Stimulant effects of muscarinic agonists in embryonic chick ventricular muscle. Are the effects mediated by protein kinase C?

Muscarinic agonists at high concentrations produce stimulant effects in the heart. We used the embryonic chick ventricle, in which muscarinic receptor activation markedly increases phosphoinositide (PI) breakdown, to investigate the possibility that receptor-induced PI metabolism mediates the stimulant effects. In preparations paced at 0.5 to 1 Hz, carbachol (CCh; 10(-6) to 10(-3) M) shifted the action potential plateau to more positive potentials and markedly prolonged the action potential duration (APD). These effects were not associated with a change in the resting potential, did not fade with time and could be obtained in the presence of propranolol (3 x 10(-7) M) or tetrodotoxin (10(-6) M). The same effects could be obtained with acetylcholine or oxotremorine (10(-4) M) and could be suppressed with atropine (0.9 x 10(-6) M). The sensitivity of the APD to muscarinic agonists was high during early embryonic stages and declined rapidly with age. At days 3 to 5, the APD was prolonged in the presence of CCh by greater than 80% of its control value. After day 12, CCh had no prolonging effect on the APD. In nonpaced preparations, the agonist depolarized the membrane and/or induced automaticity in quiescent preparations. When automaticity was present, spontaneously or after addition of Ba++, the frequency of the automatic activity was increased by the agonist. These effects on resting potential and automaticity were also less marked in older embryos. CCh (10(-6) to 10(-4) M) evoked an atropine-sensitive positive inotropic effect in paced ventricular tissues in the presence of propranolol.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of repetitive administration of recombinant human interleukin-1 beta, an analog or corticotropin-releasing hormone combined with lysine vasopressin on rats with glucocorticoid-induced secondary adrenocortical insufficiency.

We investigated effects of corticotropin-releasing hormone (CRH), lysine vasopressin and interleukin (IL)-1 beta[1-148], a less pyrogenic analog of human IL-1 beta, on the hypothalamo-pituitary-adrenal axis in a rat model of secondary adrenocortical insufficiency. After 2 weeks of corticosterone 21-sodium succinate treatment, hypothalamic CRH, anterior pituitary adrenocorticotropic hormone (ACTH) and the adrenal weight of the rats decreased significantly and their plasma ACTH showed a significantly smaller response to ether stress, as did plasma corticosterone level. A mixed solution of CRH (10 micrograms) and lysine vasopressin (2 micrograms) or recombinant human IL-1 beta[1-148] (1 micrograms), administered to these rats for 7 days, apparently accelerated the recovery of the pituitary and adrenocortical responsiveness to ether stress and significantly increased the recovery rate of anterior pituitary ACTH contents and adrenal weight. The IL-1 beta analog also increased hypothalamic CRH. These data indicated that, in a rat model with glucocorticoid-induced adrenocortical insufficiency, synthesis and release of hypothalamic CRH, pituitary ACTH and adrenal glucocorticoid were all considerably affected. CRH combined with lysine vasopressin or a less pyrogenic IL-1 beta analog, when administered to these rats, accelerated the recovery of the pituitary and the adrenocortical functions significantly, suggesting the potential clinical usefulness of these peptides.


Reversal of desipramine toxicity in rats using drug-specific antibody Fab' fragment: effects on hypotension and interaction with sodium bicarbonate.

The effect of drug-specific antibody Fab' fragment on desipramine (DMI) toxicity was studied in anesthetized rats to determine 1) whether DMI-induced hypotension can be reversed, and 2) whether the effect of this Fab' fragment can be enhanced by the concurrent administration of hypertonic NaHCO3. DMI (60 mg/kg) was administered i.p. to produce marked hypotension. Antitricyclic antidepressant (TCA) Fab' (molar Fab'/DMI ratio = 0.11) or control Fab' was administered 15 min later as a 10 min i.v. infusion. The mean arterial pressure was higher at the end of anti-TCA Fab' infusion than after control Fab' (58 +/- 8 vs. 17 +/- 7 mm Hg, P less than .001). In a second protocol, DMI (30 mg/kg) was administered to prolong QRS duration. Anti-TCA Fab' alone (molar Fab'/DMI ratio = 0.09) and NaHCO3 alone both reduced QRS prolongation compared to control treatment, and combined therapy was more effective than either one alone. In both protocols, anti-TCA Fab' markedly increased the total DMI concentration and the bound fraction of DMI in serum, but did not alter the unbound DMI concentration. In the low DMI dose protocol, anti-TCA Fab' also reduced the cardiac DMI concentration. Concurrent treatment with anti-TCA Fab' and NaHCO3 substantially increased urinary DMI and anti-TCA Fab' excretion compared to treatment with anti-TCA Fab' alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Dopaminergic neurotoxicity of 1-methyl-4-phenylpyridinium analogs in cultured neurons: relationship to the dopamine uptake system and inhibition of mitochondrial respiration.

Several analogs of 1-methyl-4-phenylpyridinium (MPP+) were evaluated for their affinity for the dopamine uptake system and their ability to inhibit NADH dehydrogenase (complex I) of the mitochondrial electron-transport chain. Moreover, these compounds were tested for their ability to cause selective dopaminergic neurotoxicity in cultured mesencephalic neurons. Simultaneous [3H]dopamine and gamma-amino-[14C]butyric acid uptake and immunocytochemical techniques were used as indices of neuronal damage in cultured cells. The compounds that were potent and selective dopaminergic neurotoxins had high affinity for the dopamine transport system, as measured by their ability to cause dopamine release, and were similar to MPP+ in inhibiting mitochondrial respiration. One compound (1-methyl-4-phenylpyrimidinium) had high affinity for the dopamine uptake system but was a weak inhibitor of mitochondrial respiration and, accordingly, was not neurotoxic. The 4'-alkylated analogs of MPP+, which were poor substrates for the dopamine uptake system and extremely potent inhibitors of mitochondrial respiration, caused a nonselective damage of neurons in culture. Analogs that were not substrates for the dopamine carrier and not inhibitors of mitochondrial respiration were not neurotoxic. This study describes the neurotoxicity of a number of analogs of MPP+ and highlights the importance of the dopamine uptake system and the ability to inhibit mitochondrial respiration as critical processes in conferring selectivity and neurotoxicity, respectively, to MPP+ and analogs, for dopaminergic neurons in culture.


Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.


Evaluation of LY203647 on cardiovascular leukotriene D4 receptors and myocardial reperfusion injury.

In vitro studies have shown LY203647 to be a selective antagonist of contractile responses to leukotriene (LT) D4 and LTE4 in guinea pig ileum, trachea and lung parenchyma. In pithed rat, i.v. injection of LTD4 produced pressor responses that were selectively antagonized by LY203647 in a dose-dependent manner [ED50 7.5 (6.0-9.5) mg/kg, i.v.]. In normal anesthetized rats and dogs, LTD4 reduced aortic blood flow and stroke volume in association with systemic vasoconstriction, variable blood pressure responses and no change in cardiac rate. LTD4 did not alter myocardial contractility corrected for alterations in afterload. Pretreatment of rats and dogs with LY203647 (1-10 mg/kg, i.v.) produced dose-related inhibition of the myocardial and systemic hemodynamic effects of LTD4, whereas coadministration of LY203647 reversed established myocardial depression and systemic and pulmonary vasoconstriction during continuous infusion of LTD4 in dogs. LY203647 (10 mg/kg over 90 min, i.v.) infusion in normal dogs abolished or greatly antagonized hemodynamic responses to LTD4 for 6 hr. In subsequent experiments, myocardial infarct size was measured after 1 hr of occlusion of the circumflex coronary artery and 5 hr of reperfusion. LY203647 (10 mg/kg over 90 min, i.v.) treatment did not alter cardiovascular parameters when compared to time-related alterations observed in control dogs. ST segment deviation and the intensity and duration of cardiac arrhythmias associated with coronary artery occlusion and reperfusion also were similar between groups. Resultant infarct sizes were 46 +/- 1 and 45 +/- 1% of the left ventricular mass placed at risk in control and LY203647-treated dogs, respectively. Present data illustrate the prominent cardiac and systemic hemodynamic effects of LTD4 and indicate that LY203647 produces selective and sustained antagonism of cardiovascular LTD4 receptors. Lack of containment of infarction by LY203647 suggests that endogenous cysteinyl-LT do not contribute to reperfusion injury of ischemic myocardium.


High-affinity dextromethorphan and (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites in rat brain. Allosteric effects of ropizine.

Dextromethorphan (DM) binds to high- and low-affinity sites in the rat brain. The high-affinity DM binding is inhibited by nonnarcotic antitussives, opipramol and sigma ligands with nanomolar affinities. Computer-assisted modeling of homologous and heterologous competition studies between DM and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] were performed at pH 8.4. These experiments confirmed the existence of the common high-affinity DM1/sigma 1 site (R1) for which DM and (+)-3-PPP have Kd values of 20 and 10 nM, respectively. DM also binds to a second high-affinity site (R2, Kd, 20 nM) for which (+)-3-PPP has only micromolar affinity. Similarly, (+)-3-PPP binds to another high-affinity site (R3, Kd, 60 nM) for which DM has micromolar affinity. The common high-affinity DM1/sigma 1 site is allosterically modulated by the anticonvulsant ropizine, and is (+)-pentazocine sensitive, as is the homologous site in the guinea pig. However, in the rat the common DM1/sigma 1 site is 10 times smaller than in the guinea pig. This explains the apparently different effects of the allosteric modifiers in both species. The multiplicity of binding sites for DM and (+)-3-PPP resolved in this investigation will help to establish the physiological role and the pharmacological potential of the different sites. Meanwhile, the pharmacological effects of DM and sigma ligands cannot be summarily attributed to any particular binding site or receptor. This investigation also demonstrates that the use of multiple labeled and unlabeled ligands, combined with computer-assisted modeling, is essential to resolve multiple binding sites with similar affinities and to characterize the complex effects of allosteric modifiers.


Heterogeneity for allelic loss in human breast cancer.

Loss of heterozygosity (LOH) at specific chromosomal regions in the tumor cells implicates the presence of tumor suppressor genes. However, it is also possible for an LOH to be randomly acquired and irrelevant to tumor development. To determine whether a particular LOH in human breast carcinomas represents a loss of tumor suppressor gene or merely a random loss, we analyzed untreated primary breast cancers for LOH. Ninety-eight primary human breast cancers from previously untreated patients were analyzed for LOH at 12 chromosomal regions including five randomly selected regions and seven regions previously reported in other cancer types and/or breast cancers. The baseline incidence of LOH was five out of 124 tests (4%) using randomly selected probes on chromosomes 1p, 2p, 4p, 11q, and Xq. Incidences of LOH significantly greater than background were seen in the following chromosomal regions: 22q (10 of 26 cases, 38%); 18p (five of 24 cases, 21%); 17p (30 of 59 cases, 51%); 13q (five of 14 cases, 36%); 3p (13 of 28 cases, 46%); and 1q (18 of 70 cases, 26%). In contrast to previous reports, the incidence of LOH was not significantly different from background for 11p15. In all cases, results were the same for metastatic lymph nodes and primary tumors, suggesting that the losses occurred early in the malignant progression. In cases with LOH at more than one locus, the same DNA sample often varied in degree of signal reduction for the missing alleles. These observations indicate the presence of both intertumor and intratumor heterogeneity for LOH.


Levamisole potentiation of fluorouracil antiproliferative activity mimicked by orthovanadate, an inhibitor of tyrosine phosphatase.

Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU. Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro. Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates. We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity. Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro. Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.


Stimulation of growth by both androgen and estrogen of the EMP-K1 transplantable tumor with androgen and estrogen receptors from human extramammary Paget's disease in nude mice.

Experimental approaches using transplantable human tumors in nude mice have greatly improved studies on their pathogenesis and treatment. We studied the establishment of a transplantable tumor, EMP-K1, derived from extramammary Paget's disease and the morphology and unique hormone dependence of this tumor. Tissue fragments from a metastatic skin tumor of an 80-year-old man with primary genital extramammary Paget's disease were implanted into male nude BALB/c mice. Tissue fragments of the established tumor were implanted into 50 castrated male 8-week-old nude mice, which were given injections of 100 micrograms testosterone propionate (TP), 100 micrograms 5 alpha-dihydrotestosterone (DHT), 10 micrograms diethylstilbestrol (DES), or 1, 10, or 100 micrograms 17 beta-estradiol (E2). All injections were administered intramuscularly once daily, starting from the day of transplantation. The established tumors were examined immunohistochemically and biochemically. A transplantable tumor (EMP-K1) was established in the nude mouse. EMP-K1 tumor cells expressed antigens such as carcinoembryonic antigen and epithelial membrane antigen, cytokeratin, and c-erbB-2 protein and contained androgen, estrogen, and progesterone receptors. The growth of the EMP-K1 tumor was stimulated by TP, DHT, DES, and E2. These results suggest that both androgen and estrogen may stimulate the growth of the same tumor by both androgen receptor and estrogen receptor pathways. The EMP-K1 tumor is a useful tool for studies on the biology of extramammary Paget's disease, and further studies using these tumors will provide useful information concerning proper hormone therapy.


[Splenic lymphoma of Ki-1 positive B-cell type treated with CHOP].

A 75-year old man was admitted to our hospital by complaining of his left supraclavicular lymphadenopathy. Physical examination also revealed splenomegaly and cervical lymphadenopathy. An abdominal CT scan showed paraaortic lymphadenopathy and splenomegaly with multiple low density area. A pathological diagnosis of Ki-1 lymphoma was made by the biopsied supraclavicular lymph node because the node consists of large cells with clear cytoplasms and conspicuous nucleoli and large cells were positive for IgM, lambda, CD20 and CD30 (Ki-1). The primary lesion of lymphoma was suspected to be spleen because of the radiological finding of the spleen. The patient responded to systemic chemotherapy (CHOP) with complete remission. Pathological and clinical characteristics of this B-cell Ki-1 lymphoma were discussed.


[Cytomegaloviral interstitial pneumonia after autologous bone marrow transplantation in a case of acute lymphoblastic leukemia. Bone Marrow Transplantation Team].

A 22-year-old man with T cell type acute lymphoblastic leukemia in first remission underwent autologous bone marrow transplantation (BMT). The preparative regimen included cytosine arabinoside, cyclophosphamide and fractionated total body irradiation. His harvested bone marrow cells were purged with 4-hydroperoxycyclophosphamide. His serological test was positive for cytomegalovirus (CMV) before the BMT. On day 53 after the BMT, he developed dry cough and his chest X-ray film showed bilateral basilar infiltration. Bronchoalveolar lavage was performed and cytology of the specimen revealed typical cytomegaloviral inclusion bodies. DNA analysis and viral culture of the specimen were also positive for CMV. The patient was started on ganciclovir and immunoglobulin with high dose methylprednisolone. His respiratory status deteriorated, however, and the patient expired because of respiratory failure. Autopsy revealed severe interstitial pneumonitis with suppression of CMV replication from the treatment. Interstitial pneumonitis due to CMV should be considered as a significant complication of autologous BMT.


[Suppression of in vitro hemopoiesis by the monocytes in a patient with aplastic anemia associated with hepatitis].

The cause of aplastic anemia associated with hepatitis (AAH) is as yet still unknown. There is a supposed relation to the immune mechanisms, however few reports have shown the effects of monocytes on the pathogenesis in the patients with AAH. We have reported a case of a 12-year-old boy with AAH related to cytomegaloviruses, and studied the hemopoietic progenitors. He showed pancytopenia and hypoplasia of the bone marrow on admission to our hospital. The culture studies showed that mononuclear cells (MNC) of the bone marrow produced few hemopoietic colonies in all cell lineages. However, the depletion of adherent cells from the MNC increased numbers of erythroid, neutrophil-macrophage and megakaryocyte colonies. Furthermore, the addition of adherent cells of the peripheral blood suppressed the colony formation in the aforementioned cell lineages by marrow MNC from which adherent cells, phagocytic cells and T-cells were abrogated. The results way suggest that monocytes play some soles in the pathogenesis of aplasia through inhibitor of hemopoiesis.


[Detection of cytomegalovirus in urine specimens from renal transplant recipients using polymerase chain reaction].

Human cytomegalovirus (CMV) in this study, a polymerase chain reaction (PCR) assay was developed to detect human cytomegalovirus (CMV) from urine specimens taken from renal transplant patients and one congenital CMV infection. Twenty eight urine specimens of 20 renal transplant patients were analyzed by the PCR. CMV infection was detected in 11 specimens (8 recipients). The incidence (40.0%) of PCR positive in the renal transplant patients was highest among the other conventional methods including fluorescent antibody study, anti-IgM EIA and complement fixation test. In two cases, CMV infection could be identified by PCR before antibody development. The PCR procedure is more sensitive and rapid than conventional methods for the diagnosis of CMV. Rapid diagnosis of CMV infection using PCR may be useful in clinical diagnosis and have therapeutic value.


[Evaluation of proposed criteria of disk susceptibility testing for tosufloxacin and lomefloxacin in NCCLS guidelines and WHO standards].

Disk diffusion zone diameter breakpoint criteria for Tosufloxacin and Lomefloxacin were tentatively established by correlating MICs with 1-, 5- and 10 micrograms of Tosufloxacin disk inhibitory zone diameters and with 10 micrograms of Lomefloxacin disk of those to 418 clinical isolates representing 32 species. We recommend 5 micrograms disks for Tosufloxacin with the following breakpoints: Susceptible (MIC, less than or equal to 0.5 microgram/ml), greater than or equal to 22 mm; intermediate, 17 to 21 mm; and resistant (MIC, greater than or equal to 2.0 micrograms/ml), less than or equal to 16 mm. We recommend 10 micrograms disks for Lomefloxacin with the following breakpoints: Susceptible (MIC, less than or equal to 2.0 micrograms/ml), greater than or equal to 21 mm; intermediate, 16 to 20 mm; and resistant (MIC, greater than or equal to 8.0 micrograms/ml), less than or equal to 15 mm. Using these criteria for Tosufloxacin and Lomefloxacin, the occurrence rate of major errors in judging susceptibility and resistance was 0.48%.


Resection of brain metastases from non-small-cell lung carcinoma. Results of therapy. Memorial Sloan-Kettering Cancer Center Thoracic Surgical Staff.

The treatment of patients with a solitary brain metastasis has been evolving, with most centers recommending resection in patients with good performance status. To evaluate the results of resection of brain metastases from non-small-cell lung cancer, we reviewed our 16-year experience with 185 consecutive patients undergoing resection of brain metastases from 1974 to 1989, inclusive. There were 89 men and 96 women; ages ranged from 34 to 75 years (median 54). Sixty-five (35%) had synchronous and 120 (65%) metachronous brain metastases. Discounting the brain metastasis, 68 patients (37%) had stage I, 13 (7%) stage II, 62 (33%) stage IIIA, 30 (16%) stage IIIB, and 12 (6%) stage IV carcinoma. There was no significant difference in age, locoregional stage (TN), or histologic features in patients with synchronous versus metachronous lesions. The overall survival rates (n = 185) were as follows: 1 year, 55%; 2 years, 27%; 3 years, 18%; 5 years, 13%; and 10 years, 7% (median 14 months). There was no significant difference in survival between patients with synchronous and metachronous lesions. To evaluate the impact of locoregional stage and treatment of the primary site, we analyzed only those patients with synchronous brain metastases. Multivariate analysis demonstrated that locoregional stage had no significant effect on survival (p = 0.97), but complete resection of the primary disease significantly prolonged survival (p = 0.002). Therefore complete resection, and not stage, of the locoregional primary lesion is the primary determinant of survival in patients undergoing resection of brain metastases from non-small-cell lung cancer.


High production of catalase in hydrogen peroxide-resistant human leukemia HL-60 cell lines.

The catalase activities of HP50-2 and HP100-1 cells, which are H2O2-resistant cell lines derived from human leukemia HL-60 cells, were 3 and 18 times higher, respectively, than that of HL-60 cells. These catalase activities of the resistant cells were precipitated with anti-catalase serum. The glutathione peroxidase activity of HP50-2 cells was about twice that of HL-60 or HP100-1 cells. The superoxide dismutase activities of HP50-2 and HP100-1 cells were, respectively, about 4 and 2 times that of HL-60 cells. In addition, both the resistant cell lines were completely devoid of myeloperoxidase activity. Pulse-labeling experiments showed that the syntheses of catalase in HP50-2 and HP100-1 cells were, respectively, 2 and 4 times that in HL-60 cells, and that, unlike the parent cells, neither line synthesized myeloperoxidase. Thus the alteration of catalase, glutathione peroxidase, and superoxide dismutase activity could be linked to the resistance of H2O2 of human leukemia cells.


Mayo Clinic experience with allogeneic and syngeneic bone marrow transplantation, 1982 through 1990.

Between April 1982 and July 1990, 101 patients underwent allogeneic or syngeneic bone marrow transplantation at the Mayo Clinic. This patient population consisted of 30 with acute nonlymphocytic leukemia, 25 with acute lymphoblastic leukemia, 29 with chronic granulocytic leukemia, and 17 with other diseases (aplastic anemia in 7, myelodysplastic syndrome in 5, and lymphoma in 5). The results achieved in our patients who underwent transplantation in first complete remission of both acute nonlymphocytic leukemia and acute lymphoblastic leukemia compare favorably with previously reported results. Only 1 of 15 patients (7%) with acute nonlymphocytic leukemia and 2 of 8 patients (25%) with acute lymphoblastic leukemia who underwent transplantation in first complete remission had a relapse. Thus, we recommend early bone marrow transplantation during initial complete remission for patients with either of these disorders who have adverse prognostic factors. In contrast, of 12 patients with either acute nonlymphocytic leukemia or acute lymphoblastic leukemia who underwent transplantation during relapse, 11 died within 6 months. Therefore, such patients should be offered new experimental treatments. Our patients with chronic granulocytic leukemia fared better when they underwent transplantation early during the course of their disease rather than during the accelerated or blast phase. Prospective studies are needed to determine the best approach in these patients.


Experimental applications of porous (coralline) hydroxylapatite in middle ear and mastoid reconstruction.

Hydroxylapatite is a ceramic implant material which has been extensively investigated in the orthopedic and oral surgical literature. A set of animal experiments was carried out to determine the feasibility of the use of porous (coralline) hydroxylapatite for middle ear and mastoid reconstruction. Coralline hydroxylapatite was placed in the mastoid bulla of dogs and was carved into struts which were placed into the middle ear and against the tympanic membrane. Coralline hydroxylapatite is a highly biocompatible material which showed abundant ingrowth when in contact with host bone. There was also good soft-tissue ingrowth and fibrous tissue fixation within the middle ear and against the tympanic membrane. Hydroxylapatite is generally accepted to be osteoconductive, but not osteogenic. However, one of the specimens described with chronic inflammatory mucosal changes, shows de novo new bone formation within the pores of the graft. While normally the bone formed in coralline hydroxylapatite is secondary (lamellar), in this specimen primary (woven) bone was found. Porous (coralline) hydroxylapatite appears to be an excellent otologic graft material. It biointegrates with the host, is osteoconductive, and under certain circumstances may be osteogenic.


Comparison of pulsed and continuous wave light in photodynamic therapy of papillomas: an experimental study.

Papillomavirus infection of the upper aerodigestive tract is a serious and potentially life-threatening disease which can result in complete respiratory obstruction. Papillomas are the most common benign neoplasm of the larynx in children but have been shown to have an equal propensity to occur in adults. The hallmark of this disease is one of multiple recurrences despite adequate surgical removal, often resulting in a frustrating and prolonged clinical course. Multiple treatment modalities have been used throughout the years, unfortunately with limited success. Recent reports on the treatment of virally induced lesions with photodynamic therapy (PDT) using hematoporphyrin derivative (HPD) or its newly purified form dihematoporphyrin ether (DHE) as photosensitizing agents have appeared in the literature. The successful treatment of papillomas in both the animal model and humans holds great promise. The most commonly used activating light source has been the argon pumped-dye laser (ADL) which produces a continuous wave of coherent light at 630 nm. Although the ADL has proved efficacious in most studies, its cost, size, special cooling water requirements, and large electrical power requirements with limited power output have made it a less than desirable clinical tool. The gold vapor laser (GVL) has been recently proposed as a possible alternate light source for PDT. This laser appears to be more efficient and offers greater power output while requiring less electrical energy than the ADL. The GVL does not produce the same continuous wave light as the ADL but emits pulsed light with high peak power pulses. Some reports have shown a greater tumor response using the GVL at 628 nm, possibly because of greater tissue penetration from the high-peak power pulses. At this time, no studies have investigated the effects of pulsed light during PDT on virally induced papillomas. The present study was conducted to evaluate the efficacy of pulsed light from a GVL as compared to continuous wave light of the ADL in the treatment of papillomas with DHE phototherapy. Statistical analysis of the rate of tumor response, histological changes, and molecular analysis of viral DNA from the involved tissues were performed. Results have shown that the GVL did produce a greater initial rate of tumor response during the first 3 weeks after PDT but did not improve the overall cure rate. Histological and molecular analysis of the treated tissues demonstrated that similar results were obtained when either the ADL or GVL were used as activating light sources.


The role of mu1 receptor in physical dependence on morphine using the mu receptor deficient CXBK mouse.

It is known that the CXBK inbred strain of mouse is deficient in mu1 opioid receptors, whereas the strain has a delta opioid receptor population that is less consistently altered. In the present study, we compared physical dependence on morphine between CXBK and C57BL/6 mice. Both strains of mice were treated with morphine-admixed food for 5 days. During the treatment, the two strains of mice showed no signs of toxicity. There was no significant difference in morphine intake during the treatment between CXBK and C57BL/6 mice. After the treatment, the withdrawal was precipitated by injecting naloxone (0.01-30 mg/kg, s.c.). CXBK mice showed weight loss, diarrhea and ptosis, but not jumping and body shakes after low dose of naloxone. Whereas, C57BL/6 mice showed weight loss, diarrhea, ptosis, body shakes and jumping. These results suggest that naloxone-precipitated weight loss, diarrhea and ptosis may be mediated by mu2 and/or delta opioid receptor, while naloxone-precipitated jumping and body shakes may be mediated by mu1 opioid receptors.


Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.


The effect of immune mediators (cytokines) on the release of endothelium-derived relaxing factor (EDRF) and of prostacyclin by freshly harvested endothelial cells.

The effect of recombinant tumor necrosis factor and other cytokines stimulated by LPS (lipopolysaccharide), on the release of endothelial-derived relaxing factor and of prostacyclin was investigated using freshly harvested endothelial cells attached to plastic microcarrier beads. The results show that the cytokines failed to interfere with the release of EDRF and prostacyclin under the conditions of these experiments.


An ESR-CT imaging of the head of a living rat receiving an administration of a nitroxide radical.

Three-dimensional ESR imaging of a living rat has been performed by an L-band ESR system, which is composed of an L-band ESR spectrometer, a field gradient coil, and a data processor. The imaging was carried out by Lauterbur's method. A nitroxide, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL), was used as an imaging agent in saline solution at a concentration of 0.2 M and administered intraperitoneally to obtain a constant concentration in the head for about an hour. It took about 40 min to obtain one set of ESR-CT images. The cross-sectional images were made, both as coronal and horizontal images. In the images of the rat head the nitroxide-rich region was clearly distinguished from the deficient region. The nitroxide-deficient areas corresponded well to the brain of the rat.


Differential regulation of striatal preproenkephalin mRNA by D1 and D2 dopamine receptors.

The effect of administration of subtype selective dopamine (DA) agonists on the 6-hydroxydopamine (6-OHDA) lesion-induced increase of striatal preproenkephalin (PPE) mRNA was examined by dot-blot hybridization. Eight days following a unilateral 6-OHDA lesion of the substantia nigra pars compacta (SNc), PPE mRNA levels in the ipsilateral striatum were increased approximately two-fold. Administration of the D2 DA agonist, quinpirole, dose-dependently attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA. The effect of quinpirole was blocked by coadministration of the D2 DA antagonist eticlopride. In contrast, administration of the D1 DA agonist, SKF 38393, either dose-dependently augmented or had no effect on the 6-OHDA lesion-induced increase in striatal PPE mRNA. In the contralateral striatum, administration of quinpirole decreased PPE mRNA, while administration of SKF 38393 increased PPE mRNA compared to sham lesioned control levels. These data suggest the action of DA at D1 and D2 DA receptors differentially regulates striatal PPE mRNA levels and the apparent inhibition of ENK biosynthesis by DA is mediated via an interaction with D2 DA receptors.


Differential regulation of muscarinic and nicotinic cholinergic receptors and their mRNAs in cultured sympathetic neurons.

Mechanisms regulating expression of neuronal muscarinic and nicotinic receptors were examined in cultures of neonatal rat sympathetic neurons. Two factors known to stimulate cholinergic transmitter development in sympathetic neurons were examined for their effects on cholinergic receptor expression. A membrane associated factor (MANS46) and a diffusible factor produced by cultured rat fibroblasts (RFCM) each decreased muscarinic receptor number. By contrast, neither treatment altered levels of nicotinic receptors. Levels of muscarinic (m2) receptor mRNA were decreased by MANS but not by RFCM, indicating that effects of the two treatments were mediated by different mechanisms. Neither MANS nor RFCM altered levels of nicotinic alpha 3 or beta 2 mRNAs, consistent with the lack of change in numbers of nicotinic receptors. These observations indicate that receptor phenotype in developing neurons is subject to regulation by multiple epigenetic factors. Further, the same signals which regulate transmitter development may also regulate receptor expression in sympathetic neurons.


Ontogeny of D1 and DARPP-32 gene expression in the rat striatum: an in situ hybridization study.

D1 dopamine receptor (D1R) and DARPP-32 (a dopamine and adenosine 3',5'-monophosphate regulated phosphoprotein), gene expression was studied in the rat striatum in adults and during ontogeny by in situ hybridization. D1R mRNA was first detected in the striatal primordium at day 17 of gestation. At day 18, D1R mRNA was found throughout the striatum. Before birth, the striatal neurons had neuroblastic aspect and were close together, giving homogeneous and compact labelling. After birth, the topography and aspect of the neurons containing D1R mRNA and DARPP-32 mRNA were similar. The two mRNAs were detectable in the caudate-putamen, accumbens nucleus and olfactory tubercle. The microautoradiographic analysis demonstrated that D1R and DARPP-32 genes are massively expressed by the medium-sized striatal neurons. The proportion of medium-sized neurons containing the DARPP-32 mRNA was however higher than that of the neurons containing the D1R mRNA. Furthermore, an unexpected proportion of large-sized neurons express these genes. This proportion varies with development. Comparison between the appearance, topography and frequency of choline-acetyltransferase immunoreactive neurons and large-sized neurons containing D1R or DARPP-32 mRNA suggest that these large-sized neurons containing D1R and DARPP-32 mRNAs are cholinergic ones.


Induction of transcription factors in somatosensory cortex after tactile stimulation.

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 min period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-fos. The different transcription factors showed slightly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus.


Glucocorticoid-mediated down regulation of c-fos mRNA in C6 glioma cells: lack of correlation with proenkephalin mRNA.

We investigate the linkage between the transcriptional factor, c-fos, and expression of proenkephalin in rat C6 glioma cells. C6 cells contained abundant levels of c-fos mRNA. Treatment of cells with dexamethasone resulted in a 10-fold decline in c-fos transcripts and a small increase in proenkephalin mRNA. Combined exposure to dexamethasone and isoproterenol also induced a decrease in c-fos mRNA while proenkephalin mRNA increased 8-fold. Treatment of the C6 cells with phorbol 12-myristate 13-acetate caused a 13-fold increase in c-fos expression 0.5 h after administration and a decrease in proenkephalin mRNA. These data indicate that c-fos and proenkephalin mRNA are not regulated in a sequential, parallel manner, that newly synthesized c-fos is not the determining factor controlling proenkephalin gene regulation, and that c-fos expression is under negative control by glucocorticoids.


Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells.

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.


Characterization of the major 68 kDa heat shock protein in a rat transformed astroglial cell line.

The heat shock response in a transformed astrocyte line was compared with nontransformed astrocytes. The synthesis of HSP 68, the major inducible heat shock protein (HSP 68) was induced by a non-lethal 45 degrees C, 10 min heat shock. Although the incorporation of [35S]methionine into HSP 68 suggested that similar amounts of protein were being synthesized after heat shock, Western immunoblotting demonstrated striking differences in the HSP immunostaining between the two cell types. By one- and 'two-dimensional gel electrophoresis the major 68 kDa heat shock protein (HSP 68) was similar in both cell types. However, HSP 68 from heat shocked, transformed astrocytes did not immunostain with the monoclonal antibody, C-92, which is specific for the major inducible heat shock protein of HeLa cells. In contrast HSP 68 from heat shocked, nontransformed astrocytes immunostained quite well. A polyclonal antibody raised against the inducible 72 kDa heat shock protein of HeLa cells immunostained the HSP 68 from both astrocytes and transformed astrocytes. Analysis of the mRNA from the two cell types after heat shock revealed two bands of approximately 2.5 and 2.8 kb in astrocytes but only a single 2.5 kb band in the heat shocked transformed astroglia. These results suggest that structural differences in the HSP 68 may be present in the transformed astrocytes compared to the normal astrocytes.


Preprotachykinin gene expression in the human basal ganglia: characterization of mRNAs and pre-mRNAs produced by alternate RNA splicing.

The nature and distribution of preprotachykinin (PPT, i.e. substance P/neurokinin A-encoding) gene expression in human basal ganglia was determined. Northern blot analysis visualized a single band of approximately 1300 bases, confirming the postmortem stability of PPT mRNA. Gross anatomical analysis indicated that PPT gene expression was relatively evenly distributed throughout the human caudate and putamen, but absent in the globus pallidus and substantia nigra. Nuclease protection analysis of these tissues established that human PPT mRNA consisted of approximately 80-85% beta-PPT (exon 1-7 derived) mRNA and 15-20% gamma-PPT (minus exon 4), with no alpha-PPT (minus exon 6) mRNA detected; these data contrast with the proportions of PPT mRNAs seen in non-human species. The incompletely spliced PPT RNA species detected in basal ganglia accounted for approximately 8% of total human PPT RNA and suggested a fixed order of exon splicing. Since various PPT mRNAs encode different combinations of tachykinin peptides with distinct biological activities, the markedly different proportions of PPT mRNAs seen in human basal ganglia compared to non-human tissues may be of physiological significance.


Long-term expression of the c-fos protein during the in vitro differentiation of cerebellar granule cells induced by potassium or NMDA.

Levels of the c-fos protein were assayed in mouse cerebellar granule cells during their in vitro development under different culture conditions. When grown in media favoring both their survival and differentiation, i.e. in the presence of 30 mM K+ or 12.5 mM K+ plus 100 microM N-methyl-D-aspartate (NMDA), the c-fos protein becomes detectable in the nucleus of granule cells on and after 6 days and persists to high levels until the culture begins to decline. The protein c-fos appears therefore after the critical period described for the survival effect of K+ depolarization or NMDA receptor stimulation which corresponds to days 2-5 after plating. The c-fos protein remains however scarcely detectable or undetectable throughout the life-span of cells cultured under conditions providing poor survival and differentiation, i.e. in the presence of low K+ (5 or 12.5 mM) alone or when the effect of NMDA is blocked by the NMDA receptor antagonist MK-801. Interestingly, in cortical and striatal neurons, the survival and differentiation of which being not affected by depolarizing media, no c-fos protein is detected whatever the culture conditions tested at least during the first 18 days in vitro. This suggests that long-term expression of the c-fos gene might be related to some aspect of the late in vitro differentiation process of cerebellar granule cells.


Differential dopaminergic regulation of proenkephalin and prodynorphin mRNAs in the basal ganglia of rats.

Proenkephalin and prodynorphin mRNA levels in the caudate-putamen and in the nucleus accumbens of rats were measured by in situ hybridization 2, 4 and 8 weeks following unilateral lesion of the medial forebrain bundle by 6-hydroxydopamine (6-OHDA). After 2 weeks a 60% increase of the levels of proenkephalin mRNA in the ipsilateral caudate-putamen was observed which declined to 20% above control after 8 weeks. A smaller increase in the levels of proenkephalin mRNA of about 20% was observed in the nucleus accumbens after 2 weeks and no significant alteration could be observed 4 and 8 weeks after lesioning. The levels of prodynorphin mRNA in the ipsilateral caudate-putamen decreased 20% below control and returned to control levels 4 and 8 weeks post-lesion. In contrast, in the nucleus accumbens a persistent ipsilateral decrease of prodynorphin (20-30%) was found 2, 4 and 8 weeks post-lesion. These findings indicate, that lesions of the mesostriatal dopamine (DA) system differentially influences opioidergic gene expression in distinct areas of the caudate-putamen. The lesions cause an increase in proenkephalin mRNA levels which was higher in the caudate-putamen than in the nucleus accumbens and tend to be reversible. Conversely, the lesion caused a persistent decrease in the levels of prodynorphin mRNA in the nucleus accumbens and a small and transient decrease in the caudate-putamen.


Quantitative in situ hybridization evidence for differential regulation of proenkephalin and dopamine D2 receptor mRNA levels in the rat striatum: effects of unilateral intrastriatal injections of 6-hydroxydopamine.

Nigrostriatal (NS) dopaminergic (DA) neurons are thought to exert an inhibitory influence on striatal enkephalinergic systems through their DA D2 receptors. In order to investigate the effects of partial lesions of the NS DA on striatal proenkephalin (PEK) and D2 receptor mRNAs, animals were allocated to High, Intermediate, and Low rotators on the basis of amphetamine-induced rotation observed after intrastriatal injections of 6-hydroxydopamine (6-OHDA). On the ipsilateral side of the lesions, there were significant increases in PEK mRNA in the total aspect of the caudate-putamen (CPu) of the High (+204%), the Intermediate (+125%), and of the Low (+67%) rotation groups in comparison to controls; these changes correlated positively with increases in rotation rate. Unexpectedly, there were also significant increases in striatal PEK mRNA on the contralateral side although these changes were much less prominent than those observed on the side of the lesions. Conversely, only the High rotation group showed significant increases (+112%) in D2 receptor mRNA which occurred only on the lesioned side. Interestingly, the low rotation group actually showed some non-significant decreases (-25%) on the side of the lesions. These results indicate that partial lesions of the NS DA projections are sufficient to cause substantial increases in PEK mRNA but not in D2 receptor mRNA. These data also provide evidence that the two nigrostriatal DA projections and the systems which they modulate might be under interdependent sets of controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation of a calreticulin-like calcium binding protein from bovine brain.

Intracellular calcium levels are stringently regulated in all cells. The nature of this regulation is incompletely understood, but recent evidence indicates that the endoplasmic reticulum plays an important role in sequestering intracellular calcium. Using methods for isolating both calsequestrin and calreticulin, we have isolated a 58 kDa, high capacity calcium binding protein that exists in microsomes that shift their density in an oxalate-mediated density shift assay. This protein which we call CBP-58 bears similarities to the endoplasmic reticulum protein, calreticulin, in that it has a pI of 4.7 containing approximately 30% glutamate and aspartate, has a high capacity for calcium, and stains blue with the carbocyanine dye, 'Stains-all'. Peptide, amino acid, nucleotide and immunochemical analyses reveal further similarities between CBP-58 and calreticulin, but also some marked differences. Its tissue distribution suggests it is highly enriched in brain versus other tissues. We believe that CBP-58 is a calreticulin-like protein and that differences in the amino acid composition and sequences may reflect species diversity in calreticulin.


Dopaminergic regulation of cholecystokinin mRNA content in rat striatum.

The nigrostriatal dopaminergic activity was pharmacologically changed to assess whether dopamine (DA) regulates cholecystokinin (CCK) mRNA steady state in rat striatum. Cocaine and benztropine, two dopaminergic agonists known to induce DA release and to block its re-uptake, produced a time dependent increase in CCK mRNA content in rat striatum. A significant increase in striatal CCK mRNA was observed 8 h after a single injection of cocaine (15 mg/kg, i.p.) or benztropine (15 mg/kg, i.p.) whereas a two-fold increase was observed after a daily treatment for one week with these two dopaminergic agonists. Cocaine and benztropine failed to change CCK mRNA content in the cerebral cortex. Haloperidol, a dopaminergic receptor blocker, injected at 1 mg/kg, i.p., daily for 7 days, decreased CCK mRNA content in striatum but not in the cerebral cortex. Moreover, haloperidol blocked the effect of cocaine and benztropine, suggesting that the stimulation of striatal dopaminergic receptors is necessary for the induction of CCK biosynthesis. The neurotoxin 6-hydroxydopamine injected into the medial forebrain bundle, elicited a 50% decrease in striatal CCK mRNA, supporting the hypothesis that DA tonically regulates CCK biosynthesis in postsynaptic neurons. To characterize the dopaminergic receptor subtype involved in this regulation, BHT 920, a specific D2 receptor agonist and SKF 38393, a specific D1 receptor agonist were used. While one week treatment with BHT 920 (1 mg/kg, i.p.) increases striatal CCK mRNA content, SKF 38393 (3 mg/kg, i.p.) failed to change this parameter. These data suggest that the increase of striatal CCK mRNA is mediated by the activation of the D2 receptor subtype.


In situ hybridization of calcium/calmodulin dependent protein kinase II and tau mRNAs; species differences and relative preservation in Alzheimer's disease.

Abnormal phosphorylation of the microtubule associated protein tau component of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) may result from alterations in protein kinase expression. Calcium/calmodulin dependent protein kinase II (CaM kinase II) has been shown to phosphorylate tau in vitro in such a way to decrease its electrophoretic mobility. A68, apparently a modified form of tau in AD brain, also shows abnormal phosphorylation and slower mobility than tau. To further examine the role of CaM kinase II in AD, in situ hybridization studies were performed on tissues from rat, monkey and human to examine and compare the patterns of CaM kinase II mRNA expression in different brain regions. The most notable differences among the three species were observed in dendrites in layer I of isocortex, in the molecular layer of the dentate gyrus and stratum radiatum and stratum lacunosum-moleculare in hippocampus, where hybridization was detected in rat, but not in monkey or human brain. In addition, comparisons between tau and CaM kinase II mRNA expression were made in tissue from normal aged adults and AD patients, especially in areas prone to NFT formation. CaM kinase II and tau mRNAs were co-expressed in many neuronal populations, both those which are prone to NFT formation as well as those which are rarely affected by AD changes. No major differences in the relative abundance of either CaM kinase II or tau mRNA within particular neuronal populations was noted between normal aged and AD brain. Diminished hybridization was associated with serve neuronal pathology and cell loss.


Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector.

Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system. We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3. This vector, denoted Cgal delta 3, contains the E. coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome. We studied the efficiency of Cgal delta 3 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer. Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS. Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia. Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expression of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression. Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.


Anti-Hu--associated paraneoplastic encephalomyelitis/sensory neuronopathy. A clinical study of 71 patients.

We studied 71 patients with "paraneoplastic" encephalomyelitis, sensory neuronopathy, or both associated with the presence of the anti-Hu antibody in their serum. Most (78%) had small-cell lung cancer. In 9 patients no tumor was detected. Fifty-two patients (73%) had signs and symptoms of multifocal involvement of the nervous system; in 28 (39%), 2 areas, and in 24 (34%), 3 or more areas were clinically affected. Sensory neuronopathy was present in 52 patients (74%), but in only 44 (62%) did it dominate the course of the disease. Other predominant findings were: motor neuron dysfunction (14 patients, 20%), limbic encephalopathy (14, 20%), cerebellar symptoms (11, 15%), brainstem encephalopathy (10, 14%), and autonomic nervous system dysfunction (7, 10%). The presence of the anti-Hu antibody prompted a search for the tumor in 60% of the patients; the tumor when found was usually small and remained localized until death, or was demonstrated only at autopsy. Treatment using steroids and plasmapheresis, immunosuppressants, or both, did not improve the paraneoplastic symptoms. Autonomic and respiratory failure, either of central origin or secondary to neuromuscular weakness, were the principal causes of death. Patients with rapidly developing sensory neuropathy or symptoms of encephalomyelitis should be studied for the presence of the anti-Hu antibody; if the antibody is found, the possibility of small-cell lung cancer should be investigated. If a tumor is not found in the initial search, one may become evident in several months.


Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis.

The arginine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCo1, with the highest affinity for protein, is located within the 5' promoter sequences, whilst the other, argCo2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCo1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCo2 in vivo forming a repression loop similar to those observed for the E. coli Lac repressor.


Cervical cancer--what role for human papillomavirus?

To review the role of human papillomavirus (HPV) as a causative agent of cancer of the cervix. DATA SOURCES, data synthesis, study selection: Medical journals, recently published text books related to cancer of the cervix and HPV and Papillomavirus Reports were examined to review the pathology of cervical cancer and its precursor lesions, its epidemiology in Australia and overseas, methods of detection of HPV (in particular molecular biology techniques used to diagnose HPV) and evidence linking HPV with genital neoplasia. While there is compelling evidence strongly linking certain HPV types with genital cancer, a causative role is yet to be proven and the aetiology is most likely multifactorial. Detection and typing of high risk genotypes of HPV in the genital tract as a diagnostic exercise to identify those women most at risk of developing genital neoplasia is not currently recommended.


Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum.

Endothelin receptors (ETRs) display subtype heterogeneity and are widely distributed throughout the tissues of the periphery and central nervous system. In order to gain further insight into the potential molecular differences of ETRs, we initiated molecular cloning of ETR genes by screening for the appearance of 125I-ET-1 binding activity in COS cells transfected with pools of a porcine cerebellum cDNA expression library. Two independent clones (pPCETR 1.1 and pPCETR 5.6) were identified and isolated by repeated rounds of pool enrichment and COS cell expression. DNA sequence analysis of pPCET 1.1 and pPCET 5.6 indicated that both clones have the same nucleotide sequence; the deduced amino acid sequence indicated that the porcine cerebellum ETR is 443 residues in length and consists of seven potential transmembrane domains, with homology to members of the GTP-binding protein-coupled receptor superfamily. Northern analysis indicated a single mRNA species of about 5 kilobases, which is expressed significantly in cerebellum, lung, kidney, and pituitary. Expression of functional receptor was demonstrated by endothelin-1 (ET-1)-mediated Ca2+ mobilization in COS cells transfected with pPCETR 1.1 (COS/ETR 1.1) and ET-1-mediated electrophysiological responses in Xenopus oocytes injected with RNA derived from pPCETR 1.1. Quantitative comparison of saturation binding of 125I-ET-1 to either porcine cerebellum or COS/ETR 1.1 membranes indicated an identical apparent dissociation constant. The relative efficacy of ET-related peptides to compete for binding of 125I-ET-1 to receptor from porcine cerebellum and COS/ETR 1.1 indicated that both preparations encode a nonselective or ETBR subtype. Chemical cross-linking of 125I-ET-1 to receptor derived from cerebellum or COS/ETR 1 revealed two bands, with apparent molecular masses of 47 and 35 kDa. These data demonstrate that the pPCETR 1.1 encodes the major ETR subtype in the porcine cerebellum.


Maitotoxin effects are blocked by SK&F 96365, an inhibitor of receptor-mediated calcium entry.

The dinoflagellate toxin maitotoxin (MTX) elicited a sustained increase of [Ca2+]i in C6 glioma cells. This response was inhibited by SK&F 96365, a blocker of receptor-mediated calcium entry. In C6 cells, endothelin-1 elicited a rapid but transient increase in [Ca2+]i, followed by a smaller sustained increase. SK&F 96365 inhibited the sustained increase in [Ca2+]i. In both C6 glioma cells and RIN insulinoma cells, MTX elicited a marked influx of 45Ca2+. SK&F 96365 inhibited MTX-induced 45Ca2+ influx by 95% at 30 microM. The L-type calcium channel blocker nifedipine, even at 10 microM, inhibited MTX-induced calcium uptake by only 20% in RIN cells and by only 10% in C6 cells. MTX elicited calcium-dependent phosphoinositide breakdown in both C6 and RIN cells. In both cell lines, the MTX-induced phosphoinositide breakdown was inhibited by 90% by SK&F 96365 at 30 microM. Endothelin-1 and carbamylcholine elicited phosphoinositide breakdown in C6 cells and RIN cells, respectively. The stimulations were unaffected by the presence of SK&F 96365 up to 100 microM. In RIN insulinoma cells, MTX elicited calcium-dependent release of insulin. SK&F 96365 at 30 microM inhibited MTX-induced insulin release by 75%, whereas nifedipine, even at 30 microM, inhibited release by only 10%. The blockade of MTX-induced responses by SK&F 96365 indicates that MTX increases intracellular calcium by interacting directly with a calcium-entry system that is similar, in its sensitivity to SK&F 96365, to the calcium-entry system activated by receptors that elicit phosphoinositide breakdown. Activation of phospholipase C and hormone release by MTX also are blocked by SK&F 96365 and, thus, may be secondary to the activation of such a calcium-entry system.


Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand.

The mutual effects that a hormonal ligand (H) and a guanine nucleotide regulatory protein (G protein) exert on each other when simultaneously occupying distinct sites of the receptor molecule (R) can be viewed as the molecular mechanism of drug efficacy. These effects are predictable on the basis of a model assuming that the ternary complex between the three partners (HRG) reaches equilibrium in the membrane [J. Biol. Chem. 255:7108-7117 (1980)]. Ligands can be classified as agonists, neutral antagonists, or negative antagonists, depending on whether they enhance, leave unchanged, or reduce, respectively, the spontaneous tendency of R to interact with G. Using this model and the assumption that the G protein response observed in membranes reflects the sum of ligand-independent (RG) and ligand-dependent (HRG) receptor-G protein complexes, we can explain virtually all the phenomenology reported earlier for opioid receptor-mediated stimulation of GTPase, i.e., 1) existence of ligands with both "positive" and "negative" intrinsic activity (the latter termed negative antagonists), 2) equipotency of neutral antagonists for the competitive blockade of the responses elicited both by agonists and by negative antagonists, and 3) apparent heterogeneity of binding sites for the binding isotherms of negative antagonists. The ternary complex model can also explain the differential effects of sodium on ligand binding and ligand-dependent GTPase activity, if we assume that this ion reduces the stability constant between receptor and G protein in membranes. Computer simulations predict that a negative antagonist exhibits a discrepancy between "biological" Ki (obtained by Schild plots) and true dissociation constant for the receptor, which increases as the fraction of "precoupled" receptors in the membrane increases. The demonstration of negative antagonism is definitive evidence for the existence of receptor coupling (hence activity) in the absence of ligand. Using this experimental paradigm, we show here that spontaneous receptor activity occurs in isolated membranes but not in intact NG108-15 cells.


Competitive regulation of phospholipase C responses by cAMP and calcium.

DDT1-MF2 smooth muscle cells demonstrated a robust phospholipase C response to norepinephrine, as detected by inositol phosphate accumulation. A selective A1-adenosine receptor agonist, cyclopentyladenosine, caused only a minor stimulation of phospholipase C, which was eliminated in the absence of added extracellular calcium. The simultaneous addition of norepinephrine and cyclopentyladenosine resulted in a synergistic increase in phosphoinositide hydrolysis either in the absence or in the presence of external calcium. In the presence of external calcium and a calcium ionophore, and adenosine agonist caused a significant stimulation of phosphoinositide hydrolysis without the addition of norepinephrine. Influx of extracellular calcium through voltage-sensitive calcium channels did not appear to be required to observe an effect of cyclopentyladenosine, because neither calcium channel antagonists (nifedipine, verapamil, and LaCl3) nor a chelator of extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were able to alter the degree of potentiation of norepinephrine-stimulated phosphoinositide hydrolysis due to the adenosine agonist. On the other hand, buffering of intracellular calcium concentration with the membrane-permeant calcium chelator quin2 blocked the potentiation. This blockade of potentiation by quin2 was reversed by the addition of extracellular calcium. Agents that stimulated cAMP production or membrane-permeable analogues of cAMP also blocked the action of the adenosine agonist to potentiate norepinephrine-stimulated phosphoinositide hydrolysis. This effect of cAMP was less pronounced in the presence of elevated extracellular calcium and was abolished in the presence of a calcium ionophore. When norepinephrine-stimulated calcium transients were quantitated using fura-2 fluorescence, a reduction in the amplitude of the calcium response was observed in the presence of forskolin. Conversely, both the amplitude and the duration of the calcium response were enhanced by the addition of the adenosine agonist. The results of these studies suggest that the mechanism by which adenosine receptors enhance the stimulation of phosphoinositide hydrolysis is dependent upon a rise in intracellular Ca2+ concentration resulting from the simultaneous activation of alpha 1-adrenergic receptors. The results further suggest that cAMP inhibits this mechanism by decreasing the norepinephrine-stimulated rise in intracellular Ca2+ concentration.


Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.

The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin.


HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors.

Proteins containing the basic-helix-loop-helix (B-HLH) domain have been shown to be important in regulating cellular differentiation. We have isolated a cDNA for a human B-HLH factor, denoted HEB, that shares nearly complete identity in the B-HLH domain with the immunoglobulin enhancer binding proteins encoded by the E2A and ITF2 genes (E proteins). Functional characterization of the protein expressed from this cDNA indicates that HEB is a third member of the E-protein class of B-HLH factors. HEB mRNA was found to be expressed in several tissues and cell types, including skeletal muscle, thymus, and a B-cell line. HEB, ITF2, and the E12 product of the E2A gene all bound to a similar spectrum of E-box sequences as homo-oligomers. All three factors also formed hetero-oligomers with myogenin, and the DNA-binding specificity and binding off-rates (dissociation rates) were modulated after hetero-oligomerization. Both homo- and hetero-oligomers of these proteins were able to distinguish between very closely related E-box sequences. In addition, HEB was shown to form hetero-oligomers with the E12 and ITF2 proteins. Finally, HEB was able to activate gene expression. These data demonstrate that HEB shares characteristics with other E proteins and show that HEB can interact with members of both the myogenic regulatory class and the E-protein class of B-HLH factors. HEB is therefore likely to play an important role in regulating lineage-specific gene expression.


Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.

The effect of mammary gland-specific expression of the polyomavirus middle T antigen was examined by establishing lines of transgenic mice that carry the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen resulted in the widespread transformation of the mammary epithelium and the rapid production of multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it possess an enhanced metastatic potential.


Mapping neuroinvasiveness of the herpes simplex virus type 1 encephalitis-inducing strain 2762 by the use of monoclonal antibodies.

Monoclonal antibodies (MAbs) directed against herpes simplex virus (HSV)-coded glycoproteins gB, gC, gD and gE were employed in a an in vitro model of neuroinvasiveness using sensory neurons from rat dorsal root ganglion (DRG) cells. The neurons were cultured in at two-chamber system allowing infection via the neuritic extensions exclusively. The effects of 30 MAbs on viral replication of the encephalitis-derived HSV-1 strain 2762 and its less neuroinvasive variant 2762p11 were assayed in this model. One MAb reactive with gD gave a nine-fold reduction of the virus yields of both strains. One MAb directed against gB gave an enhanced virus yield of strain 2762, but not of the 2762p11 variant. Another gB-reactive MAb decreased the virus yield of strain 2762p11, but not of 2762 after neuritic infection. The findings indicate that an alteration of gB has occurred during the passage of the strain 2762. Mutants of the same strain were derived by infecting hybridomas producing MAb reactive with gB, gC, gD and gE, respectively. The gB hybridoma mutant showed a significantly lower neuroinvasiveness in the DRG model, and was non-virulent after snout infection of mice. We suggest that the structure of gB of the strain 2762 is of importance for the neuroinvasiveness of this strain.


Detection of human cytomegalovirus in peripheral mononuclear cells and urine samples using PCR.

Samples of peripheral blood lymphocytes from 105 different blood donors were investigated for the presence of human cytomegalovirus (HCMV) DNA using the polymerase chain reaction (PCR) with primers specific for the Pst I w fragment (IE region). Viral DNA sequences were detected in 53 samples, a fifth of which had been previously serotyped as HCMV negative. In the latter cases, Western blot analysis re-determined two out of three individuals that were resampled as seropositive. PCR could therefore be used to extend existing methods employed for the identification of HCMV infected blood samples prior to transfusion to individuals in high risk groups. In addition, the value of PCR as a diagnostic test was evaluated in a small pilot study by comparing the results obtained with urine samples from babies suffering congenital infection and from other high risk patients, with data obtained by isolation of infectious virus or through the detection of immediate early antigens in infected cultures. Data from this study indicated that PCR is at least as sensitive as the other methods used in HCMV diagnosis.


Expression of type IV collagenase correlates with the invasion of human lymphoblastoid cell lines and pathogenesis in SCID mice.

An in vitro model, called the Membrane Invasion Culture System (MICS), was used to study the invasive potential of an Epstein-Barr virus (EBV) positive lymphoblastoid cell line (LCL), an EBV-negative Burkitt lymphoma (BL) cell line of American origin and an EBV-positive BL of African origin. MICS measured the ability of these cell lines to invade reconstituted basement membrane-coated filters, which correlated with their tumorigenic and metastatic capabilities in a SCID mouse model. Furthermore, the significantly greater invasive behaviour of the EBV-positive LCL was directly correlated with the cells' ability to express and secrete human type IV collagenase (72 kDa), an important metalloproteinase responsible for the degradation of collagen IV in basement membranes. The data suggest that MICS and the SCID mouse are useful tests of tumorigenicity in lymphoid cells, with measurable effects in both systems related to human type IV collagenase activity. Both models allow further exploration of malignant phenotypes associated with EBV transformation of lymphoid tissues.


DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood.

Ependymomas and papillomas of the choroid plexus occur in early childhood. The ubiquitous human polyomaviruses, BK virus and JC virus, have been associated with the induction of these neoplasms in animal models. A related monkey polyomavirus, simian virus 40 (SV40), is highly tumorigenic in rodents and also induces choroid plexus papillomas. We tested the possibility that polyomaviruses were associated with these tumors in humans. Tumors from 31 children--20 with choroid plexus neoplasms and 11 with ependymomas--were evaluated for the presence of polyomavirus T-antigen gene sequences by means of amplification with the polymerase chain reaction. Ten of the 20 choroid plexus tumors and 10 of the 11 ependymomas contained amplification products that preferentially hybridized to probes specific for SV40 viral DNA rather than BK or JC viral DNA. In two specimens, DNA sequencing demonstrated that the amplified sequence was identical to the sequence of that region of the SV40 gene. In three other specimens, amplification with SV40-specific primers revealed a 574-bp segment of the SV40 viral gene. In 7 of 11 tumors examined by immunohistochemical staining, viral T antigen was expressed in the nuclei of the neoplastic cells. Half of the choroid plexus tumors and most of the ependymomas that we studied contained and expressed a segment of T-antigen gene related to SV40. These results suggest that SV40 or a closely related virus may have an etiologic role in the development of these neoplasms during childhood, as in animal models.


Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.


Mutation of protein kinase A causes heterochronic development of Dictyostelium.

In heterochronic mutants the relative timing of developmental events is altered compared with the wild type. This generally results in a disordered embryo, though heterochronic mutations may also be an important source of evolutionary variation. In the rapidly developing (rde) mutants of Dictyostelium, stalk and spore cells differentiate before morphogenesis is complete. We have traced the lesion in one class of these mutants to the regulatory subunit of cyclic AMP-dependent protein kinase (pk-A). Inactivation of this protein results in the unrestrained activity of the catalytic subunit, so prematurely triggering terminal cell differentiation.


[Support following pregnancy termination for genetic indications: experiences of involved women and their partners].

By means of semistructured interviews with 40 women and their partners data were collected concerning on the one hand the care and support and on the other the grieving after termination of pregnancy performed because the child appeared to be genetically affected. Crucial in the grieving process is having to choose personally for or against termination of the pregnancy. This grieving process can be complicated by factors such as uncertainty concerning either the evidence of the disease or its prognosis, high recurrence risk, negative self-esteem, feelings of failure and pre-existent relational or personal problems. Clear and complete counselling, combined with structural support during the hospital stay, proves to be the basis of adequate care. Follow-up interviews after the hospital stay play a supporting part, both for the hospital workers and for the couple involved.


The pathogenesis of cerebral gliomatous cysts.

In this study, the authors have examined the mechanism of the formation of tumor cysts. Cyst fluid samples were obtained during surgery and by percutaneous aspiration from 22 patients with cystic cerebral gliomas. The concentration of protein was measured in the cyst fluid and blood plasma. Analysis of brain tumor cyst fluids revealed that plasma proteins constituted a major fraction (92%) of cyst fluid proteins; moreover, the protein fractions occurred in concentrations (relative to the plasma concentrations) that were around 50-fold of those in cerebrospinal fluid. This strongly indicates blood-brain barrier disruption. Evidence from computed tomographic and magnetic resonance imaging scans as well as from electron microscopy of tumor cyst walls suggests the transition of spongy edematous tissue in or around tumors into the contents of associated cysts. Pathophysiologically, blood-brain barrier breakdown is inherent to the occurrence of vasogenic brain edema. It is therefore plausible that the development of cysts is related to peritumoral vasogenic edema.


Combined intra-arterial and systemic chemotherapy for recurrent malignant brain tumors.

Seventy-nine patients harboring recurrent brain tumors received four cycles of infraophthalmic carotid injections of 160 mg of carmustine. Two milligrams of intravenous vincristine and 50 mg of oral procarbazine was also administered three times daily for 1 week in conjunction with each BCNU treatment. The response rate was 60% with a median survival for patients with astrocytomas, anaplastic astrocytomas, and glioblastomas of 32, 20, and 6.5 months, respectively. The median survival of the responding patients was 20 months, and the survival at 30 months was 45%. The survival in patients not responding to treatment was 5 months, reflecting the natural history of the tumor. There have been no deaths related to the treatment procedure. No incidents of severe or permanent eye complications or leukoencephalopathy were observed. Based on multivariate survival analysis, only patients with a good performance status who are not steroid dependent are candidates for this treatment.


Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia.

Sympathetic ganglia are innervated by neuropeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, neurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.


Effect of ibuprofen on the in vitro and in vivo reactivation of latent HSV-1.

Prostaglandins have been suggested to play an important role in the reactivation of latent herpes simplex virus. To further understand the role of prostaglandins in the reactivation process, we investigated the effects of ibuprofen, a nonsteroidal anti-inflammatory drug with prostaglandin synthesis inhibitory activity, on the in vitro and in vivo reactivation of latent type 1 herpes simplex virus in mouse ganglia and rabbits, respectively. Ibuprofen, at a concentration of 50 or 100 microM, did not alter the titer of reactivated virus from explanted ganglia with latent virus, but, at a concentration of 200 or 500 microM, it significantly reduced the reactivated viral titer from the ganglia. Ibuprofen also directly inhibited the replication of herpes simplex virus in trigeminal ganglia and Vero cell monolayers, which indicates that the drug reduced the recovery of reactivated viral titers from explanted ganglia with latent virus by acting on the replication process rather than on the reactivation mechanism in vitro. The systemic administration of ibuprofen failed to demonstrate any significant effect on the ocular shedding of virus after attempted reactivation by 6-hydroxydopamine iontophoresis in rabbits with latent herpes simplex virus infection. This failure in vivo could be due to the short half-life and low concentration of ibuprofen at the site of reactivation and replication of latent virus. Alternatively, in the clinical setting, it is conceivable that ibuprofen may not have an effect on in vivo reactivation of latent herpes.


The 'other' sexually transmitted diseases. Chlamydial, herpes simplex virus, and human papillomavirus infections.

The incidence of sexually transmitted diseases continues to rise across the country. Because many cases are subclinical and asymptomatic, chlamydial, herpes simplex virus, and human papillomavirus (HPV) infections continue to spread at an alarming rate. These infections can lead to serious sequelae, such as pelvic inflammatory disease, infertility, and cervical dysplasia or carcinoma, so improved disease-control strategies are needed. Preventive efforts should include use of the latest diagnostic and therapeutic methods to uncover and eradicate subclinical chlamydial and HPV infections. In addition, notification of sexual partners should be encouraged. Education about prevention of sexually transmitted diseases should be incorporated into basic primary medical care for all sexually active patients.


[Lymphocytic infiltration in cerebral gliomas].

The authors analyse the incidence, extent and prognostic significance of lymphocytic infiltration in 80 cases of operated cerebral gliomas (61 high- and 19 low-grade gliomas). The results obtained are discussed in relation to the data previously reported by the literature.


Suitability of using sieved or unsieved maize mash for production of "OGI"--a fermented cereal food.

Proximate analysis of sieved and unsieved maize mash revealed that there was a decrease in the protein and lipid content of the sieved maize mash as compared to that of the unsieved maize mash. Crude fibre and ash was completely absent in the sieved maize mash, while they were present in the unsieved mash. Chemical analysis of the fermented unsieved maize mash revealed an increase in the protein content from 9.9% (unfermented) to 13.4% after 3 days of fermentation, whereas the protein content of the sieved maize mash increased from 7.1% (unfermented) to 8.4% after the same period of fermentation. Furthermore, the results revealed that the protein content of the fermented unsieved maize mash was 32.1% higher than that of the fermented sieved maize mash indicating that the unsieved maize mash was of a better nutrient quality and should be preferred to sieved maize mash for use in "Ogi" production.


Absorption of color additives and settling volume in water of blue-green alga, ishikurage (Nostoc commune).

The properties of ishikurage (Nostoc commune) and other algae were compared. Ishikurage and suizenji nori (Aphanothece sacrum) absorbed much more amaranth than others. They have a high settling volume (SV) in water. The high SV of ishikurage is attributable to its neutral detergent fiber (NDF). The acid detergent fiber (ADF) from the alga had a high amaranth absorption. However the absorption by the ADF did not explain the absorption by ishikurage. The results suggest that ishikurage may have a nutritional significance as a source of dietary fiber.


[Evaluation of the safety and efficacy of the lisinopril + hydrochlorothiazide and captopril + hydrochlorothiazide combinations in the treatment of essential arterial hypertension].

The aim of this study was to assess the efficacy and tolerability of the combinations of lisinopril (LIS) 20 mg + hydrochlorothiazide (HCTZ) 12.5 mg and captopril (CAP) 50 mg + HCTZ 25 mg in moderately hypertensive patients not adequately controlled by LIS or CAP alone. The study was multicentre (11 centres), open, random and carried out in parallel groups. After two weeks' placebo run in patients were randomly assigned to LIS 10-20 mg/o.d. or CAP 25-50 mg/b.i.d. treatment for 6 weeks. After this, patients with supine diastolic blood pressure (SDBP) greater than 90 mmHg were treated with the combinations LIS 20 mg + HCTZ 12.5 mg/o.d. or CAP 50 mg + HCTZ 25 mg/o.d. for 4 weeks; this dose was doubled if DBP was found to be greater than 90 mmHg after 2 weeks' combined therapy. A total of 175 patients were enrolled (92 females and 83 males) of which 153 completed the study. The LIS + HCTZ association caused a significant reduction of DBP in comparison to the other combined treatment (88.1 +/- 0.7 vs 90.3 +/- 0.7; p = 0.026). The statistical analysis of mean SBP values showed no significant difference between the two groups (144.0 +/- 1.3 vs 146.8 +/- 1.3; p = 0.15). At the end of the study 79.5% of patients treated with LIS + HCTZ presented normal results (DBP less than or equal to 90 mmHg), whereas the percentage of similar results in the comparison group was 72%. The percentage of "responder" patients to therapy (DBP reduced by 10 mmHg or more in relation to basal values) was 96.3% in the LIS + HCTZ group and 86.7% in the CAP + HCTZ group. In the CAP + HCTZ group 0.6% of patients reported adverse reactions, while only 0.3% were observed in the LIS + HCTZ group.


Effect of full-fat or defatted rice bran on serum cholesterol.

Rice bran has been shown to lower serum cholesterol in hamsters. Leghorn cockerel chicks were fed 60% full-fat rice bran (FFRB) and corn/soy (CS) diets with 0.5% added cholesterol. Both diets contained 18% protein. All reported parameters are on blood serum. Significant differences (p less than 0.05) were found in total cholesterol (TC), triglycerides (TG), high-density and low-density lipoprotein cholesterol (HDL and LDL). In a second study, chicks were fed FFRB, defatted rice bran (DFRB), and CS diets balanced for 18% protein, 14.47% total dietary fiber and 10.78% lipid with 0.5% added cholesterol. Both TC and TG were significantly lower (p less than 0.05) in chicks fed FFRB and CS diets. Significant differences were found in HDL values for all diets with FFRB exhibiting the highest mean value (155 mg/dl) and CS exhibiting the lowest mean value (114 mg/dl). All diets were significantly different (p less than 0.05) in LDL, with mean values of 249, 318 and 275 mg/dl for FFRB, DFRB and CS, respectively. FFRB appears to increase HDL and to lower LDL in chicks, but does not always affect TC, whereas DFRB may increase all three serum lipid components.


[Relation between 67-gallium scintigraphy and bronchoalveolar lavage--differential cell count and superoxide anion liberation--in patients with systemic scleroderma and systemic lupus erythematosus].

The aim of the study was to determine the pulmonary 67-gallium uptake, bronchoalveolar lavage (BAL) cell differentiation and the activity of BAL cells, measured as release of superoxide anion (O2-), and to investigate the results whether there are relations. In 11 nonsmoking systemic scleroderma (SS) patients and 11 systemic lupus erythematosus (SLE) patients with lung involvement double-sided BAL were performed, mainly in regions with increased 67-gallium uptake. Release of O2- was measured by INT-assey. BAL cell differentiation was in SS and SLE pathological in 68.2% without side-difference. In contrast to SS, O2(-)-release in SLE depends on BAL cell differentiation and is most increased in normal BAL cell differentiation. There is no correlation between 67-gallium uptake and both BAL cell differentiation and O2(-)-release. The results suggest, that in contrast to SS, BAL cells in SLE with pathological differentiation are less activated than BAL cells with normal differentiation probable due to autoimmunological factors. Pulmonary 67-gallium scan and BAL seem to be independent from each other.


Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons.

A major goal of the combined effort of basic scientists and plastic and reconstructive surgeons is the development of novel bone substitutes based on osteogenic growth and differentiation factors with optimal delivery systems for skeletal repair. Osteogenin is a protein initiator of bone differentiation. The present study examined the osteogenic potential of osteogenin in combination with porous hydroxyapatite replicas obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. Bovine osteogenin, with an apparent molecular weight of 28 to 42 kDa, purified by hydroxyapatite-Ultrogel adsorption chromatography, heparin-Sepharose affinity chromatography, and HR Sephacryl S-200 molecular sieve chromatography, was delivered into rods of nonresorbable and resorbable hydroxyapatite replicas with an average porosity of 600 microns. A total of 48 rods were bioassayed for osteogenic activity by intramuscular implantation into eight adult baboons (Papio ursinus) as a prerequisite for clinical trials in humans. Bovine osteogenin fractions reconstituted with baboon insoluble collagenous bone matrix were implanted in an additional four adult baboons. Specimens were harvested at 30 and 90 days after implantation and subjected to histomorphometry and alkaline phosphatase activity determination. Differentiation of bone occurred in nonresorbable hydroxyapatite rods, both osteogenin-treated and controls. However, no bone formation was observed in resorbable rods, even in the presence of osteogenin. These results demonstrate that the surface and chemical characteristics of the substratum, independent of the osteogenic stimulus, have a profound influence on the morphogenesis of bone. The demonstration of bone induction in nonhuman primates with porous nonresorbable hydroxyapatite replicas and baboon insoluble collagenous bone matrix reconstituted with bovine osteogenin establishes the therapeutic potential of the principle of bone induction in craniofacial, periodontal, and orthopedic reconstructive surgery.


Alpha melanocyte stimulating hormone (alpha-MSH) enhances eicosanoid production by bovine retinal pigment epithelium.

Explants of bovine eyes consisting of retina, with its underlying choroid and sclera (termed retinal explants) were maintained in organ culture in the absence or presence of alpha-melanocyte stimulating hormone (alpha-MSH) for up to 19 days. The conditioned media was collected twice a week and assayed for the following eicosanoids, prostaglandin E2 (PGE2) and prostacyclin. The addition of alpha-MSH to the incubation media resulted in a 1.5 fold enhancement in the production of both PGE2 and prostacyclin. This stimulatory effect diminished after 11 days. Additionally, the three tissue components comprising the retinal explants i.e. 1. neural retina 2. retinal pigment epithelium (RPE) with its underlying vascular layer (choroid) and 3. scleral tissue were separated and incubated in the presence or absence of alpha-MSH. Hormone treatment caused an enhanced eicosanoid production by RPE tissue alone, while its production by the neuronal retina and sclera was reduced or unaffected respectively. This demonstrates that the RPE layer is the source for the alpha-MSH induced eicosanoid production observed in the whole retinal explant. Our findings demonstrate, for the first time that alpha-MSH can stimulate prostaglandin production by RPE maintained in organ culture.