Turnover of peroxisomal vesicles by autophagic proteolysis in cultured fibroblasts from Zellweger patients.

Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis.


Independent expression and assembly properties of heterologous lamins A and C in murine embryonal carcinomas.

The majority of cells derived from adult mammalian tissues contain three major species of nuclear lamin proteins, A, B and C. In contrast, embryonic cells including undifferentiated murine embryonal carcinomas, contain only B-type lamins, A and C appearing only after differentiation. Human lamins A or C have been introduced by transfection into undifferentiated P19 embryonal carcinomas. Twenty-four hours after transfection, both of these proteins were found to independently associate with the nuclear envelope as judged by immunofluorescence microscopy and at the same time were associated with a salt-resistant structure having solubility properties similar to those of the nuclear lamina. Biosynthetic experiments indicated that heterologous lamin A underwent processing to its mature molecular weight, an event which in adult type cells occurs after assembly into the lamina. Observations on mitotic cells demonstrate that either of the two human lamins will, independent of the other, become dispersed throughout the cytoplasm during prophase and subsequently reassemble at the nuclear periphery during telophase. Nuclear lamins A and C are not, however, equivalent in their abilities to incorporate into the nuclear lamina in these cells. Experiments involving cells arrested in S phase using thymidine suggest that lamin C, but not lamin A, requires progression through the cell cycle and probably mitosis for assembly into the nuclear lamina of P19 EC cells.


Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.


Effect of PDGF-AB heterodimer on a corneal epithelial cell line.

Exposure of cells of a rabbit corneal epithelial cell line (SIRC) to platelet-derived growth factor-AB heterodimer (PDGF-AB) resulted in a rapid and transient elevation of cytosolic free calcium concentration with a maximum at 2 to 3 min after stimulation. The kinetics of the calcium response were dose-dependent, e.g., higher concentrations of PDGF-AB caused a faster rise in cytosolic free calcium concentration. Maximum response was achieved with 10 ng/ml PDGF; higher concentrations up to 100 ng/ml did not further enhance cytosolic free calcium concentration. The ED50 was calculated to be 5 ng/ml PDGF-AB. After complexing extracellular calcium, PDGF-AB still caused a significant rise in cytosolic free calcium concentration indicating a mobilization of calcium from intracellular stores. This rise, however, was less pronounced than in the presence of extracellular calcium. The elevation in cytosolic free calcium concentration was not accompanied by an increased mitotic or proliferative activity of the cells as checked by [3H]thymidine incorporation and counting of cell numbers after 3 days of continuous incubation with various concentrations of PDGF-AB or by alterations in cell size and cell volume. In contrast, alterations in cell shape with a remarkable amount of rounded and partially detached SIRC cells after addition of PDGF-AB were observed within 24 h. Moreover, PDGF-AB caused a reversible distortion of cytoskeletal components such as actin-containing microfilament bundles, microtubules, and vimentin filaments. The results suggest that PDGF-AB may act only as a competence factor for the stimulation of SIRC cells via modification of the intracellular calcium homeostasis.


Coupled down-regulation of the RB retinoblastoma and c-myc genes antecedes cell differentiation: possible role of RB as a "status quo" gene.

The ability of the well known morphogen, retinoic acid (RA), as well as 1,25-dihydroxy-vitamin D3 (VD), whose receptor complex binds a DNA consensus sequence related to that of the retinoic acid receptor, to regulate expression of the retinoblastoma (RB) tumor suppressor gene in a context of induced cell differentiation was characterized. HL-60 human promyelocytic leukemia cells were induced to undergo myeloid or monocytic terminal cell differentiation by these agents. To investigate the potential coupling between down-regulation of RB and c-myc oncogene expression with cell differentiation, dose response relationships for the induced down-regulation of RB and c-myc expression were compared with each other and with induced cell differentiation. The total amount of RB protein per cell increased as cells advanced through the cell cycle, but the amount of RB protein relative to the total cell mass remained approximately constant. Treated with RA or VD, an early progressive decrease in cellular content of the RB protein occurred in all cell cycle phases well before any cell cycle modulation or phenotypic differentiation. For a differentiation-defective variant HL-60 cell line, failure to differentiate was preceded by a failure to down-regulate cellular levels of the RB protein. In dose response experiments, progressively increasing RA or VD concentrations caused progressively greater reductions in RB as well as c-myc expression with an increasing fraction of cells terminally differentiating. For both RA and VD, the dose response relationships for reductions in RB and c-myc expression were similar suggesting that their down-regulation may be coupled. These observations are consistent with a model whereby RB expression acts as a cellular brake to sustain a developmentally ordained state of differentiation (i.e., preserve the "status quo"); and the down-regulation of heterogeneously distributed RB protein per cell below a threshold is part of the metabolic cascade culminating in terminal cell differentiation. Thus, RB may have a role in this developmental context.


Study of the function and regulation of liver N-CAM in Xenopus laevis.

The liver of Xenopus laevis is a unique exception in terms of the cell adhesion molecules (CAM) which it expresses. In most species, hepatocytes are characterized by the expression of the epithelial Ca(2+)-dependent CAM E-cadherin or of closely related variants of this molecule (e.g., L-CAM); in Xenopus liver, however, the levels of expression of epithelial cadherins is very low while a thyroxine-inducible isoform of N-CAM is expressed in postmetamorphic hepatocytes. Since Xenopus liver N-CAM is localized in regions of contact between hepatocytes, it has been proposed that it might be involved in mediating hepatocyte adhesion in this species. In this study, we demonstrate that N-CAM can indeed act as a functional adhesion molecule in the liver of Xenopus and that its expression is correlated with a number of profound morphological changes of this organ. After thyroxine treatment, hepatocytes are no longer organized in long loose cords but in compact lobules of cells. Furthermore, at the ultrastructural level, plasma membranes are in much closer proximity with the appearance of electron-dense material in areas of closer contact. We have established two novel culture systems for premetamorphic Xenopus hepatocytes as adherent and non-adherent cells, and we describe the induction of expression of N-CAM in these cells. Given the difference in the profile of adhesion molecules present in the liver of Xenopus and of other species, our results are discussed in view of the importance of the expression of a specific set of cell adhesion molecules in defining the development of homologous organs in different species.


Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.


Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.


Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.

Isolated blood neutrophils from normal healthy subjects were separated into fractions by sequential countercurrent distribution (CCD) in a charge-sensitive dextran/polyethylene glycol aqueous phase system. The neutrophils separated as a broad profile, and in a charged phase procedure the separation was based upon differences in cell surface electrokinetic properties, as confirmed by electrophoretic mobility measurements of fractions across the profile using analytical cytopherometry. The CCD cell fractions were generally pooled as three or four major subfractions for analysis of functional and metabolic differences. These included measurements of chemotaxis, phagocytosis, and respiratory burst. An inverse relationship was found between the electrophoretic mobility (EPM) of the subfraction pools and their functional competence, with the less electronegative cell fraction pools often as much as 2 to 3-fold more active than the more electronegative pools. This demonstration of electrokinetic and functional heterogeneity in 'resting' neutrophil subpopulations separated by CCD may reflect changes during their sojourn in the circulation that determine selective margination and recruitment of cells to inflammatory foci and sites of infection.


Immunocytochemical localization of alkane-inducible cytochrome P-450 and its NADPH-dependent reductase in the yeast Candida maltosa.

Antibodies directed against cytochrome P-450Cm1 and the NADPH-cytochrome P-450 reductase were used to study the induction and intracellular localization of these components of the alkane monooxygenase system in the yeast Candida maltosa. Transition from glucose to n-hexadecane utilization resulted in an about 100-fold increase of the immunodetectable P-450 form whereas the reductase was only moderately induced by a factor of about 5. P-450 but not the reductase was further increased by oxygen limitation during cultivation on n-hexadecane. Using an immunogold technique on ultrathin cryosections, P-450 was found to be concentrated in the nuclear envelope during the early phase of the induction process. However, after maximal induction, the highest labeling was observed in membranes of the endoplasmic reticulum closely associated with the peroxisomes and the plasma membrane. Double-labeling experiments revealed that P-450 and its reductase were distributed in the same regions of the endoplasmic reticulum.


Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.


The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.


SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.


Role of vaginal surgery in the 1st stage endometrial cancer. Experience of the Florence School.

The role of vaginal surgery in first stage endometrial carcinoma is reviewed. Out of total of 231 patients with endometrial cancer, observed at the Gynecologic Clinic of Florence from 1977 to 1981, 187 were at Stage I and 180 of them were submitted to surgery. During the period 1977-1979, the treatment protocol recommended vaginal hysterectomy; since 1980 abdominal surgery has prevailed. Depending on risk factors, adjunctive radiotherapy was employed. No statistically significant difference was observed in the actuarial survival curves between different surgery routes. However, severe complications and operative mortality was significantly lower in patients operated by vaginal hysterectomy. The vaginal route may be considered an adequate alternative therapy for endometrial carcinoma Stage I.


Organization of the screening programme in Bialystok Province.

This paper describes the new "model" of the cervical cancer screening introduced as pilot study in the Białystok Province. The new programme was carried out to choose which "model" was better: computerized monitoring and invitation to the women to the outpatient clinic for the Pap-test performed by screeners on full integration into gynecologic health care. The new system introduced in the chosen outpatients clinics increased the acceptance (of the screening) by women especially in younger age groups.


Surgical pathological staging of endometrial carcinoma and results of treatment.

114 women with endometrial carcinoma at clinical stage 1 to 3 were treated with surgery as first line of treatment. Patients were classified as being low or high risk on the basis of the surgical pathological patterns of the tumor. Disease limited to the uterine body, G1-G2 tumors and myometrial invasion of less than 1/3, identified low risk patients which received no adjuvant therapy. All the others were considered high risk and treated with radiation therapy. Patients were retrospectively restaged according to 1988 FIGO guidelines and survival was analyzed. Cox's proportional hazards method was employed to identify independent prognostic factors. Disease free survival (DFS) was 90% for stage 1, 83% for stage 2, and 43% for stage 3 patients. Lymphatic spread was associated to the poorer prognosis. Proportional hazards model showed that tumor grading, myometrial invasion and lymphatic spread were significantly related to the time of relapsing. Low risk patients showed better outcomes despite not having received adjuvant treatment, thus post-operative therapy is not indicated in this subset of patients. Radiation adjuvant therapy for high risk patients did not give satisfactory results. Failures were observed both locally and distantly calling for new adjuvant strategies. Surgical pathological staging of endometrial cancer is currently mandatory. Retroperitoneal lymph node sampling is indicated in patients with high risk pre- (advanced clinical disease, undifferentiated tumors) or intra-operative (deep myometrial invasion, enlarged pelvic nodes) prognostic factors. All prognostic indicators must be obtained from surgery and pathology in order to assess the risk of relapse.


Cisplatin-based combination chemotherapy with or without doxorubicin in advanced epithelial ovarian cancer: 8-year update of a randomized multicentric clinical trial. The Gruppo Oncologico Nord Ovest (GONO).

One hundred and twenty-five patients with advanced epithelial ovarian cancer (EOC) were postsurgically included in a multicentric clinical trial comparing the association of cisplatin (CDDP) (50 mg/m2 iv day1) + cyclophosphamide (CTX) (600 mg/m2 iv day 1) (PC regimen) versus the combination of PC + doxorubicin (ADM) (45 mg/m2 iv day 1) (PAC regimen), repeated every four weeks. After the sixth course of chemotherapy, patients without clinical evidence of disease or with surgically resectable residual disease (RD) underwent second-look laparotomy. Afterwards patients in surgical complete response (sCR) stopped chemotherapy, while partial responders or patients with stable disease received six more courses of the same regimen used as first-line treatment. Among the 67 patients with measurable RD, PAC regimen achieved a better clinical complete response (40.6% vs 20.0%); the difference approached statistical significance. In the 75 patients who underwent second-look laparotomy, PAC regimen induced a significantly higher sCR rate (62.2% vs 39.5%, p less than 0.05). The median survival (S) and progression free survival (PFS) were better in PAC arm, even if the differences were not statistically significant. Eight-year S and 8-year PFS were 32.7% and 24.7% respectively, for PAC arm, and 23.9% and 14.1%, respectively, for PC arm. These data seem to confirm the clinical advantage provided by the addition of ADM to PC regimen in the treatment of advanced EOC.


Intracystic carcinoma of the breast. Our experience.

Malignant tumours of the breast occurring as cysts present an incidence from 0.3% to 0.7%. The Authors report their experience during 15 years in 470 patients with carcinoma of the breast. Endocystic carcinoma appeared in 0.6% of patients: the diagnosis is not always possible with cytology, but ultrasound scans make possible to identiphy cysts which have to undergo biopsy because solid areas within the cystic cavity have been detected. In this case surgery is advised even if the cytological examination has been negative.


Prognostic factors in epithelial ovarian cancer.

In the present study the Authors have statistically evaluated the influence of the main prognostic factors in 160 women affected by epithelial ovarian cancer. They were sequentially observed at Oncologic Institute of Bari, Italy from december 1981 to december 1989. The prognostic factors (age, stage, grading, histological type, performance status and residual disease), have been evaluated through an actuarial analysis of survival and compared with those reported by many authors in literature. Our data confirm the relevance, among all, of the grading, the clinical stage and residual disease as traditional parameters. Furthermore it is proposable the use of new prognostic factors such as nuclear ploidy and proliferative activity (S-phase, labeling index).


Quality of life and neoplastic diseases. Ethical issues.

The article analyses the concept of the quality of life comparing two different ethical approaches, that is "catholic" personalist bioethics and "lay" bioethics. As regards the oncological field, personalist bioethics defends the respect for life as an absolute value (sanctity of life), while "lay" bioethics focuses attention on the patient's autonomy. The authors think that in oncological therapies the subjective evaluation of the patient must be taken into consideration as the expression of his/her personal concept of the quality of life, always with priority of the respect of life.


Multiparametric discrimination of serous ovarian tumors by analytical morphometry.

In order to enhance the discrimination power in the field of serous ovarian tumors, we applied the software system SAM (Shape Analytical Morphometry) to the analytic studies of biological forms. Besides the usual dimensional evaluations (perimeter, area, maximum diameter and shape index), this procedure permits the description of the nuclear form using analytical parameters: 1) extraction of nucleus fundamental curve; that is a functional curve giving the "smoothing" of the original contour by two parametric equations (separately for x and y values as independent variables); 2) evaluation of nuclei contour irregularities by Fourier analysis; 3) evaluation of shape asymmetry by SAE (Shape Asymmetry Evaluator); that is the ratio between the length of a segment of a parabola interpolating the original curve points, and a straight line joining its extremities for a 180 degrees barycentric rotation according 10 degrees steps. All parameters resulted to be independent and were submitted to multivariate discriminant analysis. We studied 180 nuclei from 18 cases of serous ovarian tumors, (6 benign, 6 borderline and 6 malignant tumors). With respect to the dimensional parameters, the application of analytical morphometry permitted us to reduce the minimum percentage error in the discrimination of the different classes. In fact, in the distinctions of benign and malignant nuclei, the minimum percentage error was 13.30%, against the 18.3% error when using dimensional morphometry. Furthermore, in the comparison of malignant and borderline nuclei there was a reduction of error from 23.3% to 22.5%, and in the comparison of benign and borderline nuclei, the error was reduced from 37.5% to 30%.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of the prognostic factors of endometrial carcinoma. A retrospective study of 144 patients.

One hundred and fourty-four patients with endometrial carcinoma who had undergone surgery as primary treatment were retrospectively studied from January 1980 to September 1990 for the purpose of correlating the survival rate with known or presumed prognostic factors. The patients averaged 63.1 years of age (range 32-88 years); 106 were classified as having Stage I disease, 10 Stage II, 16 Stage III and 2 Stage IV. Histology confirmed pure adenocarcinomas in 77%, adenosquamous carcinoma in 3.5%, clear cell carcinomas in 2.5% and serous papillary in 17%. Surgical treatment consisted of extrafasial hysterectomy with pelvic lymphadenectomy up to the aortic bifurcation in 69 patients (48%), Wertheim's hysterectomy in 10 (7%), simple or vaginal hysterectomy in the remaining 65 patients (45%). Five-year survival rate in all the case series was 74.3% whereas for patients with Stage I carcinoma it was 79.9%. Different prognostic factors were correlated in a multivariate analysis with the outcome of the disease. Myometrial invasion presented a mortality ODDS RATIO (OR) of 3.18 (95% CI 1.25-8.06), for histologic grade OR 4.33 (95% CI 1.74-10.74) and for stage (2-3 vs 1) OR of 2.73 (95% CI 1.09-6.83) demonstrating a high significance, whereas pregnancy, excess body weight, age and histotype were not considered as relevant factors for prognosis. For age we found mortality OR 2.54 (95% CI 0.75-8.59) for women greater than 55y.


Inflammatory carcinoma of the breast.

Inflammatory breast carcinoma has to be defined by accurate clinical and pathological criteria. The prognosis is very poor and improvements made by chemotherapy are limited to the increase in the disease-free period and overall survival for a few months.


Carbon dioxide laser surgery in colposcopy for cervicovaginal intraepithelial neoplasia treatment. 10 years experience and failure analysis.

CO2 laser vaporisation is an effective method in the treatment of cervico-vaginal pre-invasive lesions. In a case-series of 334 patients no case of invasive neoplasia development were registered at longterm follow-up. The failure rate is comparable to those described with traditional surgical methods and are due to the particular topography of the disease.


Serum levels of TAG 72 and CA 15.3 in patients with gynaecological neoplasms.

In the present report we have evaluated two new serum tumour markers identified by monoclonal antibodies: TAG 72 and CA 15.3 in 62 patients with gynaecological carcinomas, in prevalence ovarian and 36 women with benign gynaecological diseases. Serum levels of both markers were determined using two immunoradiometric assays and the cut-off values were set at 5 U/mL for TAG 72 and at 40 U/mL for CA 15.3. In our study the sensitivity was 57% for TAG 72 and 43% for CA 15.3 with a specificity of 97% and 89% respectively. Even if these two markers show a sensitivity lower than CA 125, these preliminary results seem to suggest the possible role of TAG 72 and CA 15.3 both as confirmatory tests and as adjunctive tests, especially the first for its excellent specificity.


Quality of life in gynaecological oncology. The personalist perspective of bioethics.

Quality of life in gynaecological neoplastic diseases assumes a specific role within the therapeutic choice. Gynaecologists must give attention to the possibility of the patient's identity crises: a woman's reconstruction of her psycho-physical identity as regards gynaecological cancer is particularly difficult. Psychological assistence acquire a relevant role in improving the patient's quality of life. Personalist bioethics proposes a concept of corporeity which considers body not only in the physical aspect but also as an expression of subjectivity and totality of the person.


Tumor necrosis factor and soluble interleukin-2 receptor: two immunological biomarkers in female neoplasms.

In this study we have evaluated two new immunological parameters, soluble IL-2 receptor (s IL-2 R) and TNF, in 119 patients with female solid neoplasms (47 ovarian and 72 breast cancer). Our data demonstrate that both these markers have mean serum levels in cancer patients higher than in normal population, particularly in ovarian cases. Also the overall positivities were higher in ovarian (68%) than in breast cancer (51%). Finally we observed no relevant differences according to the status of disease in both groups of cancer patients. These preliminary results could suggest the possible usefulness of an immunological monitoring in cancer patients, above all when an immunotherapy with biological responder modifiers is proposed.


Regional surgery in advanced ovarian carcinoma.

Regional surgery can be defined as "the maximum cytoreduction of all macroscopic tumors developing in the abdominal cavity". The Authors present their experiences with this difficult technique and conclude that regional surgery can improve prognosis.


Surgical treatment of invasive carcinoma of the vulva. A reappraisal.

The Authors studied three groups of patients affected by invasive vulvar carcinoma. The first group (19 cases), treated by radical vulvectomy and bilateral inguinal lymphadenectomy, had a survival rate at 5 years of 89% in Stage I and 56% in Stage II. The second group (9 cases) which presented poor general health conditions, had a survival rate at 4 years of 33% and 14% in Stage I and in Stage II, respectively. The third group of patients (7 cases), who refused any type of treatment, died within 12 months. Nodal involvement influenced survival rate. In fact, regardless of the stage, a survival rate at 5 years of 92% and 26% was seen in patients with negative nodes and positive nodes, respectively. In conclusion, the study confirms that radical surgery is the therapy of choice in advanced carcinoma of the vulva. However, early diagnosis remains the most important agent in reducing the extension of the surgical treatment.


Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.


Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan.

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.


Variable lymphocyte responses in rats after space flight.

Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.


The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.


Effects of acrylamide on germinal vesicle migration and dissolution in oocytes of the frog, Rana pipiens.

We have demonstrated that when Rana oocytes are treated with 10 mM acrylamide, germinal vesicle migration (GVM) is promoted while meiosis reinitiation by progesterone is inhibited. A number of other specific alterations result from the acrylamide treatment: (i) A dense band of fibrillar material appears adjacent to the oolemma in acrylamide-treated oocytes. Furthermore, (ii) the fibrillar material reacts with an intermediate filament antibody using immunogold techniques applied to transmission electron microscopy. Moreover, (iii) acrylamide inhibits progestogen-induced annulate lamellae breakdown. In addition, (iv) the cortical mitochondria-rich layer appears to be thickened by acrylamide, which also (v) affects oocyte microvillar retraction and organization. Finally, (vi) electrophysiological measurement of membrane voltage indicates that acrylamide does not significantly affect cell viability during the incubation period used in this study. In summary, acrylamide exerts profound effects on the physiological event of GVM, and these are consistent with the hypothesis that changes in the cytoskeleton are a contributing factor in meiosis reinitiation.


An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.


A nuclear protein associated with lethal heat shock of HL-60 cells.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.


Retinoic acid- and staurosporine-induced bidirectional differentiation of human neuroblastoma cell lines.

The differentiation pattern of two related human neuroblastoma cell lines, SK-N-SHF and SK-N-SHN, induced by retinoic acid and staurosporine was studied. Immunohistochemical and electron microscopic examination of the cells indicated that the SHF variant could undergo differentiation along a melanocytic route when treated with retinoic acid and to neuronal cells when treated with retionic acid and staurosporine together. Treatment of SHN cells with either or both these agents caused neuronal differentiation. The melanocytic pathway was characterized in part by the flattening of the cells, the appearance of melanocytic antigens and various forms of melanosomes, an increase in tyrosinase activity, and the absence of neuronal marker proteins. The neuronal route was typified by the development of long neuritic processes containing microtubules and numerous neurosecretory granules as well as by immunohistochemical reactions for neural cell adhesion molecule, synaptophysin, and neurofilament proteins. The significance of these results is discussed in terms of the differentiation responses of neuroblastoma cells to chemical agents as well as some of the factors involved in the regulation of phenotype expressions of these cells.


Reversible desensitization of fibroblasts to cadmium receptor stimuli: evidence that growth in high zinc represses a xenobiotic receptor.

The xenobiotic Cd2+ triggers the production of inositol trisphosphate and releases stored Ca2+ in certain cell types, apparently by binding to a zinc site in the external domain of an "orphan" receptor (no known endogenous stimulus). Cd2+ and bradykinin evoke similar spikes in cytosolic free Ca2+. Growth in high Zn2+ (100-200 microM) abolished the free Ca2+ spike evoked by Cd2+ without affecting the spike produced by bradykinin. Growth in high Zn2+ almost abolished Cd(2+)-evoked production of [3H]inositol mono-, bis-, and trisphosphate. Bradykinin-evoked [3H]inositol phosphate production was not affected by growth in high Zn2+. Growth in high Zn2+ nearly prevented the stimulation of 45Ca2+ efflux by Cd2+ without affecting the stimulation of 45Ca2+ efflux by bradykinin or histamine. Removing Zn2+ from the culture medium and incubating the cells for several hours fully restored responsiveness to Cd2+. Cycloheximide, actinomycin D, or tunicamycin prevented the restoration of Cd2+ responsiveness, indicating that resensitization requires macromolecular synthesis. Growth in high Zn2+ reversibly abolished Ca2+ mobilization evoked by two additional stimuli: a decrease in extracellular pH or Na+ concentration. These findings support the hypothesis that the three stimuli (Cd2+ or a decrease in external pH or Na+ concentration) activate the same orphan receptor. Growth in high Zn2+ apparently desensitizes the cells to the Cd2+ receptor stimuli by repressing receptor synthesis.


Localization of the nuclear matrix protein mitotin in mouse cells with a mitotic or endomitotic cell cycle.

Immunofluorescence staining was used to study the precise subcellular distribution of the nuclear matrix antigen, mitotin, in mouse cells characterized by either a mitotic or an endomitotic organization of the cell cycle. In mitotically dividing cells, mitotin showed a speckled distribution within interphase nuclei. In addition, some interphase cells exhibited a weak, focused signal adjacent to the nucleus, reflecting a possible staining of the centrosome region. Using digital contrast-enhanced immunofluorescence microscopy, a distinct association of mitotin to the centrosome, pole microtubules, and midbody could be revealed in cells at different stages of mitosis. In parallel, trophoblast giant cells characterized by an endomitotic cell cycle were derived from blastocyst outgrowths and analyzed likewise. In all giant cells examined so far, mitotin was restricted to the nuclear compartment alone, although different patterns of intranuclear staining could be detected. The present study provides further information about the precise localization of mitotin in mitotic cells, especially during mitosis. In view of the results, the staining pattern observed in endomitotic cells may allow for a better understanding of the origin and the organization of the endomitotic cell cycle.


Ets-1 and Ets-2 protooncogene expression in theca cells of the adult mouse ovary.

We have investigated the mRNA expression of the Ets-1 and Ets-2 genes in murine gonads and found expression in adult ovaries. In situ hybridization experiments show that the Ets genes are predominantly expressed in theca cells and cells of ovarian interstitium. By gel retardation experiments we detected DNA binding proteins in ovaries that specifically bind to the ETS motif, suggesting the expression of Ets or Ets-related proteins. Our results raise the possibility of Ets-2 involvement in ovarian pathology seen in patients with Down's syndrome.


Induced cell surface expression of functional alpha 2 beta 1 integrin during megakaryocytic differentiation of K562 leukemic cells.

The pluripotential hematopoietic cell line K562 was studied as a model of inducible integrin expression accompanying differentiation. Differentiation along the megakaryocytic pathway was induced with phorbol 12,13-dibutyrate and differentiation along the erythroid pathway with hemin. Induction of megakaryocytic differentiation was associated with changes in cell morphology and with increased cell-cell and cell-substrate adhesion and spreading. Erythroid differentiation was not associated with changes in morphology or adhesion. Cell surface expression of the IIb-IIIa and alpha 2 beta 1 integrins increased markedly with phorbol treatment but decreased with hemin treatment. Phorbol-treated K562 cells, but not control cells or hemin-treated cells, adhered to collagen substrates in a Mg(2+)-dependent manner which was specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1 integrin. Northern blot analysis revealed that megakaryocytic differentiation of K562 cells was accompanied by de novo expression of the alpha 2 integrin mRNA with no change in the level of mRNA for the beta 1 subunit. K562 cells provide a model of differentiation-dependent, regulated integrin expression in which expression is up- or down-regulated depending upon the differentiation pathway selected.


Platelet-derived growth factor in combination with collagen promotes the migration of human skin fibroblasts into a denuded area of a cell monolayer.

Since we have found previously that adult donor skin fibroblasts (TIG-114) migrated more slowly in serum-depleted medium than in medium supplemented with 10% FBS, we tried to identify a factor(s) which promotes fibroblast migration from the edge of a denuded area in a monolayer. In medium supplemented with 10% FBS, the effects of both suramin, a competitor of growth factors at the receptor level, and monensin, an inhibitor of the secretion of extracellular matrix, were examined. Both substances suppressed cell migration, suggesting that growth factors and matrix substances are important for cell migration. Then, we examined the effects of growth factors and extracellular matrix on fibroblast migration in serum-free medium. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, acidic fibroblast growth factor, and transforming growth factor-beta did not stimulate cell migration. Type I collagen, plasma fibronectin, and heparin also did not promote cell migration. However, the combination of PDGF and type I collagen did promote cell migration. Addition of anti-PDGF antibody reduced the stimulatory effect induced by the combination of PDGF and type I collagen. These results suggest that the copresence of growth factors and extracellular matrix regulates fibroblast migration into a denuded area in a monolayer.


How is rat liver S-adenosylmethionine synthetase regulated?

The in vivo regulation of S-adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed.


The gene structure of tetranectin, a plasminogen binding protein.

The gene for human tetranectin was isolated from a genomic library with a mixture of degenerate oligonucleotide probes. The gene is about 12 kbp and contains two intervening sequences. The gene encodes a protein of 202 amino acid residues, with a signal peptide of 21 amino acid residues, followed by the tetranectin sequence of 181 amino acid residues. Northern blot analysis revealed that tetranectin mRNA was present in all eight tissues tested with the highest concentration in lung. Southern blot analysis showed hybridization to two genes. Further investigations are needed to determine whether the genes are allelic or non-allelic.


Thrombin-induced human platelet aggregation is inhibited by protein-tyrosine kinase inhibitors, ST638 and genistein.

We have investigated the involvement of protein-tyrosine kinases in thrombin-induced aggregation of human platelets, using ST638 and genistein which are known inhibitors of protein-tyrosine kinase. Preincubation of platelets with 50 microM of ST638 or 25 micrograms/ml of genistein completely blocked the platelet aggregation induced with 0.05 unit/ml of thrombin. The increase of protein-tyrosine phosphorylation bands (135-, 124-, 76-, 64-, and 60-kDa) induced with thrombin was also inhibited by these inhibitors in a dose-dependent manner. These inhibitors also blocked the platelet aggregation and protein-tyrosine phosphorylation induced with thrombin in aspirin-treated platelets. Increase of the intracellular Ca2+ concentration induced by thrombin was also inhibited by higher concentrations of genistein. These results suggest that the protein-tyrosine phosphorylation plays a certain role in platelet activation having some relation to the intracellular Ca2+ concentration.


Membrane inserted APP fragments containing the beta A4 sequence of Alzheimer's disease do not aggregate.

Previously we have shown that the COOH-terminal fragment (A4CT) of the Alzheimer amyloid protein precursor (APP), which at the NH2-terminus carries the sequence of the amyloid beta A4 protein, forms highly insoluble aggregates [EMBO J. (1988) 7, 949-957]. Here we report that aggregation is prevented if A4CT is expressed in vitro with a signal sequence at the NH2-terminus (SPA4CT) under conditions which allow membrane insertion. Aggregates from SPA4CT are obtained after removal of membranes by chloroform/methanol extraction or heating.


The zinc finger motif. Conservation of chemical shifts and correlation with structure.

Zinc fingers of the transcription factor IIIA (TFIIIA) type, in which zinc is co-ordinated by two cysteine and two histidine ligands (Cys2/His2), contain a length of helix packed against a beta-hairpin. These zinc fingers comprise the widest range of structurally homologous proteins for which 1H chemical shifts are available. A number of key resonances have chemical shifts that are highly sensitive to tertiary structure and are conserved between these peptides. The high conservation of these fingerprint chemical shifts is correlated with the common global fold of Cys2/His2 zinc fingers. These chemical shifts are largely independent of primary structure and should facilitate NMR assignments for future zinc finger proteins, as well as provide a diagnostic signature for the characteristic Cys2/His2 zinc finger fold.


Co-transcription of the gastrin and cholecystokinin genes with selective translation of gastrin mRNA in a human gastric carcinoma cell line.

So far, no cells have been found to synthesize both of the homologous hormones, cholecystokinin and gastrin. Northern analysis and reverse transcription PCR showed, that the human gastric carcinoma cell line (AGS) expresses both a gastrin mRNA of 0.7 kb and a cholecystokinin transcript of 0.8 kb. A library of sequence-specific radioimmunoassays, cleavage with processing-like enzymes and chromatography subsequently revealed that the gastrin mRNA was translated into progastrin that was constitutively secreted into the medium (45 +/- 3 pmol/l). Neither procholecystokinin nor any of its processing products were detectable in cells and media. The results suggest that differentiation into gastrin- or cholecystokinin-producing cells may be regulated at the translational level. The gastric cell line, AGS, provides a model for studies of translational regulation of cell differentiation.


Distinct and specific GAP activities in rat pancreas act on the yeast GTP-binding proteins Ypt1 and Sec4.

Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide-binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyces cerevisiae. Ypt1, another ras-like 23 kDa guanine nucleotide-binding protein in yeast has been found to be involved in ER-Golgi transport. A mammalian homologue of Ypt1 called rab1 has also been identified. Recent studies using purified Sec4 protein have identified a component of yeast lystate that specifically stimulates the hydrolysis of GTP bound to Sec4. In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E. coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas. Our studies showed that 65% of Sec4-GAP activity was associated with the 150,000 x g pancreatic particulate fraction with approximately 35% being found in the cytosol. On the other hand, more than 95% of Ypt1-GAP activity was found to associate with the particulate fraction. Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs. The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.


Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

An adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands. The protein, mol.wt. 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase. Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis. In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast. These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.


Crystal structure of human recombinant interleukin-4 at 2.25 A resolution.

The crystal structure of human recombinant interleukin-4 (IL-4) has been solved by multiple isomorphous replacement, and refined to an R factor of 0.218 at 2.25 A resolution. The molecule is a left-handed four-helix bundle with a short stretch of beta sheet. The structure bears close resemblance to other cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF). Although no sequence similarity of IL-4 to GM-CSF and other related cytokines has been previously postulated, structure-based alignment of IL-4 and GM-CSF revealed that the core of the molecules, including large parts of all four helices and extending over half of the molecule, has 30% sequence identity. This may have identified regions which are not only important to maintain structure, but could also play a role in receptor binding.


The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.


Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.


Inhibitory effect of human chorionic gonadotropin (hCG) on HIV-1 transmission from lymphocytes to trophoblasts.

It has been demonstrated that human chorionic gonadotropin (hCG) inhibits HIV production in vitro, suggesting that this soluble placental glycoprotein can control viral replication and spread in vivo. hCG--the major product of fetal trophoblasts--was tested on an in vitro model consisting of choriocarcinoma-derived ENAMI trophoblasts exposed to HIV-infected MOLT-4 lymphocytes. The results show a U-shaped antiviral dose-effect and suggest that hCG may contribute to protection against intrauterine transmission of HIV-1.


Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non-hydrolysable GTP analogues.

Treatment of reticulocyte polysomes with elongation factor eEF-2 and GTP led to an increased sensitivity of peptidyl-tRNA for puromycin as a result of the translocation from the ribosomal A-site to the P-site. Upon addition of an excess of the non-hydrolysable GTP analogue, GuoPP[CH2]P, the puromycin sensitivity decreased rapidly. The decrease in sensitivity required high concentrations of eEF-2 with half maximal effect at an eEF-2 concentration of around 1 microM. The data suggest either that peptidyl-tRNA had re-translocated back to the A-site due to the higher affinity of eEF-2 for the pre-translocation than for the post-translocation ribosome, or that the eEF-2-GuoPP[CH2]P complex blocks the peptidyl-transferase activity.


The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values.

We examined the effects of histidine residues that were artificially introduced into complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values. Using a monoclonal antibody specific for hen egg-white lysozyme and three mutant antibodies that contained a histidine residue, we measured binding constants for antibodies and lysozyme at different pH values (pH 5-8). No gross conformational changes were evident over this range of pH values, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH-dependent antigen-binding patterns observed between the wild-type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH-dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.


[Discovery of pancreatic diabetes in Strasbourg].

Although many clinical aspects of diabetes were already known in the second half of the XIXth century, its pathogenesis remained mysterious. The German Medical School of Strasbourg, rich of numerous eminent teachers and researchers such as A. Kussmaul and B. Naunyn in the 1880ies, represented by this time one of the leading centers in diabetes research. The studies of Joseph Freiherr Von Mering on phloridzin-induced glycosuria are an example between others, but the discovery by Oskar Minkowski of the pancreatic origin of the disease undoubtedly stands as the most famous. This discovery was partly indebted to luck since Von Mering and Minkowski were studying a problem of digestive absorption of lipids, but it was greatly due to the excellent clinical and experimental training of Minkowski, as well as his operatory skill. These works, initially criticized by some authors, turned out to stimulate in several countries researchs on the isolation from pancreas of an hypoglycemic substance. Several investigators gave up or failed near the end, which was reached in 1921 by F. Banting and C. Best, subsequently opening the therapeutic era of diabetology.


Effect of oral contraceptive use on the incidence of impaired glucose tolerance and diabetes mellitus.

Combined estrogen-progestin high-dose oral contraceptives increase the risk of impaired glucose tolerance which is estimated at approximately 12% of oral contraceptive current users. Glucose tolerance is adversely affected by the chemical structure of the progestins contained in oral contraceptives such as estrane (norethindrone, ethynodiol) and particularly gonane (norgestrel). The women at high risk to develop an impaired glucose tolerance on high-dose, oral contraceptives are those with previous gestational diabetes, with a positive family history of diabetes mellitus in a first-degree relative, or who are obese or older. Low-dose oral contraceptives with a reduced content of estrogen and progestogen such as levonorgestrel or desogestrel as well as the triphasic oral contraceptives containing a low daily dose of levonorgestrel or gestodene, are associated with a significantly lower risk of impaired glucose tolerance, even in women with previous gestational diabetes. Noteworthy however is the ability of low-dose progestogen containing oral contraceptives, such as low dose gonane or estrane oral contraceptives, to induce glucose intolerance by increasing the plasma insulin response to oral glucose. Impaired glucose tolerance is often reversible within 6 months of discontinuing oral contraceptives, except for oral contraceptive users with previous gestational diabetes. Development of diabetes mellitus in high-dose oral contraceptive users is rare.


Medical complications in pregnancy and delivery of gifted children.

A questionnaire on medical complications in pregnancy, labor and delivery was given to 195 families of gifted children. 156 (80%) answered the questionnaire. The incidence of medical disorders in this group did not differ from the accepted incidence in a normal obstetric population. There was a sex ratio of 71.5% boys and 28.5% girls, and a higher probability of another gifted child in these families.


Vitamin B12 among parturients and their newborns and its relationship with birthweight.

Vitamin B12 (Cobalamin) is an essential nutrient in the diet of humans, particularly during pregnancy, nevertheless very few epidemiological studies have been reported, particularly concerning variation factors. The purpose of this study is to assess the role of vitamin B12 on birthweight after taking into account potential confounders, such as obstetrical, socioeconomic, and biological factors which will be identified. This study was conducted on 188 single births occurring during a 5-month period in an obstetrical care unit in Paris. Vitamin B12 plasma levels and folate plasma levels of mothers and cords along with haematological parameters were measured. Social and obstetrical features of the mothers as well as their tobacco exposure and alcohol consumption were recorded. Cord blood vitamin B12 levels were highly correlated with maternal levels (r = 0.63, P less than 0.001) and were 2-3-fold higher. Three variation factors of vitamin B12 were identified: maternal age, ethnicity and tobacco exposure. Negative correlations between birthweight and vitamin B12 levels were observed only among the smoker group (r mothers = -0.46, P less than 0.05 and r cords = -0.42, P less than 0.05). After adjustment for ethnicity and parity, birthweight remained negatively linked to vitamin B12 in smokers. This relationship could suggest that the availability of vitamin B12 for fetal development depends on certain biological factors present only in mothers who smoked. We hypothesized this relationship could be explained, in part, by lipid metabolism, particular to smokers.


Effect of 50 and 100 g glucose loads on peripheral muscle glucose metabolism in normal man.

The present study was designed to determine the effects of 50 and 100 g glucose loads on forearm muscle glucose uptake, oxidation and nonoxidative glucose metabolism in normal man. Ten healthy male subjects were studied during the postabsorptive state (12-14 h overnight fast) and for 3 hours following glucose ingestion. Peripheral glucose metabolism was analysed by the use of the forearm technique to estimate muscle exchange of substrate combined with indirect calorimetry. Greater forearm muscle uptake and nonoxidative metabolism of glucose were observed in the subjects during the G.100 study than during the G.50 study (G.100 = 178.9 +/- 19.7 and 155.3 +/- 23.0 vs 103.5 +/- 16.6 and 85.2 +/- 16.7 mg/100 g forearm muscle. 3 h, respectively). the muscle glucose oxidation did not show significant difference after the two glucose loads. Insulin levels reached after 100 g glucose ingestion were significantly higher than after the 50 g glucose load. In conclusion, this study revealed a dose-dependent metabolic response in the muscular tissue of normal subjects to oral glucose loads of 50 and 100 g, with respect to forearm muscle glucose uptake and nonoxidative glucose metabolism. The oxidative responses of the muscle tissue to the oral glucose challenges seem not to be directly proportional to the ingested meals.


Comparison between metal cup and silicone rubber cup vacuum extractor.

A total of 400 primiparous patients who were delivered by vacuum extractor were studied. The patients were divided in two groups: Group A included 200 patients who were delivered by metal cup vacuum extractor and Group B consisted of 200 patients who were delivered by rubber cup vacuum extractor. There were no statistically significant differences in the mean maternal age, mean gestational age, mean neonatal birth weight, indications for operative delivery, occipital positions and head stations between the two groups. No differences were found in the rates of birth canal trauma (11% vs. 12.5%), major neonatal scalp trauma (6.5% vs. 5.5%), neonatal jaundice (15.5% vs. 13.5%) and Apgar score. From this study we can conclude that both metal and silicone cups are equally satisfactory for vacuum extraction.


Interaction of lipid and carbohydrate metabolism after infusions of lipids or of lipid lowering agents: lack of a direct relationship between free fatty acid concentrations and glucose disposal.

The present work was planned to study the effects of changes in lipid metabolism irrespective of FFA concentrations (FFA) on the regulation of oxidative and nonoxidative disposal of a glucose infusion during hyperinsulinaemia. Fifteen normal volunteers participated in the 3 protocols, in which 1) Intralipid 2) beta-pyridylcarbinol or 3) isotonic saline were infused during 2 hours. Thereafter, these infusions were discontinued and a two-hour euglycaemic hyperinsulinaemic clamp was performed. All three studies were carried out in combination with indirect calorimetry to measure glucose uptake, and oxidative and nonoxidative glucose disposal (corresponding essentially to glucose storage). Plasma FFA concentrations were 508 +/- 34, 601 +/- 43 and 546 +/- 45 mumol/l in the basal state during the Intralipid, beta-pyridylcarbinol and control protocols. It increased to 960 +/- 71 mumol/l after the Intralipid infusion, fell to 246 +/- 17 mumol/l after the beta-pyridylcarbinol infusion, vs 600 +/- 48 mumol/l in the control. At the end of the glucose-insulin clamp the values were low in the 3 protocols: 263 +/- 17, 233 +/- 19 and 204 +/- 14 mumol/l. Intralipid infusion prior to the clamp protocol induced a suppression of both insulin-mediated glucose uptake (4.91 +/- 0.46 (Intralipid) vs 6.83 +/- 0.63 mg.kg-1.min-1 (saline)) and storage (1.61 +/- 0.34 vs 2.99 +/- 0.53 mg.kg-1.min-1) while beta-pyridylcarbinol infusion induced an increased insulin-mediated glucose uptake (8.58 +/- 0.37 mg.kg-1.min-1) and in glucose storage (4.29 +/- 0.31 mg.kg-1.min-1) (p less than 0.5 vs Intralipid). These changes occurred even though FFA plasma concentrations were similar in the 3 experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Spontaneous diabetes in bio-breeding rats: evidence for insulin dependent liver microsomal delta 9 stearic acid desaturation.

We studied liver microsomal delta 9 stearic acid desaturase activity and fatty acid composition of liver phospholipids and microsomal total lipids in the insulin-dependent spontaneously diabetic adult male Bio-Breeding rat. The diabetic Bio-Breeding rats (3 weeks of diabetes) were killed 48, 17 and 3 h after the last insulin injection (1.0 IU, 100 g body weight-1 day-1). Under these experimental conditions, delta 9 desaturase activity was defective during the normo- and hyper-glycaemic periods and restored during the hypoglycaemic period which followed the insulin injection to the diabetic rats. The fatty acid composition of diabetic rat liver phospholipids and microsomal total lipids were not consistent with delta 9 desaturase activity at the different periods of glycaemia and may be explained by factors other than disturbances of this desaturation.


Quantitation of breathing-related modulation of umbilical arterial and venous flow velocity waveforms in the normal term fetus.

The effect of fetal breathing activity on umbilical arterial and venous flow velocity waveforms was studied in 11 normal pregnant subjects at term during fetal behavioural state 2F (Nijhuis et al. Early Hum Dev 1982;6:177-195). A total of 1041 breathing cycles was evaluated. Our data suggest a net reduction in venous flow velocity during fetal breathing activity. In contrast to venous flow velocity, the magnitude of breathing-related modulation of arterial pulsatility is small and independent of breathing amplitude. Nevertheless, for clinical purposes umbilical arterial flow velocity waveform recording during fetal apnea is recommended.


Sulfate metabolism in experimental diabetes.

Previous studies have indicated altered rates of sulfate clearance in experimental diabetes, but have disagreed on the magnitude and direction of change of serum sulfate, or its impact on tissue sulfate concentrations. The present study was undertaken in order to investigate the temporal effects of streptozotocin-induced diabetes on sulfate pools in the rat. In contrast to some earlier reports, we found no effects of diabetes on serum sulfate concentrations throughout 12 weeks of disease, as measured by ion chromatography. However, marked increases in sulfate concentration were found in liver cytosol (3.5-fold; p less than 0.0005) and kidney (1.7-fold; p = 0.002) by 7 weeks. The measurement of inulin space demonstrated that the changes in hepatic sulfate were due to increased concentrations of the ion in the cell cytosol. Normal serum sulfate concentrations, together with reports of increased renal clearance of sulfate in diabetes, suggest an intracellular origin of the excess cytosolic sulfate. Glucose did not affect the uptake of sulfate by renal mesangial cells in culture. These aberrations in sulfate metabolism are not such as to compromise biological sulfation reactions, and cannot account for reported changes in the incorporation of sulfate into basement membrane proteoglycans during diabetes.


Abnormal fetal behaviour in first trimester spontaneous abortion.

The behaviour of 5 fetuses at 10 weeks of gestational age that later underwent a spontaneous abortion during the first trimester of pregnancy was compared to similar aged normal fetuses (n = 116). Marked abnormalities of behaviour were noted in three of the pregnancies that spontaneously aborted. These behavioural abnormalities were not identified in any of the pregnancies which continued to term. We conclude that fetal behaviour, which reflects the neural integrity of the individual, and thus fetal well-being, can be used during the first trimester to give an indication as to the viability of the on-going pregnancy.


Routine previous cesarean scar exploration following successful vaginal delivery. Is it necessary?

Among the 960 women who successfully delivered by vaginal route after previous cesarean section, 10 (1.04%) had complete lower segment scar rupture over a 9 year period. All were sufficiently symptomatic in order to suspect a ruptured uterus prior to scar exploration. We conclude that lower uterine segment palpation should be reserved only for symptomatic patients.


Severe ovarian hyperstimulation: a case report and essentials of prevention and management.

A case of ovarian hyperstimulation syndrome is presented occurring in a young woman with polycystic ovary-like disease after induction of ovulation with the combined treatment of a luteinizing hormone releasing hormone analog and human menopausal gonadotrophins. Prevention and management based on pathophysiological considerations are reviewed.


Determinants of hormonal replacement therapy in recently postmenopausal women.

Although the efficacy of hormonal replacement therapy (HRT) on the consequences of the menopause is not questioned, it appears that in Europe and in the USA only a small proportion of women are users of HRT. In this study, we examined the prevalence and the determinants of HRT among 1986 French menopausal women, aged 45 to 55 years, presenting to a preventive medicine centre. Overall, 8.1% of women reported current use of HRT. The estrogen preparation most commonly reported was transcutaneous 17 beta-oestradiol. The first determinant of current HRT was birth-place. Women born in France were nearly four times more likely to be on treatment than foreign-born women. A surgical menopause multiplied the probability of current HRT by 2, as did a high level of education. An age at first pregnancy of more than 20 and less than 4 children were also positively linked with HRT use. Even in this population of recently menopausal women, volunteering to undergo health evaluation, the prevalence of HRT was low. The reservations towards HRT may be partly due to the women themselves, and partly due to the physicians. It seems very important to inform the medical profession about the risks and benefits of HRT, and to understand more precisely the reasons why so few women use HRT.


First steps in the development of an information and counselling service for women with an abnormal smear result.

A study was conducted to develop and evaluate an information leaflet and to investigate the acceptability of a counselling service for women with an abnormal smear result. A newly constructed information leaflet which addressed concerns related to receipt of an abnormal smear result and its medical management was compared for ease of readability with five other leaflets in current use and found to be superior. 144 women were given the leaflet either prior to a colposcopy examination or outpatient ablative treatment and randomly assigned to two recruitment strategies to examine the acceptability of a counselling interview. There was no significant difference between the two strategies and an uptake rate of 80% suggested that women welcomed such an opportunity. An interview evaluation schedule yielded a 98% positive response rate which indicated that the women were satisfied with the counsellor and found the interview useful. Significant differences emerged before and after medical intervention. Patients reported less anxiety, improved mood, and less distress related to the smear result and subsequent medical procedures immediately after the medical interventions (P less than 0.001).


Pregnancy and protein C deficiency.

This report examines a patient with recurrent attacks of thrombo-embolism due to a protein C deficiency. Alterations in the coagulation during pregnancy and the possible consequences of an altered coagulation during pregnancy will be discussed.


Prenatal ultrasound diagnosis of an intracranial teratoma influencing management: case report and review of the literature.

Of seventeen cases of prenatally diagnosed intracranial teratomas, fourteen babies were delivered through a cesarean section. All children died before or shortly after delivery. In this report another case is presented of ultrasound diagnosis of an intracranial teratoma. The teratoma filled the whole fetal cranium, and eroded through the skull base into the left maxillary region. In full agreement with the wishes of the parents, conservative management resulted in the spontaneous vaginal delivery of a fetus that died during labor as a result of brain compression and acidosis during the second stage. In contrast with other cases in the literature, no encephalocentesis was performed. In order to minimize maternal morbidity, it is advised to induce or accept labor as soon as the diagnosis is clear and to pursue a vaginal delivery.


Serum concentrations of magnesium and parathyroid hormone in randomly selected hospital in-patients and out-patients, and in in-patients with dementia.

The relationship between hypomagnesaemia and the secretion of parathyroid hormone was studied. Serum parathyroid hormone (PTH) concentrations were measured in 63 randomly selected patients and 54 hospitalized demented patients using carboxyl-terminal PTH53-84 (h-PTH53-84), mid-region PTH44-68 (m-PTH44-68) and intact PTH1-84 kits. Both groups of patients were further subdivided into those with near normal serum magnesium concentrations (1.7 - 3.2 mEq/l) and those with hypomagnesaemia (1.2 - 1.6 mEq/l). Serum h-PTH53-84, m-PTH44-68 and PTH1-84 concentrations in both the randomly selected and the demented patients with hypomagnesaemia were significantly lower than those in the groups with near normal serum magnesium concentrations. The results suggest that parathyroid hormone secretion was inhibited in randomly selected patients and dementia patients with hypomagnesaemia.


Caffeine reduces the hepatotoxicity of paracetamol in mice.

Paracetamol causes extensive liver damage when taken in overdose quantities; however, it is less hepatotoxic when administered in combination with caffeine. The present work in mice was undertaken to study the effect of caffeine on mortality rates and biochemical and histological parameters of liver damage after administration of toxic doses of paracetamol. It was found that caffeine markedly increased the survival rate after administration of a dose of paracetamol that was lethal to 50% and 100% of mice, reduced liver damage as assessed by serum glutamic pyruvic and glutamic oxaloacetic transaminase activities, partially prevented the depletion of reduced glutathione and reduced histological changes to the liver accompanying paracetamol intoxication. The results support the possibility that caffeine might be useful for the treatment of paracetamol intoxication in humans.


Inhibitory effect of nafamostat mesilate on metastasis into the livers of mice and on invasion of the extracellular matrix by cancer cells.

Although many agents that interfere with clotting mechanisms have been investigated for their potential to inhibit metastasis, their toxicity has prevented administration of sufficiently high doses to achieve inhibition of metastasis in clinical trials. Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, inhibited liver metastasis in a CDF1 mice model with colon 26 adenocarcinoma cells. The apparently dose-dependent inhibitory effect was seen 21 days after all of the doses tested (0.3, 1.0, 3.0 and 10.0 mg/kg for 7 days) but the effect was only statistically significant (P less than 0.01) at the highest dose. The blood concentrations 3 min after dosing were less than 10(-6) M for all of the doses tested. At a concentration of 10(-5) M or less nafamostat mesilate was not cytotoxic towards colon 26 cells in vitro. The results indicate that it may not be difficult to achieve blood nafamostat mesilate concentrations that inhibit metastasis in mouse liver. Possible mechanisms of nafamostat mesilate are inhibition of extravasation and invasion of cancer cells, inactivation of collagenase due to inhibition of plasmin activity and inhibition of the formation of the cancer cell thrombus, and arrest in the capillaries through inhibition of thrombin activity. These preliminary results suggest that peri-operative administration of nafamostat mesilate may prevent metastasis into the liver after surgery for gastrointestinal malignancies.


Effects of sodium hyaluronate on tympanic membrane perforations.

The effects of topically applied 1% sodium hyaluronate as an alternative treatment to surgery for tympanic membrane perforations was studied. In an open study, the inclusion criteria were increased by accepting patients previously treated by surgery or with placebo and, compared with a previous study, the frequency at which sodium hyaluronate was applied was reduced. Reduction in the size of the perforation was detected in 12/16 (75.0%) patients, six (37.5%) of whom showed complete tympanic healing, and in the remaining four (25.0%) there was no healing. The average percentage reduction in the perforation area was 80.5%. The two factors altered in this trial, i.e. inclusion criteria and frequency of administration of treatment, had little influence on the degree of membrane healing.


Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells.

To dissect the functional architecture of the rat gonadotropin releasing hormone (GnRH) gene promoter and its regulation by estrogen, gene transfer studies were performed in a placental cell line. 5'-Deletional constructs demonstrated that cis-acting elements important for rGnRH promoter activity in placenta were contained within a region of the proximal promoter between -73 and -16 bp upstream of the transcription initiation site. In addition, an inhibitory region was found from -903 to -424. Deletion of sequences to -424 which removed the inhibitory region, produced a promoter fragment which exhibited a 50% inhibition of GnRH promoter activity in the presence of estrogen (E) and cotransfected estrogen receptor. Negative regulation by E was retained in constructs deleted to -73 bp. We conclude that sequences important for placental cell expression and estrogen regulation of rGnRH lie within the region from -73 to -16. In addition, potential repressor sequences active in placental cells are present between -903 to 424.


Transfected insulin-like growth factor II modulates the mitogenic response of rat thyrocytes in culture.

Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.


Androgens increase insulin receptor mRNA levels, insulin binding, and insulin responsiveness in HEp-2 larynx carcinoma cells.

Androgen receptors have been found in human larynx and androgens have been supposed to play an important role in promoting the growth of laryngeal carcinomas. The molecular mechanism underlaying this phenomenon is not at all understood. Aim of this work was to investigate the effects of two androgens (testosterone and dihydrotestosterone) on insulin receptor mRNA levels and insulin binding activity as well as on either metabolic or growth-promoting actions of insulin in a human larynx carcinoma cell line (HEp-2). We found that HEp-2 cells express a high affinity insulin receptor. Both androgens significantly increase insulin receptor mRNA levels and insulin receptor number in HEp-2 cells. Insulin action, evaluated either as total glucose utilization or as [3H]thymidine incorporation into DNA, significantly increased in HEp-2 treated with androgens in comparison to control cultures. Altogether, our data allow us to speculate that the increased insulin effectiveness we observed in the larynx carcinoma cell line HEp-2 after androgen treatment might be involved in the regulation of larynx cancer cells growth.


Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus.

Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein with a molecular weight of 9000. CaBP-9k is mainly expressed in intestine, uterus and placenta, with intestinal levels controlled by vitamin D and uterine levels controlled by estrogens. CaBP-9k mRNA levels were measured in rat uterus throughout the estrous cycle. On the morning of proestrus, estrus and diestrus animals were sacrificed. Serum 17 beta-estradiol concentrations were determined using a radioimmunoassay. Whole uterus was used for preparation of total RNA. Northern blot analysis was performed to quantify CaBP-9k and beta-actin mRNA. CaBP-9k levels were highest at proestrus, dropped 10-fold at estrus and were not detectable at diestrus. beta-Actin levels did not change significantly throughout the estrous cycle. Peak 17 beta-estradiol concentrations coincided with maximum CaBP-9k mRNA expression at proestrus. Despite minimal concentrations of 17 beta-estradiol at estrus, CaBP-9k mRNA was still present at 10% of the proestrus level. At diestrus, CaBP-9k mRNA was not detectable despite increasing 17 beta-estradiol. It is concluded that CaBP-9k is subject to 17 beta-estradiol regulation during the estrous cycle. Correlation between CaBP-9k mRNA and 17 beta-estradiol levels indicates a lag period for CaBP-9k induction in diestrus following a rise in steroid hormone levels.


Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells.

Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [125I]oGH molecules at high affinity (Kd = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes (Kd = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865-1872). The expressed receptor also binds ovine placental lactogen (oPL, 205,000 binding sites per cell) with high affinity (Kd = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis (M(r) 148,000 and M(r) 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M(r) 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species (M(r) 133,000 and M(r) 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c-fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148-1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265-274).