Perceptual and premotor factors of unilateral neglect

Abstract: Right-brain-damaged patients showing unilateral neglect underwent a specifically devised line-bisection task that allowed uncoupling of the direction of visual attention from that of hand movement. This made it possible to isolate and separately assess perceptual and premotor factors of the disorder. Comparison of experimental and radiologic data suggested that premotor factors were more pronounced in patients with lesions involving the frontal lobes than in patients with lesions confined to postrolandic areas. The technique employed is compatible with bedside examination and provides data useful for standard assessment of neglect symptomatology for both clinical and experimental purposes.

The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment.

Abstract: The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.

Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase

Abstract: Somatic mutations in the α-chain (αs) of the stimulatory regulatory protein of adenylyl cyclase (Gs) causing constitutive activation of the enzyme have been identified in a subset of human GH-secreting pituitary adenomas. This study reports on the differences between acromegalic patients bearing tumors without (group 1; n = 51) or with (group 2; n = 29) this alteration. No difference in age, sex, clinical features, duration of the disease, or cure rate was observed between the two groups. By contrast, group 2 patients had higher basal GH levels than group 1. Moreover, a significant difference in sellar morphology was found; group 2 patients more frequently showed sellas of normal size (grade I) than group 1. Hypersecretory activity of group 2 tumors was also apparent at electron microscopy; contrary to those of group 1, cells of group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to group 1, group 2 patients were less responsive to GH-rel...

A case-control study of gastric cancer and diet in Italy: II. Association with nutrients

Abstract: A case-control study involving interviews with 1,016 gastric cancer (GC) patients and 1,159 population-based controls in high- and low-risk areas was conducted to evaluate dietary factors and their contribution to the marked geographic variation in mortality from this cancer within Italy. Risks of GC were found to vary significantly with estimated nutrient intake. Risk rose with increasing consumption of nitrites and protein, and decreased in proportion to intake of ascorbic acid, beta-carotene, alpha-tocopherol, and vegetable fat. The associations with nitrite and beta-carotene tended to fade, however, in multivariate analyses adjusting for intake of other nutrients. Ascorbic acid showed the strongest geographic gradient, with highest consumption in low-risk areas. The findings suggest that the protective effects we previously reported for consumption of fresh fruit, fresh vegetables and olive oil may be linked to the vitamins C and E contained in these foods. The findings are consistent with the hypothesis that N-nitroso compounds are involved in GC risks, since elevated risks were apparent for agents (nitrites, protein) that promote nitrosation, while decreased risks were found for nutrients (ascorbic acid and alpha-tocopherol) which inhibit the process.

Scaling properties of band random matrices.

Abstract: It is shown on the basis of numerical data that the normalized localization length of eigenvectors of band random matrices follows a scaling law. The scaling parameter is ${\mathit{b}}^{2}$/N, where b measures the bandwidth and N is the size of the matrix.

Complement-Mediated Demyelination in Patients with IgM Monoclonal Gammopathy and Polyneuropathy

Abstract: We investigated the role of complement in the pathogenesis of the demyelinating polyneuropathy that occurs in some patients with IgM monoclonal gammopathy. Seven patients with chronic sensorimotor polyneuropathy and IgM monoclonal gammopathy were examined. In six patients, the monoclonal protein recognized an epitope shared by myelin-associated glycoprotein and two peripheral-nerve glycolipids, whereas in one patient, IgM bound to an unidentified myelin antigen. Direct and indirect immunofluorescence and immunoperoxidase assays showed colocalization along the myelin sheaths of peripheral-nerve fibers of monoclonal protein with complement components C1q, C3d, and C5. In addition, terminal-complement complex that was not associated with S protein was detected in myelin sheaths. It appeared that alterations in myelin geometry caused by the separation of myelin lamellae corresponded to sites at which terminal-complement complex was deposited. We conclude that demyelination in polyneuropathy associate...

Immunomodulatory effects of two extracts of Panax ginseng C.A. Meyer.

Abstract: The effect of Panax ginseng extracts on cell-mediated immune functions in man has been investigated Three groups, each consisting of twenty healthy volunteers, were treated under conditions of double blindness with capsules containing lactose (Control Group B), with capsules containing 100 mg of aqueous extract of the drug (Group A), and with capsules containing 100 mg of standardized extract of the drug (Group C) All the patients took one capsule every 12 h for 8 weeks Blood samples were withdrawn before beginning the treatment, at the fourth week and at the eighth week The immune parameters examined were the following: chemotaxis of PMNs, phagocytosis index (PHI), phagocytosis fraction (PHF), intracellular killing, total lymphocytes (T3), T helper (T4) subset, suppressor cells (T8) subset, blastogenesis of circulating lymphocytes, natural killer-cell activity (NK) Chemotaxis proved to be enhanced (p less than 005) already at the fourth week in Group A as well as in Group C; the increase became even more marked (p less than 0001) at the eight week in subjects belonging to Group C PHI and PHF proved to be enhanced (p less than 005) at the eighth week in subjects of Group A; these increases were found to be higher in subjects of Group C (p less than 0001) already starting at the fourth week Intracellular killing was shown to be significantly increased (p less than 005) already at the fourth week in Groups A and C; the increase becomes highly significant in both groups (p less than 0001) at the eighth week; however, a significant increase (p less than 005) at the eighth week was also noticed in the placebo group (Group B)(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential spectral analysis of 24-hour blood pressure and pulse interval in humans

Abstract: Blood pressure and pulse interval are characterized not only by erratic variations but also by rhythmic fluctuations at low-, mid-, and high-frequency (0.025-0.07, 0.07-0.14, and 0.14-0.35 Hz, respectively). However, information on these phenomena has largely been derived from analysis of short-term recordings taken in standardized laboratory conditions. In seven normotensive and 10 untreated mild essential hypertensive subjects, power spectrum analysis was performed on the intra-arterial blood pressure and pulse interval signal collected over a 24-hour period using the fast Fourier transform algorithm and splitting the recording into contiguous segments of 256 beats. About 70% of the segments were suitable for the analysis; the segments excluded for a nonstationary signal amounted to only 30%. All powers were characterized by a high segment-to-segment variability, but in each subject the mid- and high-frequency powers of diastolic blood pressure and the mid-frequency power of systolic blood pressure were markedly reduced during the night as compared with the daytime period, whereas the opposite occurred for the low- and high-frequency powers of the pulse interval. Over the 24-hour period, mid- and high-frequency powers of blood pressure were positively correlated to each other, but both accounted for less than 25% of the 24-hour blood pressure variance. No difference between mean normalized power values of normotensive and hypertensive subjects was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Salt-induced fast aggregation of polystyrene latex.

Abstract: We study fast, salt-induced aggregation of polystyrene spheres by means of low-angle static light scattering covering two decades of scattered wave vector q. The measurements are taken at a fixed salt concentration, varying the monomer concentration from ${\mathit{c}}_{0}$=1\ifmmode\times\else\texttimes\fi{}${10}^{9}$ to 5\ifmmode\times\else\texttimes\fi{}${10}^{10}$ ${\mathrm{cm}}^{\mathrm{\ensuremath{-}}3}$. During each run, the fractal dimension ${\mathit{d}}_{\mathit{f}}$ of the clusters and the time evolution of the average radius 〈${\mathit{R}}_{\mathit{G}}$〉 and average mass 〈M${\mathrm{〉}}_{\mathit{w}}$ are determined. At larger concentrations we consistently find ${\mathit{d}}_{\mathit{f}}$=1.61\ifmmode\pm\else\textpm\fi{}0.02, but, as the concentration is decreased, ${\mathit{d}}_{\mathit{f}}$ grows to 1.83\ifmmode\pm\else\textpm\fi{}0.02. The value of ${\mathit{d}}_{\mathit{f}}$ is determined both from the asymptotic behavior of the scattered intensity I(q) and from the relation between the average mass and radius, I(0)\ensuremath{\propto}${\mathit{R}}_{\mathit{G}\mathit{f}}^{\mathit{d}}$. Good agreement is found between the two sets of data. The earlier portions of the curves for ${\mathit{R}}_{\mathit{G}}$ as a function of time t are compatible with a power-law growth and collapse onto a master curve when the reduced time T=${\mathit{c}}_{0}$t is used. Noticeable deviations from power-law growth are, however, observed at later times. Finally, we show that for the lowest concentrations the reactions stop when the clusters attain a maximum diameter. We present arguments showing that reaction termination is due to sedimentation, the time required to diffuse across intercluster distance becoming longer than the settling time through the sample due to sedimentation.

Enantioselective oxidations of sulfides catalyzed by chloroperoxidase.

Abstract: The chloroperoxidase-catalyzed and horseradish peroxidase catalyzed oxidations of sulfides by tert-butyl and other peroxides have been investigated. The former metal enzyme afforded the corresponding sulfoxides having R absolute configuration in up to 92% enantiomeric excess (ee), whereas the latter gave racemic products. The various factors that control the enantioselectivity of the oxygenation have been examined.

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.). An ultrastructural and morphometric study.

Abstract: In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers. In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance. Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life. The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a "flattened" profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.

Specific binding of endothelin on human bronchial smooth muscle cells in culture and secretion of endothelin-like material from bronchial epithelial cells.

Abstract: Endothelin, synthesized by endothelial cells, is the most potent vasoconstrictor and bronchoconstrictor agent known. We investigated endothelin release from human bronchial epithelial cells and the binding of the peptide to autologous bronchial smooth muscle cells in culture. Epithelial and smooth muscle cells were isolated by enzymatic digestion of bronchial tissue obtained on surgery, and cultured to confluency by standard methods. Epithelial cells stained positively for cytokeratin filaments. Smooth muscle cells stained uniformly for alpha-smooth muscle actin. Immunoreactive endothelin contents in the supernatants of epithelial cells extracted on C8 Amprep columns were evaluated by radioimmunoassay. Epithelial cells released appreciable amounts of immunoreactive endothelin into the culture medium (from 0.65 to 2.1 pmol/ml). A single specific binding site for [125I]endothelin 1 was identified on bronchial smooth muscle cells with an apparent Kd of 113 pM and a maximal binding capacity of 22.1 fmol/106 c...

Intraperitoneal radio‐localization of tumors pre‐targeted by biotinylated monoclonal antibodies

Abstract: We describe 2-step and 3-step strategies for intraperitoneal tumor radio-localization by means of monoclonal antibodies (MAbs). Nude mice bearing intraperitoneal human colon carcinoma tumors were injected i.p. with biotinylated MAb AUAI, followed 24 hr later by radioiodinated streptavidin (2-step). The uptake of radioactivity in tumor and normal tissues was measured 4 hr after injection of radioactive compound. A 3-step strategy consisted in administering biotinylated antibody, cold avidin after 24 hr and 111In-labelled biotin after a further 4 hr; mice were then killed 2 hr later. Tumor localization of intraperitoneally-administered biotinylated antibody and direct targeting of radioactive streptavidin to biotinylated antibody bound to tumor sites were demonstrated using immunohistochemistry and autoradiography. Our results show that (i) the 2-step approach increased the percentage of radioactivity uptake by tumor with respect to directly labelled antibodies (24% vs. 6%) and improved the tumor/non-tumor ratio; (ii) the 3-step approach allowed faster blood clearance of the radioactive probe (111In-biotin) and yielded high tumor/non-tumor ratios. "Pre-targeting" methods appear to have advantages over the conventional 1-step approach with directly radiolabelled antibody.