Expression of connexins in the developing olfactory system of the mouse. To gain insight into the function of gap junctions' connexin43, connexin32 and connexin26 in a neural structure that retains neuronal turnover capacities throughout adulthood, the expression of these molecules has been investigated in the developing and adult olfactory system by immunocytochemical and biochemical methods. Connexin43 was detectable from the olfactory placode stage. During early embryonic development, the levels of connexin43 expression remained low. An increase in the expression of this connexin occurred perinatally. Expression of connexin43 became very high during the postnatal stages and adulthood. Electron microscopy (EM) immunocytochemistry of the olfactory system showed connexin43 expression in non-neuronal cells. Strong regional differences in the expression of connexin43 in the olfactory epithelium were observed. No apparent relationship between connexin43 expression and turnover activity of olfactory neurons was detected. Western blots of olfactory tissues revealed the presence of three different isoforms of connexin43. Connexin32 was detected in the olfactory bulb at late postnatal stages including adulthood. Connexin32 was observed on some cells tentatively identified as oligodendrocytes. Connexin26 was localized onto leptomeninges. Some immunofluorescence was also obtained in the periglomerular region and in the subependymal layer of the bulb. Northern blot analysis revealed the presence of mRNA of connexin32 and connexin26 in the adult olfactory system. Our results substantiate the cell specific expression of these three types of connexins and they document the primary of connexin43 in olfactory tissues. Moreover, our findings indicate that although expression of connexin43 in the olfactory system is developmentally regulated, it is not directly associated with the neuronal cell turnover of the olfactory epithelium.


The effects of cortisol infusion upon hormone secretion from the anterior pituitary and subjective mood in depressive illness and in controls. The aims of this study were to determine whether the administration of cortisol has a significant effect on mood in patients with depression and whether the effects of cortisol on changes in plasma hormone concentrations are like those of synthetic corticosteroids. Twelve patients had major depression and one each had dysthymic disorder and a depressive adjustment disorder. Five were male and nine were female. All were in-patients. Eight normal subjects, two females and six males, were used as controls. Basal beta-endorphin concentrations were 2- to 3-fold higher in depressed patients than in control subjects, but there were no significant differences between the patient and control groups in the basal (pre-infusion) plasma concentrations of ACTH, cortisol, growth hormone or prolactin. Cortisol, but not saline infusion resulted in a significant improvement in self rated mood. Surprisingly, cortisol infusion at first increased plasma beta-endorphin concentrations. At later times after cortisol infusion, plasma beta-endorphin concentrations decreased as did the plasma concentrations of ACTH and growth hormone; prolactin levels were increased. These results show (i) that cortisol infusion raises mood significantly in major depression, (ii) that plasma beta-endorphin concentration is a potential marker of major depression (iii) that rather than blunting of corticosteroid effects, responses to cortisol may even be enhanced in depressive illness. The unexpected, initial increase in beta-endorphin stimulated by cortisol, suggests that the action of cortisol is not simply one of negative feedback inhibition, but may involve mineralocorticoid, as well as glucocorticoid receptors.


Diethylbenzene inhalation-induced electrophysiological deficits in peripheral nerves and changes in brainstem auditory evoked potentials in rats. Motor and sensory conduction velocities (MCV and SCV), amplitude of the sensory action potential (ASAP) of the tail nerve and parameters of brainstem auditory evoked potentials (BAEP) were studied in male Sprague-Dawley rats after prolonged inhalation exposure to a commercial isomer mixture of diethylbenzene (DEB mixture) containing 6% 1,2-DEB. The MCV, SCV and ASAP were studied in one control group (10 rats) and three groups of 12 rats exposed to 500, 700 or 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. Rats used for recording BAEP (one control group and two other groups of 15 rats) were exposed to 600 and 800 ppm DEB mixture. The exposure time was the same. Rats exposed to DEB mixture exhibited a time- and concentration-dependent decrease in MCV, SCV and ASAP and a time- and concentration-dependent increase of both the peak latencies of all BAEP components and the interpeak (I-V) differences.


[Cyclic AMP in the kidneys of adrenalectomized and nonadrenalectomized normal rats and rats with reflex dystrophy against a background of organ denervation and a total block of the body's beta-adrenoreceptors by propranolol]. Using the radioimmunological assay, renal cAMP content was studied in adrenalectomized and non-adrenalectomized rats with reflex dystrophy resulting from chronic ischiatic nerve stimulation against the background either of organ denervation or of general beta-adreno-blockade with propranolol. It was shown that the surgical denervation of kidney resulted in an increase in renal cAMP both in norm and especially in reflex dystrophy. The beta-adrenoceptor blockade with propranolol was accompanied by a decrease in renal nucleotide being more pronounced in animals with intact ischiatic nerve. Based on own and literature data we have suggested that one cause for the damage to renal aldosterone receptors in reflex dystrophy under pathological stimulation from the focus of damage in ischiatic nerve as well as for their improved functioning in surgical or pharmacological (beta-adrenoceptor blockade) desympathization which discontinues this stimulation, may be change in cAMP content. Via cAMP-dependent protein kinases, this nucleotide triggers a complex system of intracellular metabolic transformations which is able to change the protein spectrum of cytoplasm, nucleus, and chromatin and, therefore, the protein complex of aldosterone receptors. A participation of other biochemical systems of aldosterone stimulus transmission, including cAMP-independent ones, in processes of damage and normalization of tubule cells of the kidneys undergoing reflex dystrophy cannot be excluded.


[Low-molecular heparin complex formation with the blood coagulation proteins--thrombin and fibrinogen]. It has been found that low molecular heparin (LMH) forms complexes with fibrinogen and thrombin. The formation of the heparin-fibrinogen and heparin-thrombin complexes has been testified by cross-linked electrophoresis. The reaction of complex formation was carried out at variable weight ratios of the components, i.e. 1:3 and 1:6, respectively. This complex causes lysis of unstabilized clots of fibrin. All these complexes manifested a slight anticoagulative activity.


A new human cholangiocellular carcinoma cell line (KMC-1). We have recently established a cholangiocellular carcinoma (CCC) cell line, designated KMC-1, from a nude mouse subcutaneous tumor which developed after inoculation of a surgically resected peripheral type CCC from a 62-year-old Japanese male patient. KMC-1 cells grew over a 26-month period and passaged 57 times. These cells retained the morphologic characteristics of both the original tumor and the subcutaneous tumor in the nude mouse, which mainly consisted of irregular tubules and invaded surrounding interstitial tissue in part with an indurate pattern. KMC-1 cells grew in a monolayer pavement-like cell arrangement with tubular formation in part. Some cells and/or glands had a mucin-like substance inside. The doubling time of KMC-1 cells growing in serum-containing medium was 54 h at passage 31. Cell growth in serum-free medium was slow but steady. The number of chromosomes was distributed in range from 73 to 83 with modes of 76 and 78. KMC-1 cells secreted some tumor markers such as DUPAN-2, CA125, TPA, hCG, CA19-9 and ferritin, however, the secretion of DUPAN-2, and CA19-9 and ferritin were only detectable in serum-containing and serum-free medium, respectively. These findings suggest that KMC-1 cells will provide a variety of experimental models for research on CCC and the mechanisms of tumor marker secretion.


[3'-->5'-exonucleases of the rat liver and the correction of replication errors]. Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.


Surgical resection and survival in Western patients with hepatocellular carcinoma. In a retrospective study the survival of 28 patients with hepatocellular carcinoma, 25 of them with underlying cirrhosis, submitted to surgical resection was compared with the survival of 28 untreated patients, matched for variables known to bear independent prognostic value and therefore sharing the same baseline prognosis. Diagnosis was made in the same time period for both groups of patients. In addition, to further evaluate the effects of tumor resection on survival, the outcome of operated patients was also compared to their expected survival. This was derived from a mathematical model which takes into account the regression coefficients of the variables previously shown to be independently related to the survival of untreated patients with hepatocellular carcinoma. The median survival for resected patients was 27.1 months, which was significantly better than untreated controls (12.4 months; p less than 0.003). Median survival for patients submitted to resection and with tumors smaller than 5 cm was 35.8 months, while the median survival for untreated cases was 14.6 months p less than 0.0005. The comparison of observed survival (82% at one year and 73% at two years) and statistically expected survival (58% and 34%, respectively) further indicated that surgical resection effectively improves prognosis in Western patients with hepatocellular carcinoma. Thus, early detection of small tumors in the population at risk appears to be justified.


Sodium ipodate increases triiodothyronine action in vivo. Sodium ipodate (IPO) has been shown to bind nuclear T3 receptors (NT3R) in vitro, but previous studies have conflicted in regard to demonstration of this interaction in vivo. We sought evidence for IPO-NT3R binding in vivo by giving large doses of IPO to thyroidectomized (TDX) rats replaced with low doses of T3. We predicted that IPO-NT3R binding would inhibit T3 induced increases in mitochondrial alpha glycerophosphate dehydrogenase activity (alpha-GPDH) in kidney, heart and liver. Three groups of ten euthyroid rats each received 13 daily injections of vehicle, or 6 or 12 mg/100 g body weight of IPO, respectively. Both doses of IPO resulted in decreases in serum T3 and increases in serum TSH. Liver and kidney alpha-GPDH, however, were decreased only in the group receiving 6 mg IPO. In addition, three groups of 30 TDX rats were implanted with osmotic minipumps that contained T3 in the following concentrations: 33, 69 and 96 ng/ul. Ten rats in each group received 13 daily injections of vehicle, or IPO (vide supra). The alpha-GPDH responses were complex in that there was significant interaction between T3 and IPO effects in the kidney (AxB F ratio 5.13, p less than 0.001) and liver (AxB F ratio 2.85, p less than 0.05). The major finding, however, was that alpha-GPDH was not significantly reduced by IPO in any T3 replaced group. Rather, in all three organs, alpha GPDH was significantly increased above that produced by T3 alone by at least one combination of IPO and T3. Changes in serum TSH also suggested that IPO could enhance T3 effects. We conclude that IPO-NT3R binding is not a prominent mechanism via which the drug attenuates T3 effects in vivo. The data suggest that IPO may enhance T3 effects at the cellular level and that this enhancement may not be reflected by routinely monitored serum TSH. The latter observation may have clinical importance.


Infection of peripheral mononuclear blood cells by hepatitis C virus. We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.


Non-radioactive hybridization with hepatitis C virus-specific probes created during polymerase chain reaction: a fast and simple procedure to verify hepatitis C virus infection. All routinely available assays to detect hepatitis C virus (HCV) infection are based on the specific reaction of serum antibodies to corresponding viral antigens. The only method to detect viral nucleic acids in serum or liver is the enzymatic amplification of reversely transcribed HCV-RNA by polymerase chain reaction. Here, we describe a fast and simple method to confirm the HCV specificity of amplified fragments by hybridization with a non-radioactive cDNA probe created during polymerase chain reaction. A sequenced 521 bps HCV fragment derived from serum of an anti-HCV/EIAII-positive patient A with post-transfusion hepatitis C was used to verify HCV infection diagnosed by nested polymerase chain reaction in an anti-HCV/EIAII-seronegative patient B who did not produce anti-HCV-specific antibodies during the observation period of 1 year. The method is applicable to RNAs derived from serum or liver tissue and combines the sensitivity of nested polymerase chain reaction with the specificity of Southern blot hybridization. The procedure is independent of cloned viral sequences. In clinical use it permits fast and accurate confirmation of HCV infection, especially when antibodies detectable with conventional methods are absent.


A survey of cytomegalovirus (CMV) DNA in primary sclerosing cholangitis (PSC) liver tissues using a sensitive polymerase chain reaction (PCR) based assay. Reactivation of cytomegalovirus (CMV) has been implicated as a possible etiological agent in primary sclerosing cholangitis (PSC) partly because of the ability of CMV infection to cause hepatobiliary damage, and further because of the recent recognition of a PSC-like syndrome in AIDS patients, many of whom have hepatobiliary infection with CMV. Direct evidence of CMV infection in PSC has come from a study detecting CMV DNA in 7/7 PSC livers, but only 5/20 controls. We have developed an assay for CMV-DNA by amplification of the immediate early region of CMV using the polymerase chain reaction, followed by Southern blotting and 32P oligoprobing of the amplification product. This system has an average sensitivity of at least 25 copies of CMV-DNA per 5000 formalin-fixed paraffin-embedded cells. 37 PSC and 19 control samples of formalin-fixed paraffin-embedded hepatobiliary tissues were studied. Amplification for the beta-globin in each sample was used as an amplification control, and fetal lung with known CMV infection as the CMV-positive control. 37/37 PSC tissues amplified for beta-globin, and one of these was positive for CMV-DNA. All 19 controls amplified for beta-globin, with none being positive for CMV. The lack of CMV-DNA in 35/36 PSC samples at a level of 25 copies per 5000 cells, we believe, rules out any significant CMV reactivation in these tissues, and suggests that CMV replication and re-activation is not responsible for the progression of PSC.


Hepatitis B X-gene expression in hepatocellular carcinoma. A RNA-PCR method and different sets of primers were used to investigate the expression of different hepatitis B (HB) virus genes at the RNA level. We tested paired samples (tumor and non-tumor) from the liver tissues of 48 Taiwanese patients with primary hepatocellular carcinoma (HCC). By using a set of primers which spanned the sequences of the S-gene, we found expression in only 2 patients. In one HBs-RNA was only detected in the tumor tissue and in the other only in the surrounding non-tumor tissue. Using primers covering the C-gene, expression was found in 7 patients. In 2 of these RNA was detected in both the tumor and the surrounding tissue, in 2 in the tumor tissue, and in 3 in the surrounding tissue only. Finally, when primers spanning the X-gene sequences were used, RNA was detected in 40/48 patients. In 33 of these cases HBx-RNA was detected in both tumor and non-tumor tissue, in 3 patients in tumor tissue only, and in 4 in the surrounding tissue. Among the cases in which HBc and HBs-RNA was expressed, all showed HBx expression also. These data indicate that the expression of the HBx gene in HCC may play an important role in hepatocarcinogenesis.


Transganglionic effects of Nocodazole, a new synthetic microtubule inhibitor, upon the histochemical structure of the spinal upper dorsal horn. Perineural application of Nocodazole (R 17934 of Janssen Pharmaceutica), a new synthetic microtubule inhibitor, induces transganglionic degenerative atrophy of central terminals in the spinal cord of the inflicted primary sensory neurons. This drug offers new possibilities for clinical trials aiming to relieve chronic pain by subcutaneous injection of a microtubule inhibitor.


Influence of hypothalamic-pituitary-adrenocortical hormones on reproductive hormones in gray wolves (Canis lupus). The release of hypothalamic-pituitary-adrenocortical hormones was studied in intact and neutered gray wolves (Canis lupus) to determine how these hormones interact and affect reproductive hormones. Experiments were performed on adult wolves anesthetized with 400 mg ketamine and 50 mg promazine. Intravenous (i.v.) injections with 50 micrograms ovine corticotropin releasing factor (oCRF) significantly increased adrenocorticotropin (ACTH; P < or = 0.01), cortisol (CORT; P < or = 0.004), and progesterone (P < or = 0.036), but not beta-endorphin (P > or = 0.52). Since neutered wolves demonstrated dose-dependent elevations in response to ACTH, it was concluded that the progesterone was secreted from the adrenal gland. Basal luteinizing hormone (LH) concentrations in neutered wolves were similar before and 60 min after i.v. injection of 1, 5, or 25 IU ACTH (P > or = 0.36) or 2.2 mg/kg cortisol (P = 0.42). Neither 25 IU ACTH (P = 0.55) nor 0.22 mg/kg dexamethasone (P = 0.49) altered the LH response to injection of LH releasing hormone in neutered wolves. Chronic administration of 0.22 mg/kg/day dexamethasone for 3 d did not alter baseline LH concentrations (P = 0.75). Injection of 1.0 mg/kg naloxone (NAL), however, increased LH concentrations relative to baseline values in both intact (P = 0.032) and neutered (P = 0.0005) female wolves, but not in intact (P = 0.19) or neutered males (P = 0.07). These results indicated that in gray wolves (1) oCRF stimulated the release of pituitary and adrenal hormones in a fashion similar to that of other mammals; (2) the adrenal cortex was capable of secreting progesterone into the systemic circulation; (3) exogenous glucocorticoids did not alter LH concentrations; and (4) endogenous opioids may modulate LH secretion in female wolves.


Platinum complexes of p- and m-aminobenzamidine. Synthesis, characterization, and cytotoxic activity. Four platinum(II) aminobenzamidine complexes have been prepared and characterized by IR and 1H and 13C NMR spectroscopy, and tested for their ability to interact with the nicked and closed circular forms of the pUC8 plasmid DNA. The results show that the complexes of formula [Pt(LH)2Cl2]2X have a cis- geometry with an amino-Pt bonding, where L is either p- or m-aminobenzamidine and where 2X is 2Cl- or PtCl4(2-). It was observed that these complexes significantly alter the electrophoretic mobility of nicked and closed circular forms of DNA and that the alteration in electrophoretic mobility due to Pt(II)-p-aminobenzamidine binding is higher than that due to Pt(II)-m-aminobenzamidine. No difference in mobility was observed whether the DNA interacted with complexes having as counteranion Cl- or PtCl4(2-). The synthesized compounds were, in addition, assayed for antitumor activity in vitro against colon (CX-1), lung (LX-1), and mammary (MX-1) human tumor cells. The results show that these complexes inhibited the multiplication of the tumor cells and that they show higher specificity for lung cells.


Famotidine, the new antiulcero-genic agent, a potent ligand for metal ions. Potentiometric, polarographic, and spectroscopic results obtained for Cu2+ and Ni(2+)-famotidine systems clearly indicated that this anti-ulcerogenic drug is a very potent chelating agent able to coordinate cupric ion that was at pH below 2. This drug exhibits excellent histamine H2 receptor blocking effects and its effective coordination to metal ions may have significant biological implications. Famotidine is found to be a very effective ligand for Ni2+ ions also.


Ubiquitin-positive intraneuronal inclusions in the extramotor cortices of presenile dementia patients with motor neuron disease. Ubiquitin-positive intraneuronal inclusions were found in the extramotor cortices of ten presenile dementia patients with motor neuron disease. There were inclusions in the hippocampal granular cells and in the small neurons of the superficial layers of the temporal and frontal cortices. Bunina bodies were present in the anterior horn cells in all cases. These results suggest that ubiquitin-related cytoskeletal abnormalities are common in cerebral non-motor small neurons in these patients.


Tibial autogenous cancellous bone as an alternative donor site in maxillofacial surgery: a preliminary report. This preliminary report reviews the use of the proximal tibial metaphysis as an alternative to other bone graft donor sites. A series of 21 cancellous grafts were procured from 20 patients using a predictable orthopedic approach. A singular finding was the overall lack of morbidity at the donor site and the apparent favorable quality and quantity of the procured bone. Data describing the use of tibial cancellous bone grafting in a wide range of maxillofacial reconstructive procedures are presented.


Calcium metabolism in Williams-Beuren syndrome. Increased 1,25-dihydroxyvitamin D levels and decreased basal and calcium-stimulated calcitonin serum levels have been found in children with Williams-Beuren syndrome (WBS). To determine whether isolated or combined disturbances of secretion or action of the calcium-regulating hormones may cause the tendency to hypercalcemia in WBS, we investigated several aspects of calcium metabolism in 27 normocalcemic children and adults, aged 2 to 47 years, with WBS. With the exception of slightly decreased 25-hydroxyvitamin D and slightly increased calcitonin in serum, all measured basal indexes of calcium and bone metabolism, including the serum levels of intact parathyroid hormone and 1,25-dihydroxyvitamin D, were comparable to control values. Total and extractable calcitonin, the latter representing the monomeric and biologically important form of the hormone, showed the same relative increase after a low-dose calcium infusion in patients and control subjects, indicating a normal capacity of the calcitonin-producing C cells of the thyroid gland in WBS. Furthermore, exogenous parathyroid hormone induced a normal response of 1,25-dihydroxyvitamin D, cyclic adenosine monophosphate, and phosphate excretion, indicating a normal response of the renal 25-hydroxyvitamin D-1 alpha-hydroxylase and the renal receptor-adenylate cyclase system to parathyroid hormone. These findings suggest that neither deficient calcitonin secretion nor increased renal sensitivity to parathyroid hormone is a feature of WBS in normocalcemic patients.


omega-[(4-Phenyl-2-quinolyl)oxy]alkanoic acid derivatives: a new family of potent LTB4 antagonists. A series of omega-[(4-phenyl-2-quinolyl)oxy]alkanoic acid derivatives was prepared which inhibited the binding of the leukotriene B4 to its receptors on guinea pig spleen membranes and on human polymorphonuclear leukocytes. A structure-activity relationship was investigated. The length of the carboxylic acid side chain was important for potent binding activity. The replacement of the oxygen atom at the beginning of the chain with other polar or nonpolar linking groups led to considerable loss of potency, indicating that the oxygen linking atom might be involved in the receptor recognition. alpha-Substitution on the carboxylic acid side chain led to substantially more potent compounds. Substitution on the phenyl ring and on the quinoline ring was also evaluated.


omega-[(4,6-Diphenyl-2-pyridyl)oxy]alkanoic acid derivatives: a new family of potent and orally active LTB4 antagonists. A series of omega-[(4,6-diphenyl-2-pyridyl)oxy]alkanoic acid derivatives was prepared which inhibited the binding of leukotriene B4 to its receptors on guinea pig spleen membranes and on human polymorphonuclear leukocytes (PMNs) and selectively antagonized the LTB4-induced elastase release in human PMNs. On the basis of these three screens, a structure-activity relationship was investigated. alpha-Substitution on the carboxylic acid side chain led to only small changes in the binding affinities but greatly enhanced the LTB4 antagonist activity. Substitution on the phenyl rings was also evaluated. The terminal carboxylic acid function can be replaced by a tetrazole ring without loss in activity. The best in vitro LTB4 antagonists of this series were investigated in vivo in the inhibition of LTB4-induced leukopenia in rabbits. Compound 9b (RP69698) displayed potent LTB4 antagonist activity, after oral administration, with an ED50 value of 6.7 mg/kg.


Opioid agonist and antagonist activities of morphindoles related to naltrindole. A series of naltrindole-related ligands (4-10) with an N-methyl,N-phenethyl,N-cinnamyl, or an unsubstituted basic nitrogen were synthesized and tested for opioid agonist and antagonist activity in smooth muscle preparations and in mice. The nor compounds (4 and 6) and the phenethyl derivatives (5 and 8) displayed full agonist activity (IC50 = 85-179 nM) in the mouse vas deferens preparation (MVD) while the other members of the series exhibited partial agonist or weak antagonist activity. In the guinea pig ileum preparation (GPI), all compounds except 8 were partial agonists. The ligands that were evaluated in mice were found to produce antinociception that was not selectively mediated via delta opioid receptors. However, two of these ligands (4 and 5) appeared to be delta-selective opioid receptor antagonists at subthreshold doses for antinociception. The finding that all of the compounds bind selectively to delta opioid receptors in guinea pig brain membranes together with the in vitro pharmacology and in vivo antagonist studies suggests that the lack of delta agonist selectivity in vivo may be due to a number of factors, including a basic difference between the delta receptor system in the MVD and in the mouse brain. Further, it is suggested that the constellation of message and address components in the morphindole nucleus may tend to stabilize delta receptors in the brain in antagonist state.


Rolling-circle replication of a high-copy BPV-1 plasmid. We investigated the replicating form of a bovine papillomavirus type 1 (BPV-1) deletion mutant by direct electron-microscopic analysis of low molecular weight cellular DNA fractions. The detection of viral plasmid DNA replication intermediates was facilitated by the isolation of a spontaneously transformed mouse cell subclone containing an unusually high viral genome copy number (approx. 1000 per cell), and by employing a slight modification of the Hirt fractionation procedure to reduce the level of contaminating linear chromosomal DNA fragments. We observed exclusively rolling-circle-type viral DNA replication intermediates, at a frequency of detection of approximately one replication intermediate per 200 monomeric circular viral DNA molecules. The demonstration of rolling-circles with longer-than-genome-length tails indicated that this high-copy viral plasmid was not subject to a strict once-per-cell-cycle mode of DNA replication. Our observations provide further evidence in favour of an alternative replication mode of the BPV-1 genome, and may help to explain earlier conflicting findings concerning the mechanism of stable BPV-1 plasmid copy-number-control.


[Cell kinetic effect of carcinostatic agents using BrdU and its monoclonal antibody]. We have investigated the cell kinetic effect of four carcinostatic agents (MMC, CDDP, ADR and 5-FU) on the human gastric cancer cell line (KATO-III; signet ring cell carcinoma) by means of flow cytometry (FCM), using bromodeoxyuridine (BrdU) and its monoclonal antibody. Cancer cells in the S phase were first labelled with BrdU and then the bivariate DNA/BrdU distribution was examined to analyze the effect on the cell cycle. Furthermore, cells were reincubated at 24 hours after labelling to evaluate the cell turnover during FCM. MMC, CDDP and ADR assembled the cells into late S phase and G2M phase, while 5-FU assembled them into S phase. after 24 hours, cells with cessation of cell cycle had inhibited their proliferation. We conclude that this technique can be usefully applied as a susceptibility test of carcinostatic agents, since it could define the phase where carcinostatic agents acted on cancer cells.


[Malignancy of hepatocellular carcinoma evaluated with nuclear DNA ploidy pattern]. Nucleus DNA ploidy analysis was performed by flow cytometry on 81 patients with hepatocellular carcinoma. DNA ploidy patterns were divided into two groups; diploid pattern and aneuploid pattern, which were estimated by the rate of the first peak channel on histograms. DNA aneuploidy significantly correlated with serum AFP level (p < 0.01), TNM stage (p < 0.01), and pathological findings: vp (p < 0.5), im (p < 0.01). The aneuploid cases showed better prognosis than the diploid cases (Generalized-Wilcoxon test, p < 0.01). In the diploid cases, recurrent tumors manifested almost solitary isolated. The disease free period after hepatectomy was longer in diploid cases (mean 24.2 months) than in aneuploid cases (mean 14.4 months). There was a higher frequency of aneuploid cases of recurrence within 2 years compared to diploid cases. Two and half year survival rates after recurrence of diploid cases (69.6%) was significantly better than those of aneuploid cases (38.2%) (p < 0.05). The DNA ploidy pattern of hepatocellular carcinoma plays an important role in predicting the malignant potentials of the disease.


[Flow cytometric analysis of cellular DNA content in epithelial ovarian cancer]. Tumor DNA content (ploidy) was determined by flow-cytometry (FCM) on tissue from 32 epithelial ovarian cancer patients. Staining for DNA analysis was achieved with Propidium Iodide. Peripheral blood lymphocytes were used as reference diploid cell population. Of the 32 patients, 26 (81.3%) had tumors which were aneuploid, whereas 6 (18.7%) were diploid. DNA aneuploid cell lines were found in 100% of serous adenocarcinoma, in 57% of mucinous adenocarcinoma, in 67% of endometrial adenocarcinoma, in 88% of clear cell carcinoma and in 75% of undifferentiated carcinoma. The DNA ploidy abnormalities differed in each histologic characteristic of epithelial ovarian cancer.


[Quantitative analysis of cytoplasmic myeloperoxidase positive leukemic cells by FCM]. Development of a fixation allowed flow cytometric analysis of nuclear and intracellular antigen in leukemic cells. In this paper the analyzing procedure of cytoplasmic myeloperoxidase by FCM was described. This procedure is more sensitive than cytochemical staining of fixed cell smears and more specific than cell surface immunophenotyping by CD13, CD14, CD33. So, this technique is useful for classification of myelogenous leukemic cells especially MO type leukemia by FAB classification. This technique also allowed two color analysis of cell surface antigens and cytoplasmic antigens. Mixed lineage leukemia can be easily and accurately classified by using of this procedure.


[Morphometric analysis of hepatocellular carcinoma: diagnostic usefulness of numerical nuclear density and its ratio]. In order to develop a conventional objective criterion for differentiating well differentiated hepatocellular carcinoma and benign hepatic lesions, morphometric analysis was made. First, we measured four variables, that is, cell size, nuclear size, N/C ratio and numerical nuclear density (number of nuclei of hepatocytes within a unit area) and examined the diagnostic values of these variables. Among these four variables, nuclear density was found to be the most diagnostic. Using only the variable of nuclear density, trabecular type and pseudoglandular type carcinomas could be well discriminated but not other types. Then we examined not only nuclear density but also nuclear density ratio of the tumor to non-tumor and derived a discriminant formula calculated by these two variables. This formula more improved the correct discrimination of small hepatocellular carcinoma.


[Therapeutic evaluation of recombinant thrombomodulin]. Thrombomodulin (TM) is an endothelium-associated glycoprotein that converts thrombin from a procoagulant protease to an anticoagulant. Thrombin, a key enzyme in thrombus formation, binds to TM molecules on endothelium with very high affinity. After binding to TM, thrombin fails to act on the coagulation factors and platelets, and its ability to activate protein C is enhanced more than 1000-fold. We expressed soluble recombinant TM (rTM) in CHO cells and evaluated its antithrombotic effect on thrombin-induced thromboembolism in mice and lipopolysaccharide (LPS) induced disseminated intravascular coagulation (DIC) in rats. Thrombin injection into mouse caused acute thromboembolism resulting instantaneous death, however preinjection of rTM neutralized the lethal effect of thrombin in a dose-dependent manner. Soluble rTM also improved the consumption of fibrinogen and platelets in experimental DIC-rats induced by LPS. The effect of rTM was confirmed in histologically. These data suggest that rTM may have a therapeutic effect on thrombosis or DIC in human.


[Characteristics and problems of flow cytometric nuclear DNA content analysis in lung cancer using bronchoscopically-obtained specimens]. Flow cytometric nuclear DNA content analysis was performed on 106 bronchoscopically-obtained specimens from 67 patients with primary lung cancer. Brushing, curettage and biopsy samples in which tumor cells were identified by microscopic examination were considered suitable for analysis. The incidence of DNA aneuploidy in small cell carcinoma (77.8%) was higher than that in any other histological type of lung cancer. Intratumoral heterogeneity was found to affect the detectability of DNA aneuploidy, suggesting that care must be taken to obtain adequate samples of tumor cells and to consider intratumoral heterogeneity. These results indicate that this method can be used for nuclear DNA content analysis not only in cases of resectable lung cancer but also for unresectable lung cancers and may provide useful biological information in the clinical management of all types of lung cancer.


[An autopsy case of pulmonary metastasis of cholangiocellular carcinoma associated with marked fibrotic change of the lungs]. An autopsy case of pulmonary metastasis of cholangiocellular carcinoma is presented. A 44-year-old woman was admitted to our hospital because of dyspnea, general fatigue and a sense of abdominal fullness on February 5, 1990. In November 1986, at an other hospital, she had been diagnosed as having diffuse metastatic lung tumor and multiple bone metastases, by transbronchial lung biopsy and other examinations. During the clinical course, she was not received chest irradiation and chemotherapy which induced fibrotic change of lungs. Chest X-ray film on December 21, 1986 showed diffuse nodular shadows in both lung fields. Chest X-ray film on February 4, 1990 showed diffuse reticular shadows with marked shrinkage of lung fields. She died two months after admission. The primary site of the carcinoma was not determined clinically, but was revealed by autopsy to be cholangiocellular carcinoma of the liver, with generalized metastasis. Microscopic findings of the autopsied lung showed markedly increased connective tissue around bronchi and blood vessels, in areas where microtubular adenocarcinoma was scattered. This is a very rare case of pulmonary metastasis of cholangiocellular carcinoma, associated with marked fibrotic change of the lungs during about 3.5 years. To our knowledge, this is the first reported case.


[The functional activity of the thrombocytes in chronic diseases of the large intestine]. Chronic diseases of the large intestine--catarrhal colitis, colonic stasis, unspecific ulcerative colitis, and amebiasis of the large intestine are attended by marked changes in the functional activity of blood platelets and factors of its regulation. They reflect the dynamics of the disease before and after operative and nonoperative treatment. A recurrence of the disease is linked with activation of blood platelets. Trental and contrycal are used for correction of the disorders, which improves the condition of some of the patients. The authors discuss the importance of biogenic amines, cAMP, and functional activity of the blood platelets as universal mechanisms of the development of chronic diseases of the large intestine.


A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel. We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.


In vivo labeling of the central GABA uptake carrier with 3H-Tiagabine. The in vivo binding of 3H-Tiagabine to the central GABA uptake carrier in mouse brain was characterized. 3H-Tiagabine in vivo bound to a single class of binding sites with a Kd = 72.5 nM and a Bmax = 640 pmol/g tissue. 3H-Tiagabine binding in vivo was regionally distributed within the CNS, and showed a good correlation with 3H-Tiagabine binding in vitro. Pharmacological characterization of 3H-Tiagabine binding in vivo revealed a binding site exhibiting specificity for GABA uptake inhibitors. Experiments examining the in vivo receptor occupancy of the GABA uptake carrier for a series of GABA uptake inhibitors revealed that 20-30% of the GABA uptake sites were occupied at the ED50 for inhibiting DMCM-induced clonic convulsions, while a 50-62% receptor occupancy in vivo was needed to inhibit rotarod performance. These data suggest that 3H-Tiagabine in vivo binding may be a useful method for assessing GABA uptake inhibitor penetration into the CNS, and may be a useful tool for studying the physiological regulation of the GABA uptake carrier.


Isolation and purification of calmodulin from the shrimp, Crangon crangon. Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.


In situ hybridization in viral hepatitis. In situ hybridization (ISH) is a sensitive and specific technique for detecting nucleic acids in intact cells. Visualization of the target sequences by autoradiography or immunohistochemistry allows their precise subcellular localization and quantitation. The application of ISH techniques has contributed to the understanding of the complex replicative cycle of hepatitis B virus. More recently, hepatitis delta and C virus replication has also been studied by this technique. ISH-based assays have finally been used to follow the replication of cytomegalovirus within the transplanted liver. Although ISH is a powerful tool for the molecular biologist, its clinical significance in the diagnosis and prognosis of human hepatitis virus infections has yet to be fully evaluated.


Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide. A non-isotopic in situ hybridization procedure was used to detect cytomegalovirus (CMV) sequences within routinely fixed tissue. A digoxigenin-tailed oligonucleotide was hybridized to sections of specimens obtained at autopsy from 2 patients with CMV infection. Hybrids were revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. Serial sections were also assayed for the presence of CMV by in situ hybridization with a biotin-labelled cDNA probe and by immunohistochemistry and routinely stained for morphological evaluation. Results show that the two in situ hybridization procedures are equally sensitive but superior to the immunohistochemical detection of the viral antigen. Most cells positive for CMV DNA had the cytopathological features characteristic of CMV infection. A minor population of infected cells lacking morphological changes was also found. We recommend the routine application of the oligonucleotide-based assay because it is specific, easy and less expensive than other similar procedures.


Detection of hepatitis C virus RNA by in situ hybridization. Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is ill-defined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridization with 125I-labelled riboprobes, representing approximately 35% of the HCV genome. Only a few positive cells were identified in the HCV-infected liver samples, but not in a normal liver sample. Hybridization with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages.


An immunohistochemical study of the blood vessels within primary hepatocellular tumours. We studied the blood vessels in routinely formalin-fixed and paraffin-processed tissue from 18 hepatocellular carcinomas, three "small" hepatocellular carcinomas, and 15 benign nodular lesions representing a spectrum of conditions with which liver cell carcinomas may be confused. These were stained for a basement membrane component (collagen IV) and endothelial markers (Factor VIII-related antigen, Ulex europaeus lectin binding, and QBEnd10). The staining pattern of normal and cirrhotic liver was also examined in tissue removed with these tumours. There was an increased expression by small blood vessels for collagen IV in carcinomas and benign lesions compared with cirrhotic nodules. All endothelial markers (Factor VIII-related antigen, QBEnd10, and Ulex europaeus binding) were best expressed in liver cell carcinomas. These differences were of degree and pattern, and no single marker distinguished benign from malignant lesions. The differences in staining pattern taken together with other clinical and pathological information should be useful in diagnosis particularly of small liver cell carcinoma. The differences between benign and malignant lesions support the idea that malignant neoplastic blood vessels in the liver are of a different basic biological type from normal hepatic sinusoids, and this difference could be exploited further in future therapy.


DNA curving and bending in protein-DNA recognition. Most biological events are regulated at the molecular level by site-specific associations between specialized proteins and DNA. These associations may bring distal regions of the genome into functional contact or may lead to the formation of large multisubunit complexes capable of regulating highly site-specific transactional events. It is now believed that sequence-specific protein-DNA recognition and the ability of certain proteins to compete for multiple binding sites is regulated at several levels by the local structure and conformation of the binding partners. These encompass the microstructure of DNA, including its curvature, bending and flexing as well as conformational lability in the DNA-binding domains of the proteins. Possible mechanisms for binding specificity are discussed in the context of specific nucleoprotein systems with particular emphasis given to the roles of DNA conformations in these interactions.


Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase. The C-terminal region (amino acid residues 236-329) of the Escherichia coli RNA polymerase alpha subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C-terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant alpha subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP-CRP. DNA sequence determination of several 'Lac-' mutant rpoA genes revealed that all had mutations clustered within a short segment near the C-terminus of alpha, between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).


High dose intravitreal ganciclovir in the treatment of cytomegalovirus retinitis. To assess the role of intravitreal administration of high doses of ganciclovir as a supplement and alternative to intravenous administration in the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS). A retrospective study of visual outcome, relapse and complications of intravenous and high dose intravitreal administration of ganciclovir alone and in combination. Twenty-three patients with AIDS and CMV retinitis (37 eyes) were examined by the authors and notes, fundal drawings and photographs reviewed. Initially patients were treated with intravenous ganciclovir alone and given supplementary intravitreal therapy for relapse or vision-threatening retinitis; however, later patients were managed with combination maintenance ganciclovir or maintenance intravitreal treatment alone. Relapse and loss of vision occurred frequently in patients treated with intravenous ganciclovir alone or in combination with intermittent intravitreal therapy. Eyes managed with maintenance high dose intravitreal ganciclovir alone or in combination with intravenous treatment did not relapse or lose vision. The most important complication of intravenous administration of ganciclovir was neutropenia (73% of patients), whereas that of intravitreal therapy was endophthalmitis (three eyes). High dose ganciclovir given intravitreally effectively suppressed CMV retinitis and preserved vision without adverse systemic effects or deterioration of quality of life.


[Blood leukotriene B4 response to endotoxin in fish oil fed rats]. Six-week-old male Wistar rats were given a low fat diet containing 15% cod liver oil (FO; n = 30) or 15% safflower oil (SO; n = 30) for 6 weeks. In the polymorphonuclear leukocytes, arachidonic acid (AA) was significantly lower in the FO group than in the SO group (p < 0.001), and eicosapentaenoic acid (EPA) in the FO group was present in large amounts showing the EPA/AA ratio of 1.76. When lipopolysaccharide (LPS; 10mg/kg) was intraperitoneally injected, white blood cell counts in the blood significantly decreased in both groups (p < 0.001) compared to the controls 2 hours later, and at 4 hours, the counts in the SO group (2,033 +/- 151/mm3) were lower than in the FO group (3,189 +/- 624/mm3), (p < 0.01). In both groups, leukotriene B4 (LTB4) in the whole blood increased at 4 hours following LPS-administration compared to the controls (1,219 +/- 167.3pg/ml in the SO group and 600.0 +/- 109.0pg/ml in the FO group, p < 0.001). It should be noted that the level of LTB4 in the SO group was significantly higher than in the FO group (p < 0.001). Changes of the metabolites including decreased LTB4 production in the arachidonic cascade related to the increase of EPA may play a role in the inhibition of the decrease in white blood cell counts.


Lidocaine: a hydroxyl radical scavenger and singlet oxygen quencher. Lidocaine, a local anaesthetic, has been shown to reduce ventricular arrhythmias associated with myocardial infarction and ischemic myocardial injury and its protective effects has been attributed to its membrane stabilizing properties. Since oxygen radicals are known to be produced during ischemia induced tissue damage, we have investigated the possible antioxidant properties of lidocaine and found that lidocaine does not scavenge O2-. radicals at 1 to 20 mM concentrations. However, lidocaine was found to be a potent scavenger of hydroxyl radicals and singlet oxygen. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic techniques. Lidocaine inhibited DMPO-OH adduct formation in a dose dependent manner. The amount of lidocaine needed to cause 50% inhibition of that rate was found to be approximately 80 microM and at 300 microM concentration it virtually eliminated the DMPO-OH adduct formation. The production of OH.-dependent TBA reactive products of deoxyribose was also inhibited by lidocaine in a dose dependent manner. Lidocaine was also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose dependent manner. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers and was detected as TEMP-1O2 adduct by EPR spectroscopy. The amount of lidocaine required to cause 50% inhibition of TEMP-1O2 adduct formation was found to be 500 microM. These results suggest that the protective effect of lidocaine on myocardial injury may, in part, be due to its reactive oxygen scavenging properties.(ABSTRACT TRUNCATED AT 250 WORDS)


A procedure for large-scale purification of domoic acid from toxic blue mussels (Mytilus edulis). Pure domoic acid is required for use in research to investigate the biological effects of this new shellfish toxin. It may also prove to be a useful tool in studies exploring the basis of Alzheimer's disease. In this paper we describe a procedure which is effective in obtaining adequate quantities of pure domoic acid from blue mussel (Mytilus edulis). The procedure involves tissue homogenization, treatment of homogenate with chloroform and methanol, and separation of different phases with the addition of water. The aqueous-methanolic phase (upper layer) contains water soluble components including domoic acid, the chloroform phase (lower layer) contains lipoid moieties, and the interphase contains denatured proteins. The aqueous phase containing domoic acid was removed, rotory evaporated to get rid of methanol, followed by ultrafiltration to remove high molecular weight contaminants. The filtrate was lyophilized, resuspended in 1 N HCl, centrifuged and the resulting clear solution subjected to column chromatography on C18 reversed phase silica gel. Fractions containing domoic acid were pooled, and lyophilized. A brownish dry powder contained pure domoic acid with 60-65% yield from the original tissue homogenate. Another 10-15% of domoic acid was mixed with its isomer, and can be further resolved to obtain an overall recovery of 75-80% of the starting material.


Transcription enhances intrachromosomal homologous recombination in mammalian cells. The influence of transcription on homologous intrachromosomal recombination between direct and inverted repeats has been examined by using Chinese hamster ovary cells. Recombination was monitored between two integrated neomycin (neo) genes, including one silent allele and a second allele regulated by the inducible mouse mammary tumor virus promoter. Transcription of mouse mammary tumor virus neo alleles was regulated with the glucocorticoid hormone dexamethasone. Alleles transcribed at high levels recombined about two- to sevenfold more frequently than identical alleles transcribed at low levels. Direct repeats recombined primarily by a gene conversion mechanism; inverted repeats produced a variety of rearranged products. These results are discussed in relation to recombinational processes that regulate gene expression, influence gene family structures, and mediate genomic instability associated with cellular transformation and tumorigenesis.


Expression of the woodchuck N-myc2 retroposon in brain and in liver tumors is driven by a cryptic N-myc promoter. The woodchuck intronless proto-oncogene N-myc2 was initially discovered as a frequent target site for hepadnavirus integration in hepatocellular carcinoma. N-myc2 possesses characteristics of a functional retroposon derived from the woodchuck N-myc gene. We have investigated the regulatory signals governing N-myc2 expression and found that a short promoter, including a variant TATA box and potential binding sites for several transcription factors, is localized in the N-myc2 sequences homologous to the 5' untranslated region of the second N-myc exon. The corresponding region in the intron-containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc genes suggests that they contain a cryptic N-myc promoter which may be unmasked in the particular context provided by the N-myc2 retroposon. Although N-myc2, like the woodchuck N-myc gene, contributes to an extended CpG island and was found constitutively hypomethylated, it presents a highly restricted expression pattern in adult animals. Whereas the intron-containing N-myc gene is expressed at low levels in different tissues, N-myc2 mRNA was detected only in brain tissue, raising questions about the functional significance of the maintenance of a second N-myc gene in the woodchuck genome.


Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses. The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.


Activation of the phosphoenolpyruvate carboxykinase gene retinoic acid response element is dependent on a retinoic acid receptor/coregulator complex. The accessory factor 1 (AF1) element is an upstream transcriptional control region that plays a role in the response of the phosphoenolpyruvate carboxykinase (PEPCK) gene to both glucocorticoids and retinoic acid. We demonstrate here that retinoic acid receptor alpha (RAR alpha) binds to a sequence within the AF1 element, TGACCT (site B), that is a consensus retinoic acid response element (RARE) half-site. A similar DNA sequence, TGGCCG (site C), located 1 bp downstream of site B, is not involved in the binding of RAR alpha monomers or dimers but is required for the constitution of a functional RARE. Site C is also required for the formation of a complex involving RAR alpha and a liver nuclear factor designated CR, for coregulator. Mutational analysis of the AF1 element shows that the RAR alpha/CR complex is the trans-acting unit that mediates the retinoic acid response of the PEPCK gene. Another member of the retinoid receptor family, retinoid X receptor alpha (RXR alpha), can also form a complex with RAR alpha and the AF1 element. Several observations, including the observation that RXR alpha antibody interacts with CR, indicate that RXR alpha and CR are identical or closely related proteins. Through RXR alpha forms a complex with RAR alpha and the AF1 element, we demonstrate that the AF1 element is functionally distinguishable from a retinoid X response element. Taken together, our results show that the AF1 element contains an RARE that mediates a retinoic acid response by binding an RAR alpha/coregulator complex; this coregulator is presumably RXR alpha.


Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.


The polyomavirus enhancer activates chromatin accessibility on integration into the HPRT gene. Recent studies suggest that enhancers may increase the accessibility of chromatin to transcription factors. To test the effects of a viral enhancer on chromatin accessibility, we have inserted minigenes with or without the polyomavirus enhancer into the third exon of the hypoxanthine phosphoribosyltransferase (HPRT) gene by homologous recombination and have prepared high-resolution maps of gene accessibility by using a novel polymerase chain reaction assay for DNase I sensitivity. In its native state, we find that the HPRT gene has low sensitivity to DNase I in fibrosarcoma cells. Insertion of the polyomavirus enhancer and neo reporter gene into exon 3 confers altered HPRT DNase I sensitivity for several kilobases on either side of the enhancer. The changes in DNase I sensitivity peak near the enhancer and decline with distance from the enhancer. The increase in HPRT DNase I sensitivity persisted when the tk promoter was deleted from the inserted construct but disappeared when the enhancer was deleted. These experiments identify the polyomavirus enhancer as a cis-acting initiator of chromatin accessibility.


Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP. The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.


The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors. Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.


Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors. Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.


RFL9 encodes an A2b-adenosine receptor.

RFL9 encodes an A2b-adenosine receptor. We recently reported the cloning of a cDNA (designated RFL9) that encodes a novel A2-adenosine receptor subtype. We now fully characterize the pharmacological properties of RFL9 in stably transfected CHO cells by examining cAMP responses to drug treatments. The pharmacological profile of cAMP responses in RFL9-transfected cells was similar to that expected for A2b-adenosine receptors and distinct from that of CHO cells transfected with the A2a-adenosine receptor. When RFL9-transfected cells were compared with VA 13 fibroblasts, the human cell line in which endogenous A2b-adenosine receptors were originally characterized, the dose-response curves of cAMP responses to drug treatments were highly correlated. Northern blot analysis of RNA prepared from VA 13 fibroblasts revealed specific hybridizing transcripts when probed for RFL9, but no hybridizing signal for A2a-adenosine receptor mRNA. Using degenerate oligonucleotide primers designed to detect adenosine receptors by the polymerase chain reaction, only one cDNA fragment homologous to the rat A2b-adenosine receptor was isolated from VA 13 cells. These results strongly suggest that RFL9 encodes the proposed A2b-adenosine receptor subtype. The identification of the cDNA for an A2b-adenosine receptor will allow more rigorous characterization of its anatomical distribution and functional properties.


The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions. The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.


Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNA- and ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligand-binding domain of the new protein and the DNA-binding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (100-250 microM).


Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.


3',5'-cyclic adenosine monophosphate-dependent transcription of the CYP11A (cholesterol side chain cleavage cytochrome P450) gene involves a DNA response element containing a putative binding site for transcription factor Sp1. The product of the CYP11A gene, cholesterol side chain cleavage cytochrome P450, catalyzes the initial step of steroidogenesis. A major mechanism whereby steroid hydroxylase gene transcription is regulated in the adrenal cortex requires the pituitary peptide hormone, ACTH, which acts via cAMP. We have previously identified a transcriptional enhancer in the 5'-flanking sequence [-183 to -83 base pairs (bp)] of the bovine CYP11A gene, which activates transcription of a beta-globin promoter/reporter gene in transiently transfected mouse Y1 adrenocortical tumor cells in response to the activator of adenylate cyclase, forskolin. Further deletion analysis has located the minimal cAMP-responsive sequence (CRS) to -118 to -100 bp. Analysis of DNA-protein interactions using nuclear extracts from Y1 cells revealed two protein binding sites, which were shown by competition analysis to be closely related to the two protein binding sites identified previously in the CRS of the human CYP21 gene. Namely, within the cAMP responsive fragment -118 to -100 bp, a sequence with a high degree of similarity to the consensus binding sequence for the ubiquitous transcription factor Sp1 is present, and binding of protein to this site was abolished by competition with excess GC box oligonucleotide. The second partially overlapping site is located 3' of the putative Sp1-binding site and binds to a protein identical or closely related to a putative adrenal-specific protein. Whereas the adrenal-specific protein binding site of the CYP21 CRS was previously shown to be sufficient to confer cAMP-responsive activation of transcription, the homologous site within the CYP11A CRS appears to have an attenuating effect on transcription.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter. The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE.(ABSTRACT TRUNCATED AT 400 WORDS)


3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line. The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.


Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane. The specific (i.e. nisoxetine-sensitive) binding of [3H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [3H]desipramine bound reversibly and with high affinity (KD = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (Bmax) was 2.1 pmol/mg protein. (2) Binding of [3H]desipramine was dependent on [Na+] and [Cl-]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake1) inhibited binding of [3H]desipramine with a rank order of potency typical for an interaction with the uptake1 carrier. The characteristics of [3H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin. The results indicate that the plasma membrane of bovine adreno-medullary cells is endowed with the neuronal uptake1 transporter.


Presynaptic A1-purinoceptor-mediated inhibitory effects of adenosine and its stable analogues on the mouse hemidiaphragm preparation. 1. The effect of adenosine or its stable analogues (2-chloroadenosine, CADO; 5'-N-ethylcarboxamidoadenosine, NECA; and N6-cyclopentyladenosine, CPA) on the release of [3H]-acetylcholine ([3H]-ACh), and on the development of force of contraction evoked by electrical stimulation of the nerve, were studied in the mouse phrenic nerve-hemidiaphragm preparation. Evidence was obtained that the release of ACh is subject to presynaptic modulation through presynaptic A1(P1)-purinoceptors. 2. Adenosine or its stable analogues (CADO, NECA, CPA) inhibited, in a concentration-dependent manner, both the release of ACh and the force of the indirectly elicited contraction of hemidiaphragm preparation, provided in the latter case that the margin of safety was reduced by (+)-tubocurarine or magnesium. The order of potency in reducing ACh release was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.08 +/- 0.01, 0.74 +/- 0.05, 9.05 +/- 0.20, and 410.2 +/- 42.5 mumol/l, respectively. The order of potency in reducing twitch tension was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.11 +/- 0.02, 0.48 +/- 0.03, 2.07 +/- 0.49, and 240.4 +/- 20.0 mumol/l, respectively. 3. 8-Phenyltheophylline (8-PT) antagonized the inhibitory effects of the adenosine receptor agonists on ACh release and twitch tension.(ABSTRACT TRUNCATED AT 250 WORDS)


Bilateral cerebral metabolic effects of pharmacological manipulation of the substantia nigra in the rat: unilateral intranigral application of the inhibitory GABAA receptor agonist muscimol. Rates of cerebral glucose utilization were measured by means of the autoradiographic 2-deoxy-D[1-14C]glucose technique in the rat brain in order to determine the metabolic effects of unilateral intranigral application of the GABAA agonist muscimol upon the substantia nigra and its targets. Intranigral injection of 1.5 microliters 0.3 M muscimol (52 micrograms total dose) induced local metabolic activation in the injected substantia nigra reticulata (by 87% as compared to the control group), and distal metabolic depressions in the nucleus accumbens, striatum, globus pallidus and subthalamic nucleus only ipsilaterally to the injected nigra. The remaining basal ganglia components, including the substantia nigra compacta and the entopeduncular nucleus were bilaterally unaffected. Among the principal efferent projections of the substantia nigra reticulata, the ventromedial and centrolateral thalamic nuclei as well as the deep layers of the superior colliculi were metabolically depressed bilaterally, whereas the ventrolateral, parafascicular and mediodorsal thalamic nuclei as well as the pedunculopontine nucleus displayed metabolic depressions ipsilateral to the muscimol-injection nigra. The ventromedial and centrolateral thalamic nuclei were depressed by 41 and 42%, respectively, in the ipsilateral side, and by 30 and 26% in the contralateral side, when compared to the respective values of the control group of rats. Furthermore, unilateral intranigral injection of 0.3 M muscimol induced metabolic depressions in reticular, intralaminar and prefrontal thalamocortical areas mostly ipsilateral to the injected nigra, as well as in limbic areas bilaterally. It is suggested that the present findings are due to a postsynaptic effect of muscimol on the nigral GABAergic cells and to a consequent metabolic depression of the basal ganglia and associated thalamocortical areas, in contrast to an earlier suggested presynaptic nigral effect of lower doses of intranigrally injected muscimol which induced metabolic activations within the same network. This suggestion is further supported by the fact that intranigrally injected substrate P19 induced similar effects to those elicited by the lower doses of intranigral muscimol and opposite to those induced at present by the higher muscimol dose. Moreover, it is further substantiated that the nigrothalamic GABAergic system is responsible for considerable transfer of information from one substantia nigra reticulata to the ipsilateral basal ganglia and associated thalamocortical components as well as to bilateral motor, intralaminar and limbic areas.


Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro. The purpose of the present study was to characterize the ionic and pharmacological basis of the actions of synaptically released and exogenously applied GABA in basolateral amygdaloid pyramidal cells in vitro. Stimulation of forebrain afferents to pyramidal neurons in the basolateral amygdala evoked an excitatory postsynaptic potential followed by early and late inhibitory postsynaptic potentials. The early inhibitory postsynaptic potential had a reversal potential near -70 mV, was sensitive to changes in the chloride gradient across the membrane and was blocked by the GABAA antagonists picrotoxin and bicuculline methiodide but not by the GABAB antagonists phaclofen or 2-hydroxysaclofen. In contrast, the late inhibitory postsynaptic potential had a reversal potential of approximately -95 mV and was markedly reduced or abolished by GABAB antagonists. Pressure application of GABA to the surface of the slice typically elicited a triphasic response in basolateral amygdaloid pyramidal neurons consisting of a short-latency hyperpolarization that preceded or was superimposed on a membrane depolarization followed by a longer latency hyperpolarization. Each of the responses was associated with an increase in membrane conductance. Determinations of the reversal potential, ionic dependency and sensitivity to pharmacological blockade of each component of the GABA-induced response revealed that the initial hyperpolarizing (Erev approximately -70 mV) and depolarizing (Erev approximately -55 mV) responses were mediated by a GABAA-mediated increase in chloride conductance, whereas the late hyperpolarizing response (Erev approximately -82 mV) to GABA arose from a GABAB-mediated increase in potassium conductance. Experiments in which GABA was applied at various locations on the cell suggested that the short-latency hyperpolarization resulted from activation of somatic GABA receptors, whereas the depolarizing and late hyperpolarizing responses were generated primarily in the dendrites. In contrast to the complex membrane response profile elicited by GABA, pressure ejection of the GABAB agonist baclofen produced only membrane hyperpolarizations. Taken together, these results suggest that inhibitory responses that are recorded in basolateral amygdaloid pyramidal cells are mediated by activation of both GABAA and GABAB receptors. Consistent with findings elsewhere in the CNS, the early inhibitory postsynaptic potential and initial hyperpolarization and depolarizing response to local GABA application appear to involve a GABAA-mediated increase in chloride conductance, whereas the late inhibitory postsynaptic potential and the late hyperpolarizing response to GABA arise from a GABAB-mediated increase in potassium conductance.


Glycine-containing terminals in the rat dorsal vagal complex. The present study examined the distribution of glycine and glycine-receptors in the dorsal vagal complex using pre-embedding immunocytochemistry. Glycine-immunoreactive terminals were present in moderate densities in the medial, intermediate, interstitial, commissural and ventrolateral subnuclei of the nucleus tractus solitarii. The dorsolateral nucleus tractus solitarii and the dorsal vagal motor nucleus contained only very few, scattered glycine-containing terminals. Glycine terminals appeared to be concentrated in regions of the dorsal vagal complex receiving primary vagal afferents, though previous studies have suggested that glycine is not present in these afferents. A conspicuously high concentration of glycine terminals was observed in the medial nucleus tractus solitarii where a population of cholinergic neurons has been identified previously. Ultrastructurally glycine immunoreactivity was principally associated with terminals containing flattened, pleomorphic vesicles and forming symmetrical synaptic contacts, mostly with dendrites. Glycine receptor immunoreactivity was present throughout the dorsal vagal complex with little evidence of subnuclear localization. With electron-microscopic examination, glycine receptor immunoreactivity was associated with dendritic membranes and was associated presynaptically with terminals containing flattened pleomorphic vesicles. Overall, the present data provide evidence consistent with a neurotransmitter role for glycine in the dorsal vagal complex. The presence of glycine in regions of the dorsal vagal complex receiving vagal afferents suggests a prominent role for this neurotransmitter in autonomic regulation.


Regulation of opioid binding sites in the superficial dorsal horn of the rat spinal cord following loose ligation of the sciatic nerve: comparison with sciatic nerve section and lumbar dorsal rhizotomy. The aim of the present study was to quantify time-related modifications in mu and delta opioid binding sites in the superficial layers (laminae I and II) of the L4 lumbar segment in a rat model of mononeuropathy induced by loose ligation of the sciatic nerve. We have shown a 28% (P < 0.01) and 24% (P < 0.01) decrease in ipsi/contralateral side binding ratios for tritiated (Tyr*-D-Ala-Gly-NMe-Phe-Gly-ol) ([3H]DAMGO) and tritiated (Tyr*-D-Thr-Gly-Phe-Leu-Thr) ([3H]DTLET) respectively, at two weeks postlesion which correspond to the delay of maximal hyperalgesia and of maximal alteration of fine diameter primary afferent fibers. In contrast, no change in [3H]U.69593 specific binding could be detected at this postlesion delay. For longer survival delays (four, eight and 15 weeks postlesion), mu and delta binding ratios return towards control values (approximately equal to 1), probably reflecting the occurrence of a long-term neuroplasticity (i.e. a new equilibrium in the metabolism of primary neurons, or collateral sprouting from intact primary afferents) following loose nerve ligation. In addition, a comparison of the results obtained in this model with those measured after sciatic nerve section and lumbar dorsal rhizotomy was performed in order to compare the degree of loss in opioid binding sites in these three types of lesion. The section of the sciatic nerve induced at eight days postlesion an 18% (P < 0.01) and 28% (P < 0.01) decrease in binding ratio for [3H]DAMGO and [3H]DTLET, respectively. At two weeks postlesion the loss was 24% (P < 0.01) for the two ligands, and at longer delays (four and 12 weeks), a progressive recovery in binding ratio was observed. Thus, it appears that both sciatic nerve lesions we have studied result in mu and delta binding modifications which have similar intensity and similar time course from two to 12-15 weeks postlesion. In contrast, the unilateral rhizotomy of nine consecutive dorsal roots (T13-S2), which is known to induce a massive degeneration of fine diameter primary afferent fibers, is followed by a dramatic decrease in binding ratios for [3H]DAMGO (53%, P < 0.001) and [3H]DTLET (45%, P < 0.001) at two weeks postlesion. These data suggest that the more deprived the dorsal horn is of fine diameter primary afferent fibers, the more dramatic is the opioid binding loss in the ipsilateral side as compared to the contralateral side.(ABSTRACT TRUNCATED AT 400 WORDS)


A formula for predicting the risk of a positive second-look laparotomy in epithelial ovarian cancer: implications for a randomized trial. To develop a formula to predict the risk of a positive second-look laparotomy. A retrospective review was performed on 89 patients who underwent second-look surgery following a complete clinical remission after cis-platin- or carboplatin-based chemotherapy. Logistic regression was used to develop a formula to predict the probability of a positive second look based on age, stage, grade of tumor, residual disease after initial surgery, and histologic type. We identified three groups based on estimated probabilities: low probability (0.25 or less), intermediate probability (0.26-0.74), and high probability (0.75 or more). The low-probability group had an 8% chance of a positive second look, the high-probability group had an 82% chance of a positive second look, and the intermediate-probability group had the correct outcome predicted only 61% of the time. Survival curves paralleled these results and were significantly different for each group. Using known prognostic factors, a formula can aid in implementation of a randomized clinical trial to test the efficacy of second-look laparotomy. This formula could exclude patients not suitable for randomization and give the investigator a better idea of the expected survival of various subgroups.