Werner protein recruits DNA polymerase delta to the nucleolus.

Werner syndrome is a Mendelian disorder of man that produces a number of manifestations resembling human aging. This disorder is caused by inactivation of the wrn gene, a member of the RecQ family of DNA helicases. The helicase and exonuclease activities of the Werner protein (WRN) suggest that it functions in DNA transactions, but the physiological function of WRN remains elusive. We present several lines of evidence that WRN interacts specifically with the p50 subunit of polymerase delta, the major DNA polymerase required for chromosomal DNA replication. P50, identified by yeast two-hybrid screening, interacts physically with the C terminus of WRN. Native WRN protein coimmunoprecipitates with p50 in a cellular fraction enriched in nucleolar proteins, and this immunocomplex also includes p125, the catalytic subunit of polymerase delta. In subcellular localization studies of cells transfected with WRN, p50 and p125 redistribute to the nucleolus and colocalize with WRN. These results suggest that one of the functions of WRN protein is to directly modify DNA replication via its interaction with p50 and abet dynamic relocalization of the DNA polymerase delta complexes within the nucleus.

Alterations of biological parameters in mice chronically exposed to low-frequency (50 Hz) electromagnetic fields.

In an experimental study we measured changes in hematological, biochemical and cortisol parameters in 6-week-old Swiss mice continuously exposed to ELF generated by a transformer station and high current bus bars. Mean daily exposure of 5.0 microT was maintained for 350 days. Hematological parameters were compared to those of control mice (n=12) exposed to a field level lower than 0.1 microT. Serum biochemical parameters (sodium, potassium, chloride, calcium, magnesium, phosphorus, amylase, creatine phosphokinase, and lactate dehydrogenase) were measured after 28 days of exposure and serum cortisol after 90 and 190 days. Granulocyte/macrophage colony-forming cells (GM-CFC) were counted at the end of the 350-day exposure. On day 20, exposed animals showed a significant decrease in leukocyte, erythrocyte, lymphocyte and monocyte counts and in hemoglobin and hematocrit values, while MCV increased. On days 43 and 63 no significant difference was observed in leukocyte and erythrocyte values, as if hemopoiesis had recovered. On day 90, a significant fall in the leukocyte, polynuclear neutrophil and eosinophil counts was observed in the exposed animals. No significant difference was noted in the biochemical parameters studied. On day 190, exposed animals had neutropenia and a decrease in the cortisol value. On day 350, no significant difference in hematological parameters was noted. Individual differences in sensitivity were observed, as 8 mice in the exposed group showed a significant decrease in the leukocyte, polymorphonuclear neutrophil and GM-CFC counts, while in two mice there was a significant increase in these same values compared to those unexposed mice.

Human T-cell lymphotropic virus-1-positive T-cell leukemia/lymphoma in a child. Report of a case and review of the literature.

Adult T-cell leukemia/lymphoma is a monoclonal T-cell neoplasm associated with human T-cell lymphotropic virus-1 (HTLV-1) that occurs almost exclusively in adults. This report concerns a Romanian girl who had recurrent skin eruptions since infancy, subcutaneous tumors in childhood, and peripheral blood lymphocytosis, which initially developed at the age of 12 years. The circulating lymphocytes were of helper T-cell immunophenotype. Serologic studies demonstrated a number of HTLV-1 antigens in the child and her mother, and molecular analyses revealed monoclonal T-cell-receptor gamma gene rearrangement and detectable HTLV-1 proviral DNA. Conventional cytogenetic studies revealed a t(3;6)(q23;q27) chromosome translocation in most of the neoplastic cells. The patient initially responded well to interferon alfa therapy and showed regression of skin lesions and diminished lymphocytosis, but 4 years later, she developed massive lymphadenopathy and leukemic infiltration of the breast. At last clinical follow-up, at the age of 17 years, the patient had stable low-level peripheral lymphocytosis and subcutaneous tumors while being continuously treated with interferon alfa. Our review of the literature revealed six additional children with HTLV-1-associated T-cell leukemia/lymphoma, including one case with a similar clinical presentation and ethnic background. To our knowledge, the t(3;6)(q23;q27) translocation identified in this patient's neoplasm has not been previously reported in adult T-cell leukemia/lymphoma cases and may explain the early onset of disease. Although adult T-cell leukemia/lymphoma is rare in Romania, the identification of healthy carriers and vertical transmission raise the possibility that Romania might be an endemic region for HTLV-1 infection.

An immunohistochemical study of P53 protein on colorectal carcinomas.

AIMS: To investigate the frequency of immunohistochemical expression of p53 protein in colorectal carcinomas; to evaluate the correlation between p53 alterations and the histopathological parameters; to determine the prognostic significance of this tumor marker. PATIENT POPULATIONS: Fourty patients with colorectal carcinomas. METHODS: Paraffin embedded normal and tumoral tissues, stained, with HES, PAS, PAS-Alcian Blue stains. For detecting the p53 protein, a three step (streptavidin-biotin immunoperoxidase) immunohistochemical method was used. RESULTS: 28 out of 40 (70%) colorectal carcinomas were positive for p53. The identification of nuclear accumulations of p53 protein in the present study was significantly correlated with the anatomic site and tumor stage. CONCLUSIONS: The mutational pattern observed in this study was substantially as expected. The p53 overexpression is present in advanced stage, thus demonstrating the prognostic value of this marker.

Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF). Treatment of astrocytes with interleukin-1 beta (IL-1) or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) increased NGF mRNA content by six- to 10-fold, followed in time by increases in cell content and cell secretion of NGF. Dexamethasone potently inhibited the effects of IL-1 and TPA on astroglial cell NGF expression. The action of IL-1 was not mediated by PKC because treatment of cells with maximal concentrations of both IL-1 and TPA gave an additive increase in NGF mRNA content and NGF secretion, and because down-regulating PKC activity failed to inhibit the stimulatory effects of IL-1. Moreover, both agents increased NGF gene transcription in nuclear run-on assays, but only IL-1 significantly stabilized the NGF mRNA. An analysis of the effects of IL-1 and TPA on immediate early gene expression indicated that IL-1 preferentially induced c-jun gene expression, whereas TPA greatly increased c-fos and zif/268 gene expression. These results suggest that IL-1 activates c-jun and NGF gene expression, and NGF mRNA stabilization in astrocytes by a distinct PKC-independent signaling pathway.

Induction of ornithine decarboxylase by N-methyl-D-aspartate receptor activation is unrelated to potentiation of glutamate excitotoxicity by polyamines in cerebellar granule neurons.

Polyamines positively modulate the activity of the N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)--the key regulatory enzyme in polyamine synthesis--and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.

Experimental study of the protective effect of intermittent hepatic pedicle clamping in the rat.

The tolerance of the liver to ischaemia during intermittent clamping of the hepatic pedicle was compared with that during a continuous Pringle manoeuvre. Three groups of rats undergoing total durations of clamping of 60, 90 and 120 min were studied. A temporary peroperative portacaval shunt was used to exclude the effects of splanchnic venous stasis and allow independent study of the effects of hepatic ischaemia. In each group, three methods of portal clamping were evaluated: a continuous Pringle manoeuvre (n = 10), a 30-min intermittent clamping (n = 10) and a 15-min intermittent clamping (n = 10). The clamp release time between the periods of liver ischaemia was 5 min. Survival at day 7 and postoperative changes in liver function (transaminase enzymes, bilirubin, bromsulphthalein elimination, liver adenosine 5'-triphosphate levels) were recorded. Intermittent clamping of the pedicle was tolerated significantly better than continuous clamping. This method optimizes the ability of the liver to tolerate extended periods of ischaemia. For a given duration of ischaemia, no additional improvement could be produced by shortening the intermittent clamping period from 30 to 15 min. These data suggest that, when the Pringle manoeuvre is used, it should be applied intermittently rather than continuously.

Transcriptional program coordination by N-methyl-D-aspartate-sensitive glutamate receptor stimulation in primary cultures of cerebellar neurons.

In primary cultures of rat cerebellar neurons, a brief stimulation of glutamate receptors results in coordinated activation of a programmed early gene response involving increases in the amount of c-fos, c-jun, jun-B, and zif/268 mRNAs. Each of these genes was induced to a different extent and showed a temporal pattern characterized by either a monophasic "early" response, occurring within 30 min of glutamate addition, or a biphasic response (c-jun), lasting for up to 6 to 8 hr after the initial stimulus. The early phase of the glutamate-induced gene expression was prevented by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, a highly selective isosteric antagonist of the N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDA receptor). The second phase of the c-jun response was not blocked when the NMDA receptors were completely inhibited after the initial pulse of agonist or when the quisqualate-kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione was added, suggesting that a brief NMDA receptor stimulation triggers a cascade of events critical for the manifestation of the delayed c-jun expression. Furthermore, gel retardation assays demonstrated that NMDA receptor activation results in a prolonged increase in nuclear DNA-binding activity specific for the AP-1 transcriptional regulatory element. Protein immunoblot analysis showed that the composition of this nucleoprotein complex changes as a function of time, reflecting a cascade that involves an increased translation of Fos and several Fos-related proteins. The coordinated induction of several different transcription factors and the variations in transcriptional complex formation initiated by NMDA receptor stimulation may be a key mechanism in the orchestration of specific target gene expression that underlies various aspects of neuronal function, including plasticity responses.

Surface immunoglobulin-positive lymphoblastic lymphoma. A report of three cases.

Lymphoblastic lymphomas (LBL) are a morphologically homogeneous group of non-Hodgkin's lymphomas which are indistinguishable in tissue sections from acute lymphoblastic leukemia (ALL). Although initial immunologic studies of LBL suggested that these lymphomas are of the T-cell phenotype, investigations have recently described patients with LBL having pre-B-cell, "common" ALL, and natural killer cell phenotypes. The authors recently reported detailed immunophenotypic characteristics in 36 cases of LBL, including a single case of LBL with a surface immunoglobulin-positive B-cell phenotype. Three additional patients with LBL whose cells expressed monoclonal serum immunoglobulins are presented here. In addition, the neoplastic lymphocytes expressed several B-cell-associated antigens. The tumor cells in the single case tested were TdT-negative. Despite the unusual immunologic phenotype, the morphologic features in all three cases were characteristic of LBL. In addition to the previously reported single case, to the authors' knowledge these are the only reported cases of a previously unrecognized variant of LBL: surface immunoglobulin-positive, B-cell LBL.

Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death.

Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/phosphatidylserine/diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by approximately 90% in the 100,000 x g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the beta-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and a marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 microM glutamate for 15 min (no Mg2+) induces the translocation of supernatant PKC immunoreactivity to the pellet. Such translocation persists after glutamate withdrawal and is followed by a progressive increase in neuronal death, which begins 2 hr later. Neuronal death approaches completion in about 24 hr. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [3H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45Ca2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). Moreover, the immediate cell-body swelling and activation of spectrin proteolysis elicited by glutamate remain unchanged. On the other hand, PMA-induced PKC down-regulation reduces any increase in 45Ca2+ uptake or Ca2(+)-dependent proteolysis (measured as spectrin degradation) after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca2+ homeostasis and neuronal death.

Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein.

The chain of events that results in increased production of nerve growth factor (NGF) following beta-adrenergic receptor (BAR) stimulation has been investigated in the C6-2B rat astrocytoma cell line. Exposure of these cells to the BAR agonist isoproterenol elicits the following cascade of events: (i) increase of cAMP content; (ii) increase of c-Fos mRNA content; (iii) accumulation of c-Fos protein immunoreactivity in the nucleus; (iv) increase of NGF mRNA content. The increase in c-Fos mRNA and its translation product are early events (15 and 40 min, respectively) and precede the accumulation of NGF mRNA, which peaks at 3 hr. The increase in the two mRNAs appears interrelated because cycloheximide inhibits the accumulation of c-Fos protein and NGF mRNA elicited by isoproterenol. Moreover, the accumulation of nuclear c-Fos protein and NGF mRNA induced by BAR stimulation is reduced by 2-aminopurine, an inhibitor of c-Fos mRNA induction. These data suggest that, in C6-2B astrocytoma cells, the nuclear accumulation of c-Fos protein is required for the induction of NGF mRNA expression by BAR stimulation.

In primary cultures of cerebellar granule cells the activation of N-methyl-D-aspartate-sensitive glutamate receptors induces c-fos mRNA expression.

L-Glutamate, the natural agonist of quisqualate- and N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors, elicits a rapid, transient, dose-dependent increase of the steady state level of c-fos mRNA followed by an accumulation of c-fos protein immunostaining in cell nuclei. This induction is prevented by 2-amino-5-phosphonovalerate, an isosteric glutamate receptor antagonist, and by Mg2+ ion and phencyclidine, two noncompetitive allosteric antagonists of NMDA-sensitive glutamate receptors. Kainate and quisqualate (up to 150 microM) failed to alter the basal expression of c-fos mRNA. Furthermore, glycine, a positive allosteric modulator of NMDA-sensitive glutamate receptors, potentiated the glutamate response in a strychnine-insensitive manner. Activation of other transmitter receptors present in these cells (gamma-aminobutyric acid(A), gamma-aminobutyric acid(B), and muscarinic) failed to increase c-fos mRNA expression. Our results provide evidence that activation of NMDA-sensitive glutamate receptors plays an exclusive role in the induction of c-fos mRNA expression and translation in primary cultures of granule cells. It can be inferred that, by this mechanism, glutamate can initiate a transcriptional program that may result in changes in the simultaneous expression of a set of target genes involved in neuron-specific responses.

Activation of specific glutamate receptor subtypes increases C-fos proto-oncogene expression in primary cultures of neonatal rat cerebellar granule cells.

In primary cultures of rat cerebellar granule cells the activation of excitatory amino acid receptors by 1-glutamate enhances the steady state level of c-fos proto-oncogene messenger RNA. This effect is blocked by magnesium (1mM) as well as by the glutamate receptor antagonist 2-amino-5-phosphono-valerate (APV). Among the other excitatory amino acid agonists N-methyl-D-Aspartate (NMDA) and quisqualate also increased c-fos mRNA content, the latter however to a significantly lesser extent, while kainate failed to modify the basal level of c-fos expression. The addition of the muscarinic agonist carbachol or of the inhibitory neurotransmitter GABA did not affect the basal level of c-fos mRNA. This data demonstrate for the first time that activation of signal transduction at a specific excitatory amino acid receptor subtype can increase the steady state level of c-fos proto-oncogene mRNA in primary culture of cerebellar neurons.

Effect of a protracted antidepressant treatment on signal transduction and [3H](-)-baclofen binding at GABAB receptors.

In membranes prepared from frontal cortex of rats receiving desmethylimipramine (10 mg/kg i.p. twice daily) or imipramine (7.5 mg/kg i.p. twice daily) for 3 weeks, the density of high-affinity gamma-aminobutyric acid (GABA)B recognition sites is increased when measured by [3H]GABA binding in the presence of an excess of bicuculline, but it is unchanged when measured by [3H](-)-baclofen binding. When the atypical antidepressant maprotiline was administered (10 mg/kg i.p. twice daily for 3 weeks), no change in the density of GABAB recognition sites was observed using either [3H]GABA or [3H](-)-baclofen as ligands. In addition, a protracted treatment with imipramine, desmethylimipramine and maprotiline failed to change GABAB receptor-coupled signal transduction as monitored by the ability of (-)-baclofen to inhibit the forskolin-stimulated adenylate cyclase activity in membranes prepared from frontal cortex and hippocampus or the cyclic AMP formation in slices from frontal cortex. Hence, after protracted antidepressant treatment, the increase of [3H]GABA binding may not reflect changes in the characteristics of the recognition sites of the GABAB receptors subclass coupled to the adenylate cyclase through a guanine nucleotide binding protein inhibitory (Ni).

Biliary metastases of breast carcinoma. The case for resection.

Obstructive jaundice developed in two patients 6 and 8 years after surgery for breast carcinoma. In both patients exploration disclosed a tumor of the hilus which was a biliary metastasis of breast cancer. Surgical resection was performed. Examination of the resected specimen showed infiltration of the duct walls by sheets of metastatic carcinomatous cells from the previous breast cancer. The postoperative course was uneventful in both patients with disappearance of all symptoms due to the biliary obstruction. The first patient died 4 years later from peritoneal deposits and the second was alive at 30 months with a metastasis to the hip. These observations differ from most of those previously reported, by the localization of the tumor at the hilus and the direct infiltration of the duct walls by the tumor. The results of this study suggest that aggressive surgical treatment may be the treatment of choice in patients with extrahepatic biliary metastases of breast carcinoma.

Intractable ascites in systemic mastocytosis treated by portal diversion.

A 50-year-old male presented with intractable ascites due to systemic mastocytosis. The diagnosis of systemic mastocytosis was established by histology of the bone marrow which showed mast cell infiltration and fibrosis. Ascites was related to portal hypertension which was documented by esophageal varices at endoscopy and by an increase of wedged-free hepatic venous pressure gradient. Liver biopsy disclosed dense fibrosis of hepatic arterial and portal venule walls, resulting in complete obstruction of some portal radicles. Peliosis hepatis and fibrous deposits in the walls of hepatic venules were also present. Because of intractable ascites and significant malnutrition, a portacaval shunt was performed which cleared ascites and dramatically improved the general condition of the patient.