Cytotoxic and genotoxic potential of geraniol in peripheral blood mononuclear cells and human hepatoma cell line (HepG2).

Geraniol is an acyclic monoterpene alcohol present in the essential oil of many aromatic plants and is one of the most frequently used molecules by the flavor and fragrance industries. The literature also reports its therapeutic potential, highlighting itself especially as a likely molecule for the development of drugs against cancer. In view of these considerations, this study was designed to evaluate the cytotoxic and genotoxic potential of geraniol, in an in vitro protocol, using two types of human cells: one without the ability to metabolize (peripheral blood mononuclear cells - PBMC), and the other with this capability (human hepatoma cell line - HepG2) through the comet assay and the micronucleus test. Four concentrations (10, 25, 50, and 100 µg/mL) were selected for the genotoxic assessment for PBMC and three (1.25, 2.5, and 5 µg/mL) for HepG2 cells based on cytotoxicity tests (MTT assay). Results showed that geraniol did not present genotoxic or clastogenic/aneugenic effects on both cell types under the conditions studied. However, caution is advised in the use of this substance by humans, since a significant reduction in viability of HepG2 and a marked decrease in cell viability on normal PBMC were verified.

Neuroprotective effects of guanosine administration on behavioral, brain activity, neurochemical and redox parameters in a rat model of chronic hepatic encephalopathy.

It is well known that glutamatergic excitotoxicity and oxidative stress are implicated in the pathogenesis of hepatic encephalopathy (HE). The nucleoside guanosine exerts neuroprotective effects through the antagonism against glutamate neurotoxicity and antioxidant properties. In this study, we evaluated the neuroprotective effect of guanosine in an animal model of chronic HE. Rats underwent bile duct ligation (BDL) and 2 weeks later they were treated with i.p. injection of guanosine 7.5 mg/kg once a day for 1-week. We evaluated the effects of guanosine in HE studying several aspects: a) animal behavior using open field and Y-maze tasks; b) brain rhythm changes in electroencephalogram (EEG) recordings; c) purines and glutamate levels in the cerebral spinal fluid (CSF); and d) oxidative stress parameters in the brain. BDL rats presented increased levels of glutamate, purines and metabolites in the CSF, as well as increased oxidative damage. Guanosine was able not only to prevent these effects but also to attenuate the behavioral and EEG impairment induced by BDL. Our study shows the neuroprotective effects of systemic administration of guanosine in a rat model of HE and highlights the involvement of purinergic system in the physiopathology of this disease.

Chronic Atrophic Gastritis with Negative Intrinsic Factor and Parietal Cell Antibody Presenting as a Severe Hemolytic Anemia.

A 28-year-old Caucasian male with Hashimoto's disease and vitiligo presented with two weeks of dizziness on exertion following pharyngitis which was treated with prednisone 40 mg by mouth once a day for five days. Initial workup revealed anemia, elevated lactate dehydrogenase (LDH), and low haptoglobin. He underwent workup for causes of hemolytic anemia which was remarkable for a peripheral blood smear with hypersegmented neutrophils and low vitamin B12 levels concerning for pernicious anemia. Parietal cell and intrinsic factor antibodies were negative, and he then underwent an esophagogastroduodenoscopy with biopsy. The biopsy was negative for Helicobacter pylori, and the immunohistochemical stains were suggestive of chronic atrophic gastritis. He was started on vitamin B12 1,000 mcg intramuscular injections daily. His hemoglobin, LDH, and haptoglobin normalized. Given the absence of the parietal cell antibody and intrinsic factor antibody, this is a rare case of seronegative pernicious anemia.

Behavioral, Neurochemical and Brain Oscillation Abnormalities in an Experimental Model of Acute Liver Failure.

Hepatic encephalopathy (HE) represents a brain dysfunction caused by both acute and chronic liver failures, and its severity deeply affects the prognosis of patients with impaired liver function. In its pathophysiology, ammonia levels and glutamatergic system hyperactivity seem to play a pivotal role in the disruption of brain homeostasis. Here, we investigate important outcomes involved in behavioral performance, electroencephalographic patterns, and neurochemical parameters to better characterize the well-accepted animal model of acute liver failure (ALF) induced by subtotal hepatectomy (92% removal of tissue) that produces ALF. This study was divided into three cohorts: (1) rats clinically monitored after hepatectomy every 6 h for 96 h or until death; (2) rats tested in an open-field task (OFT) before and after surgery and had blood, cerebrospinal fluid, and brain tissue collected after the last OFT; and (3) rats that had continuous EEGs recorded before and after surgery for 3 days. The hepatectomized rats presented significant motor behavioral changes accompanied by important abnormalities in classical blood laboratory parameters of ALF, and EEG features suggestive of HE and deep disturbances in the brain glutamatergic system. Using an animal model of ALF induced via subtotal hepatectomy, this work provides a comprehensive and reliable experimental model that increases the opportunity for studying the effects of new treatment strategies to be explored in an unprecedented way. It also presents insights into the pathophysiology of HE in a reproducible model of ALF, which correlates important neurochemical and EEG aspects of the syndrome.

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication.

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.

Expression of c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes in normal and malignant human breast epithelial cells.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Activation of the cystic fibrosis transmembrane regulator by cyclic AMP is not correlated with inhibition of endocytosis.

Based on the observation (Bradbury et al. (1992) Am. J. Physiol. 262, C752-C759) that conditions known to activate the cystic fibrosis transmembrane regulator protein (CFTR) increase the rate of exocytosis and decrease the rate of endocytosis, it was proposed that activation of the CFTR may involved cAMP-dependent fusion of CFTR containing endosomes with the apical membrane. We have tested this hypothesis in two cell lines derived from epithelia that express defective chloride transport in cystic fibrosis (CF): the human colonic cell line, T84, and the tracheal cell line 9HTEo-. The dose-dependence of forskolin- and CPT-cAMP-induced inhibition of endocytosis were compared with the dose-dependence of chloride channel activation. Endocytosis was determined from the uptake of FITC-dextran, and assayed in purified endosomes. Chloride channel activity was measured from the rate of I-efflux. If the fusion hypothesis is correct: (1) concentrations of agonist that inhibit endocytosis should activate chloride channel activity, and (2) the relationship between endocytosis and channel activation should be similar for forskolin and CPT-cAMP. Results in both cell lines were inconsistent with these postulates, suggesting that either chloride channel activation and the inhibition of endocytosis are separate effects of cAMP, or that the increase in apical CFTR resulting from agonist-dependent inhibition of endosomal fusion is minimal.

Changes in the baroreflex control of heart rate produced by central infusion of selective angiotensin antagonists in hypertensive rats.

We have recently shown that an angiotensin-(1-7) [Ang-(I-7)] analogue, D-Ala7-Ang-(1-7) (A-779), is a selective Ang-(1-7) antagonist with no significant action on angiotensin type 1 or type 2 receptors. The availability of selective angiotensin antagonists prompted us to evaluate the role of Ang-(1-7) and Ang II on central modulation of the baroreflex control of heart rate in normotensive Wistar rats and spontaneously hypertensive rats (SHR). Blood pressure recording and reflex changes in heart rate elicited by intravenous bolus injections of phenylephrine were made before and within 1 and 3 hours of intracerebroventricular (ICV, lateral ventricle) infusion of saline (8 microL/h), A-779 (4 microg/h), DuP 753 (100 microg/h), or CGP 42112A (50 mu g/h) in conscious rats. The slope of the relationship between changes in pulse interval versus changes in mean arterial pressure was used as an index of the baroreflex control of heart rate. ICV infusion of saline or any of the antagonists did not significantly change basal levels of mean arterial pressure and heart rate in SHR (170 +/- 6 mm Hg nd 360 +/- 9 beats per minute, respectively; n = 29) or Wistar rats (108 +/- 2 mm Hg and 377 +/- 6 beats per minute, respectively; n=29). Three hours of ICV infusion of A-779 markedly decreased baroreflex sensitivity in Wistar rats (from a basal slope of 1.09 +/- O.3). In contrast, A-779 did not significantly alter the depressed baroreflex sensitivity of SHR (0.61 +/- O.l). ICV infusion of DuP 753 produced a significant increase (60 percent) in baroreflex control of heart rate in both Wistar rats and SHR. Saline or CGP 42112A infusions did not significantly alter baroreflex control of heart rate. These results suggest that endogenous Ang II and Ang-(1-7) are differentially affecting central baroreflex modulation, acting probably through distinct receptor subtypes. Although the central Ang II inhibitory effect is mediated by the type 1 receptor subtype, the facilitatory effect of Ang-(1-7) might be mediated by a different, unidentified receptor.

Interaction of bradykinin and angiotensin-(1-7) in the central modulation of the baroreflex control of the heart rate.

OBJECTIVE: Previous studies have shown that angiotensin-(1-7) potentiates the vascular actions of bradykinin. In the present study, we evaluated the interaction of bradykinin and angiotensin-(1-7) in the central modulation of baroreflex control of the heart rate. MATERIALS AND METHODS: Blood pressure and reflex bradycardia, elicited by intravenous injection of phenylephrine, were evaluated in conscious male Wistar rats before and at the end of 1 h of an intracerebroventricular infusion of angiotensin-(1-7) at 0.5 or 1.0 microg/h combined with bradykinin at 2.5 microg/h; or angiotensin-(1-7) at 2.0 microg/h combined with bradykinin at 4.0 microg/h; or angiotensin-(1-7) alone at 2.0 or 4.0 microg/h; or bradykinin alone at 4.0 or 8.0 microg/h; or saline at 8 microl/h. In addition, baroreflex bradycardia was evaluated before and at the end of 1 and 2 h of intracerebroventricular infusion of angiotensin-(1-7) at 4 microg/h for 2 h; or saline at 8 microl/h in the first hour followed by HOE 140 at 90 ng/h in the second hour; or angiotensin-(1-7) at 4 microg/h in the first hour followed by angiotensin-(1-7) at 4 microg combined with HOE 140 at 90 ng/h in the second hour; or HOE 140 at 90 ng/h in the first hour followed by HOE 140 at 90th ng/h combined with angiotensin-(1-7) at 4 microg/h in the second hour; or saline at 8 microl/h for 2 h. RESULTS: The intracerebroventricular infusion of angiotensin-(1-7) or bradykinin alone required a dose of 4.0 and 8.0 microg/h, respectively, to facilitate baroreflex control of the heart. However, a simultaneous infusion of these peptides at subeffective rates was able to produce a significant increase in baroreflex sensitivity. In addition, the facilitation of the baroreflex control of the heart rate induced by angiotensin-(1-7) at 4.0 microg/h was inhibited by HOE 140. CONCLUSIONS: These results suggest that centrally, bradykinin and angiotensin-(1-7) can interact in order to modulate baroreflex control of the heart rate. In addition, our data indicate that the central modulatory effect of angiotensin-(1-7) on the baroreflex is mediated, at least in part, by the release of kinins.

[The nursing school of the Federal University of Minas Gerais: from its creation until today]. / A escola de enfermagem da UFMG: da criação aos dias atuais.

The purpose of this work was to bring back some historical aspects of the Federal University of Minas Gerais Nursing School--EEUFMG, since its creation in 1933 until 1998.

[Funeral rites in memory of the elderly]. / Ritos de morte na lembrança de velhos.

This a phenomenologial study rites in the memory of elderly people, originated from the discomfort lived by the authors in their professional life dealing with death and dying. Verbal information from elderly people was collected with the objective of recovery and decoding mortuary rites. Nine themes originated from these informations: feelings and meanings in relation to death, the time of death, the annunciation death, the body's preparations, the watcher, the funeral procession, the grave, the return to home, the remembered death. The results gave the authors opportunity to understand better the attitudes of health professionals in caring for patients and their families in this existential experience of to-be-for-death. The death rationalized by scientific knowledge and nonpersonal technological care hides new rites, transmuted by new representations which the society built.

The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 µM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.

Histopathology and the lethal effect of Cry proteins and strains of Bacillus thuringiensis Berliner in Spodoptera frugiperda J.E. Smith Caterpillars (Lepidoptera, Noctuidae).

Among the phytophagous insects which attack crops, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) is particularly harmful in the initial growth phase of rice plants. As a potential means of controlling this pest, and considering that the entomopathogen Bacillus thuringiensis Berliner demonstrates toxicity due to synthesis of the Cry protein, the present study was undertaken to evaluate this toxic effect of B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 on S. frugiperda. The following method was used. Both bacterial strains were evaluated in vitro in 1st instar S. frugiperda caterpillars, by means of histopathological assays. The Cry1Ab and Cry1Ac proteins, codified by the respective strains of B. thuringiensis, were evaluated in vivo by bioassays of 1st instar S. frugiperda caterpillars in order to determine the Mean Lethal Concentration (LC50). The results of the histopathological analysis of the midget of S. frugiperda caterpillars demonstrate that treatment with the B. thuringiensis thuringiensis strain was more efficient, because the degradations of the microvilosities started 9 hours after treatment application (HAT), while in the B. thuringiensis kurstaki the same effect was noticed only after 12 HAT. Toxicity data of the Cry1Ab and Cry1Ac proteins presented for the target-species LC50 levels of 9.29 and 1.79 microgxcm-2 respectively. The strains and proteins synthesised by B. thuringiensis thuringiensis and B. thuringiensis kurstaki are effective in controlling S. frugiperda, and may be used to produce new biopesticides or the genes may be utilised in the genetic transformation of Oryza sativa L.

Association between interleukin-1 beta polymorphism (+3953) and obesity.

It now appears that obesity is associated with a low-grade inflammation of white adipose tissue resulting from chronic activation of the innate immune system as interleukin-1 beta (IL-1). Previous investigations have described a positive association between IL-1 beta +3953 (C>T) gene polymorphism (rs 1143634) and obesity, suggesting functional effects on fat mass, fat metabolism and body mass. However, it is necessary to determine if these results occur in other populations and if they are influenced by sex and age. Therefore, we performed a case-control study using 880 Caucasian subjects (59.7+/-11.9 years old) from the Brazilian Aging Research Program (non-overweight=283, overweight=334, obese=263) previously investigated in genetic studies, in whom we analyzed the IL-1 beta +3953C/T polymorphism. We observed higher T allele (CT/TT) frequency in non-overweight than overweight and obese groups. The odds ratio showed 1.340 (95% CI: 1.119-1.605) times more chance of the obese group being CC carriers compared to non-overweight group independent of sex and age. This study corroborates the idea that the IL-1 system is linked to the development of obesity.

Histopathology and the lethal effect of Cry proteins and strains of Bacillus thuringiensis Berliner in Spodoptera frugiperda J.E. Smith Caterpillars (Lepidoptera, Noctuidae) / Histopatologia e efeito letal de cepas e proteínas Cry de Bacillus thuringiensis Berliner para lagartas de Spodoptera frugiperda J.E. Smith (Lepidoptera, Noctuidae)

Among the phytophagous insects which attack crops, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) is particularly harmful in the initial growth phase of rice plants. As a potential means of controlling this pest, and considering that the entomopathogen Bacillus thuringiensis Berliner demonstrates toxicity due to synthesis of the Cry protein, the present study was undertaken to evaluate this toxic effect of B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 on S. frugiperda. The following method was used. Both bacterial strains were evaluated in vitro in 1st instar S. frugiperda caterpillars, by means of histopathological assays. The Cry1Ab and Cry1Ac proteins, codified by the respective strains of B. thuringiensis, were evaluated in vivo by bioassays of 1st instar S. frugiperda caterpillars in order to determine the Mean Lethal Concentration (LC50). The results of the histopathological analysis of the midget of S. frugiperda caterpillars demonstrate that treatment with the B. thuringiensis thuringiensis strain was more efficient, because the degradations of the microvilosities started 9 hours after treatment application (HAT), while in the B. thuringiensis kurstaki the same effect was noticed only after 12 HAT. Toxicity data of the Cry1Ab and Cry1Ac proteins presented for the target-species LC50 levels of 9.29 and 1.79 μg.cm-2 respectively. The strains and proteins synthesised by B. thuringiensis thuringiensis and B. thuringiensis kurstaki are effective in controlling S. frugiperda, and may be used to produce new biopesticides or the genes may be utilised in the genetic transformation of Oryza sativa L.

Entre os insetos fitófagos que atacam as culturas, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) destaca-se como uma praga polífaga que causa prejuízos na fase inicial da cultura do arroz. No seu controle, o entomopatógeno Bacillus thuringiensis Berliner revela-se tóxico devido à síntese de proteínas Cry. Nesse contexto, o objetivo deste trabalho foi avaliar a toxicidade das cepas e proteínas Cry de B. thuringiensis thuringiensis 407 (pH 408) e B. thuringiensis kurstaki HD-73 sobre S. frugiperda. As duas cepas bacterianas foram avaliadas, in vitro, em lagartas de 1º instar de S. frugiperda, através de ensaios de histopatologia. As proteínas Cry1Ab e Cry1Ac, codificadas pelas respectivas cepas de B. thuringiensis, foram avaliadas in vivo, através de bioensaios com lagartas de 1º instar de S. frugiperda para determinação da Concentração Letal Média (CL50). Os resultados da análise histopatológica do intestino médio das lagartas S. frugiperda mostram que o tratamento com a cepa B. thuringiensis thuringiensis foi mais eficiente e a degradação das microvilosidade iniciou-se 9 horas após a aplicação dos tratamentos (HAT). Para B. thuringiensis kurstaki, o mesmo efeito foi observado, 12 HAT. Os dados de toxicidade das proteínas de Cry1Ab e Cry1Ac revelaram para a espécie-alvo uma CL50 de 9,29 e 1,79 μg.cm-2, respectivamente. As cepas e proteínas sintetizadas por B. thuringiensis thuringiensis e B. thuringiensis kurstaki são eficientes no controle de S. frugiperda, e poderão ser usadas na produção de novos biopesticidas ou a utilização dos genes na transformação genética de Oryza sativa L.

Effects of cis- and trans-tamoxifen isomers on RNA incorporation of human breast cancer cells.

Cultured T47-D human breast cancer cells were used to investigate growth-inhibiting effects of the antiestrogen, trans-tamoxifen, on [3H]Cyd incorporation into specific classes of nuclear and cytoplasmic RNA. The steroid agonist, 17 beta-estradiol, and the inactive cis isomer of tamoxifen were used as treatment controls to compare antiestrogen-induced changes in RNA metabolism, independent of estrogen receptor-binding properties. Using a 24-hr labeling interval, trans-tamoxifen produced a 1.4- to 4-fold enhanced incorporation into pre-rRNA species (20S, 32-45S), with slight reduction in mature 18S rRNA incorporation, and a 2- to 3-fold increased incorporation into 5S and 5.8S rRNA and 4-4.5S tRNA. Most notable, trans-tamoxifen enhanced incorporation into the less abundant low molecular weight U1, U3, and 7S RNA species by 3- to 6-fold. These findings were associated with an apparent reduction in pre-rRNA content and little change in U1, U3, or 7S RNA levels in antiestrogen-treated cells, suggesting that trans-tamoxifen independently regulates RNA transcription and turnover. The present study provides new rationale for the choice of molecular probes to study trans-tamoxifen effects on synthesis and turnover of specific nuclear and cytoplasmic RNA species.

Estrogen-induced post-transcriptional modulation of c-myc proto-oncogene expression in human breast cancer cells.

The effects of estradiol (E2) on c-myc proto-oncogene expression were studied in the estrogen receptor (ER)-positive human breast cancer cell line, MCF-7. A biphasic modulation in c-myc mRNA levels occurs during the first 24 h of E2 (1 nM) exposure and in the absence of changes in MCF-7 culture growth, with transcript levels rising 4-6-fold within 1 h, returning to near base-line level after 3-6 h, and increasing again after 24 h of exposure. In contrast, the growth-inhibiting antiestrogen, tamoxifen (1 microM), reduces c-myc to 20% of the pretreatment level within 3-6 h of exposure, and this early decline is followed by a gradual return toward base-line level after continuous 72-h treatment. In ER-negative cells there is no change in c-myc expression following E2 exposure. MCF-7 nuclear runon assays show that c-myc transcription rates remain unchanged from base line for 24 h after E2 administration; as well, cycloheximide inhibition of protein synthesis superinduces c-myc expression and prevents E2 modulation of transcript levels. These results indicate that post-transcriptional modulation of c-myc by E2 is mediated by a labile degradative protein or otherwise dependent on active protein synthesis. We also developed MCF/nm and MCF/dm sublines by transfecting normal cells with human c-myc exons 2-3, transcriptionally driven by a retroviral long terminal repeat. Expression of the transfected c-myc genes in these sublines remains constant and elevated 10-fold, while transcript levels from the endogenous proto-oncogene continue to be modulated by E2. These findings suggest that in ER-positive breast cancer cells, E2 can modulate c-myc mRNA levels by a post-transcriptional mechanism that depends on gene sequences upstream from c-myc exon 2.

Dystrophin-related protein in the platelet membrane skeleton. Integrin-induced change in detergent-insolubility and cleavage by calpain in aggregating platelets.

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 x g) but the membrane skeleton requires 100,000 x g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin alpha IIb beta 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following alpha IIb beta 3-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate an approximately 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Importance of complement source in measuring meningococcal bactericidal titers.

Complement-mediated bactericidal antibodies in serum confer protection against meningococcal disease. The minimum protective titer is estimated to be between 1:4 and 1:8 when measured by the Goldschneider assay performed with human complement, the assay used in the 1960s to establish the correlation between bactericidal antibodies and protection. A more recently described bactericidal assay standardized by an international consortium uses rabbit complement, which is known to augment bactericidal titers. To define a protective titer measured by the standardized assay, we compared bactericidal titers against serogroup C strains measured by this assay to titers measured by the assay described by Goldschneider et al. A titer of > or =1:128 measured by the standardized assay was needed to predict with > or =80% certainty a positive titer of > or =1:4 as measured by the Goldschneider assay. However, the majority of samples with titers of 1:4 measured by the Goldschneider assay had titers of <1:128 when measured by the standardized assay. Therefore, by the results of the standardized assay such persons would be falsely categorized as being susceptible to disease. In conclusion, high bactericidal titers measured with the standardized assay performed with rabbit complement are predictive of protection, but no threshold titer is both sensitive and specific for predicting a positive titer measured by the Goldschneider assay using human complement. Up to 10% of the U.S. adult population lacks intrinsic bactericidal activity against serogroup C strains in serum and can serve as complement donors. Therefore, use of the Goldschneider assay or an equivalent assay performed with human complement is preferred over assays that use rabbit complement.