4-Aminopyridine and low Ca2+ differentiate presynaptic inhibition mediated by neuropeptide Y, baclofen and 2-chloroadenosine in rat hippocampal CA1 in vitro. 1. Presynaptic inhibition is mediated by several receptors at the stratum radiatum-CA1 synapse of rat hippocampus. We tested whether the same mechanism is activated by neuropeptide Y (NPY), baclofen and 2-chloroadenosine (2-CA), reasoning that if the receptors all activated the same process, then they should all respond to indirect manipulations of transmitter release in the same manner. 2. The effects on presynaptic inhibition by the potassium channel blocker, 4-aminopyridine (4-AP) and low extracellular concentrations of Ca2+ in the presence of 4-AP were compared using evoked population excitatory postsynaptic potentials (p.e.p.s.p.) responses in the rat hippocampal slice in vitro. 3. Log concentration-effect relationships for the inhibition of excitatory transmission were constructed for all 3 drugs in normal saline, and in the presence of 30 and 100 microM 4-AP. 4-AP reduced the inhibition mediated by all three substances, 100 microM 4-AP was only slightly more effective than 30 microM. 4. Lowering extracellular Ca2+ from 1.5 to 0.75 mM in the presence of 30 microM 4-AP restored the presynaptic inhibition caused by all effective concentrations of NPY and baclofen. By contrast, inhibition caused by 2-CA was not restored by lowering Ca2+, except at concentrations of 2-CA greater than 10 microM. 5. The results are consistent with the hypothesis that presynaptic NPY Y2 and GABAB receptors both inhibit transmitter release by the inhibition of voltage-dependent Ca2+ influx, but that the A1 adenosine receptor may activate a different presynaptic mechanism.


Interaction of pinaverium (a quaternary ammonium compound) with 1,4-dihydropyridine binding sites in rat ileum smooth muscle. 1. The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L-type) calcium channel blockers was investigated in rat ileum smooth muscle. 2. Pinaverium inhibited [3H]-(+)-PN200-110 ([3H]-(+)-isradipine) specific binding to tissue homogenates incompletely (Ki 0.38 microM; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin-treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [3H]-(+)-isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside-out vesicles. 3. Pinaverium bromide increased the apparent KD of [3H]-(+)-isradipine binding to saponin-treated homogenates but did not significantly affect the Bmax value. Moreover, the dissociation rate constant of [3H]-(+)-isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [3H]-diltiazem binding to rat brain membranes (at 30-37 degrees C). 4. Although Bmax values of [3H]-(+)-isradipine were similar in homogenates prepared from tissue and cells (collagenase-treated), the KD value was significantly higher in cell homogenates (166 vs 95 pM). Similarly, the Ki value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 microM). Thus, collagenase can significantly modify the dihydropyridine recognition site.5. The competitive interaction of pinaverium, a permanently charged drug, with [3H]-(+)-isradipine bound to intact cells and its absence of interaction with [3H]-(+)-isradipine bound to sealed inside-out vesicles imply that the dihydropyridine receptor lies near the external surface of the plasma membrane.


Peripheral and central delays in the cortical projections from human truncal muscles. Rapid central transmission of proprioceptive input from the hand but not the trunk. In contrast to the cortical connections to and from the muscles of the hand, the transmission of an afferent volley from the intercostal muscles to the cerebral cortex takes approximately 10 ms longer than it takes a cortical motor volley to reach the muscle. This disparity in afferent and efferent cortical transmission times could be due to a slower peripheral conduction velocity of intercostal muscle afferents or a slower afferent conduction within the central nervous system. The present study derived peripheral and central conduction times for the truncal muscles from the onsets of the mechanically evoked intercostal and abdominal spinal reflexes and the onsets of the cortical sensory potentials. Mean latencies of the ipsilateral intercostal and abdominal reflexes (evoked and recorded in the mid-clavicular line) were 11.9 +/- 0.7 (SEM) ms and 13.7 +/- 0.9 ms, respectively; calculated peripheral conduction velocities were 69.4 +/- 4.1 m/s and 56.2 +/- 2.3 m/s (assuming equal velocities for the sensory and motor axons and an intraspinal delay of 1 ms). Central sensory conduction time (spinal cord to cortex) was calculated by subtracting the peripheral conduction times for the intercostal and abdominal afferents (5.5 +/- 0.3 ms and 6.4 +/- 0.4 ms) from the onsets of the cortical sensory potentials (19.4 +/- 0.8 ms and 25.3 +/- 12.3 ms); central sensory conduction times (14.2 +/- 1.7 ms and 18.6 +/- 2.3 ms) were 8-11 ms longer than central motor conduction times. These results demonstrate that peripheral conduction velocities of intercostal and abdominal afferents are not slow, and that, when compared with the extremities, there is a relatively long central conduction time for proprioceptive information from the trunk to the cerebral cortex.


[Protective activity of monoacetone glucose 3-butyrate, prodrug of n-butyric acid, against the fatal effect of encephalomyocarditis virus in mice]. A comparative study was made, in the mouse, on antiviral properties of a prodrug of n-butyric acid derived from monosaccharides: monoacetone glucose 3-butyrate (MAG = 3but), and of a free form of n-butyric acid: arginine butyrate (BuO Arg). Preventive injection of MAG = 3but protected the mice twice as effectively as BuO Arg against the lethal effect of 100 LD50 of encephalomyocarditis virus. Under the same experimental conditions, monoacetone glucose (MAG) used as carrier of the biologically active moiety, was inactive on its own. Antiviral activity of MAG = 3but was shown not to be virucidal, but rather could involve stimulation of the immune system. This capability supplements known anticellular and antitumoral properties. In total these results indicate a prime therapeutic importance for this new molecule, pharmacokinetically suitable, with its very low toxicity, for clinical application. Combined use of MAG = 3but with biological response modifiers which have similar affinity, such as Interferon, is discussed.


A mouse model of coxsackievirus myocarditis. To develop a mouse model of coxsackievirus B3 (CVB3) myocarditis. Preliminary studies have indicated that mice infected with CVB3 alone erratically responded with viral myocarditis. Prospective evaluation of the effect of cyclophosphamide at two time intervals resulted in consistency for the development of myocarditis. Juvenile five- to nine-week-old male mice CD-1 type (Charles River Canada Limited). Infection with coxsackie B3 enterovirus. Pretreatment with cyclophosphamide two days and 4 h before inoculation of 0.2 or 0.15 mg/g. Animals were killed seven, nine, 28 and 63 days post infection. Following cyclophosphamide conditioning, a tissue response infection with CVB3 was uniformly observed. Virus, however, was infrequently recovered from the myocardium whereas myocarditis became chronic after 28 days. Pretreatment of juvenile mice with cyclophosphamide results in a reproducible model of viral myocarditis with chronic changes in the myocardium.


Prolonged supramaximal stimulation of canine efferent sympathetic neurons induces desensitization of inotropic responses without a change in myocardial beta-adrenergic receptors. To investigate whether desensitization of inotropic responses elicited during prolonged efferent sympathetic neural stimulation is due to decreased responsiveness of myocardial beta-adrenergic receptors or to alterations in the efferent sympathetic neurons innervating the heart. Increasing doses of noradrenaline and isoproterenol were administered intravenously before and during prolonged (20 mins) stimulation of the intrathoracic efferent sympathetic nervous system of eight dogs. Cardiac augmentor responses were correlated with liberation of catecholamines by the heart. In a second group of experiments (nine dogs), right and left ventricular beta-adrenergic receptor number and affinity were determined before and during such stimulations. Similar ventricular augmentations were induced when isoproterenol or noradrenaline was administered before and after 20 mins of efferent sympathetic neural stimulation. During the early peak stimulation response, no further augmentations were induced by isoproterenol or noradrenaline. Liberation of noradrenaline by the heart followed a similar course after an initial peak, while noradrenaline values fell to levels which were 6% of those attained during peak response after 20 mins of continuous stimulation. The Bmax and Kd of ventricular beta-adrenergic receptors were similar before and after 20 mins of efferent sympathetic neural stimulation. Desensitization of ventricular inotropism that occurs during prolonged cardiac efferent sympathetic nervous system stimulation is not primarily due to alteration of myocyte cell surface beta-adrenergic receptors or to a change in myocyte responsiveness to beta-adrenergic agonists, but rather to a reduction in noradrenaline release by sympathetic efferent post ganglionic neurons presumably reflecting a reduction in the activity of these neurons despite continued stimulation.


Biochemical and biological studies on 2-desamino-2-methylaminopterin, an antifolate the polyglutamates of which are more potent than the monoglutamate against three key enzymes of folate metabolism. Biochemical and biological studies have been carried out with 2-desamino-2-methylaminopterin (dmAMT), which inhibits tumor cell growth in culture but is only a weak inhibitor of dihydrofolate reductase (DHFR). Since it was possible that the species responsible for growth inhibition are polyglutamylated metabolites, the di-, tri-, and tetraglutamates of dmAMT were synthesized and tested as inhibitors of purified recombinant human DHFR, murine L1210 leukemia thymidylate synthase (TS), chicken liver glycinamide ribonucleotide formyltransferase (GARFT), and murine L1210 leukemia aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT). The compounds with three and four gamma-glutamyl residues were found to bind two orders of magnitude better than dmAMT itself to DHFR, TS, and AICARFT, with 50% inhibitory concentration values in the 200 to 300 nM range against all three enzymes. In contrast, at a concentration of 10 microM, dmAMT polyglutamates had no appreciable effect on GARFT activity. These findings support the hypothesis that dmAMT requires intracellular polyglutamylation for activity and indicate that replacement of the 2-amino group by 2-methyl is as acceptable a structural modification in antifolates targeted against DHFR as it is in antifolates targeted against TS. In growth assays against methotrexate (MTX)-sensitive H35 rat hepatoma cells and MTX-resistant H35 sublines with a transport defect, dmAMT was highly cross-resistant with MTX, but not with the TS inhibitors N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-ox-oquinazolin-6-yl)-N- methylamino]thenoyl)-L-glutamic acid, implicating DHFR rather than TS as the principal target for dmAMT polyglutamates in intact cells. On the other hand, an H35 subline resistant to 2'-deoxy-5-fluorouridine by virtue of increased TS activity was highly cross-resistant to N10-propargyl-5,8-dideazafolic acid and not cross-resistant to MTX, but showed partial cross-resistance to dmAMT. Both thymidine and hypoxanthine were required to protect H35 cells treated with concentrations of dmAMT and MTX that inhibited growth by greater than 90% relative to unprotected controls. In contrast, N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)-N-methylamino] thenoyl)- L-glutamic acid required only thymidine for protection. Like MTX, therefore, dmAMT appears to inhibit purine as well as pyrimidine de novo synthesis, and its effect on cell growth probably reflects the ability of dmAMT polyglutamates to not only block dihydrofolate reduction but also interfere with other steps of folate metabolism, either directly or indirectly via alteration of reduced folate pools.(ABSTRACT TRUNCATED AT 400 WORDS)


Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4'-(9-acridinylamino)methane-sulfon-m-anisidide. The effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.


Expression and regulation of retinoic acid receptors in human breast cancer cells. Retinoic acid is known to inhibit mammary carcinogenesis in rodents and to inhibit proliferation and steroid hormone receptor gene expression in human breast cancer cells. Since these effects are likely to be mediated by nuclear retinoic acid receptors (RARs) the present study was initiated to determine the expression and regulation of RARs in human breast cancer cell lines. Differential cellular gene expression of the RARs was determined by Northern blot analysis of total RNA prepared from 5 ER+ and 6 ER- cell lines. RAR alpha was detected as mRNA species of 2.7 and 3.4 kilobases in all cell lines and the level of gene expression was greater in ER+ cell lines (P less than 0.001). RAR beta mRNA (3.7 kilobases) was detected in seven of the eleven lines tested and was expressed most commonly in ER- cell lines. RAR gamma mRNA was expressed in all cell lines as a transcript of 3.4 kilobases at levels that were similar in both ER+ and ER- cell lines. Retinoic acid failed to regulate the expression of the RAR alpha and RAR gamma genes. The effect of steroid hormones on RAR alpha and RAR gamma mRNA levels was also examined. In four PR+ cell lines (T-47D, BT 474, MCF-7M, and MDA-MB-361), progestins markedly decreased RAR alpha mRNA levels. The progestin effect on RAR alpha levels in T-47D cells was detectable at concentrations of 0.05 nM and was maximal at 1 nM 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione ORG 2058, whereas dihydrotestosterone and dexamethasone were without effect. RAR alpha and RAR gamma mRNA levels were rapidly decreased by progestin, and the effect was maximal 3-6 h after ORG 2058 treatment. However, the mRNA loss was transient, and recovery of RAR alpha and RAR gamma mRNA levels was noted 12-24 h after retinoic acid treatment. Although RAR gamma mRNA returned to control levels by 24 h, the decrease in RAR alpha mRNA was maintained at around 50% control until at least 48 h. In summary, RAR alpha and RAR gamma were expressed in all human breast cancer cell lines and were regulated by progestins in the PR+ T-47D cell line. The previously reported ability of retinoic acid to down-regulate PR mRNA and the present demonstration that progestins down-regulate RAR alpha and RAR gamma mRNA suggest that mutual regulation may be a mechanism through which PR and the RARs interact in human breast cancer cells.


Time dependence of the selective modulation of cisplatin-induced nephrotoxicity by WR2721 in the mouse. 2-(3-Aminopropylamino)ethylphosphorothioic acid (WR2721; ethiofos) was shown to selectively protect nontumor tissues from cis-diamminedichloroplatinum(II) (cisplatin)-induced toxicity, when administered 30 min prior to the platinum drug. Selectivity of protection by WR2721 is probably due to the preferential formation and uptake of the thiol metabolite 2-(3-aminopropylamino)ethanethiol (WR1065), which can inactivate toxic platinum-species inside the cell. We investigated the protective potential of WR2721, when administered at different time points relative to cisplatin. BALB/c mice treated with WR2721 (200 mg/kg i.p.) either 30 min or 5 min prior to cisplatin (i.p.) allowed a 2.2-fold increase in cisplatin dose to 19 mg/kg before the occurrence of nephrotoxicity as expressed by an increase in plasma urea. A small part of the protection could be ascribed to the mannitol (200 mg/kg), present in the formulated WR2721. WR2721 (200 mg/kg) 30 min after 14.5-16-mg/kg cisplatin did not offer any protection against the rise in plasma urea. WR2721 (200 mg/kg) 5 min before 19-mg/kg cisplatin did not cause liver toxicity (increase in serum glutamic pyruvic transaminase or serum glutamic oxaloacetic transaminase). Furthermore, WR2721 (200 mg/kg) 5 min prior to cisplatin did not reduce antitumor activity in nude mice bearing well-established human ovarian cancer xenografts. Under protection of WR2721, the dose of cisplatin could be increased by a factor of 1.6 to 8 mg/kg (administered twice weekly), resulting in an increased antitumor activity.


Cytogenetic analysis of 124 prospectively ascertained male germ cell tumors. We report the cytogenetic analysis of 124 adult male germ cell tumors ascertained consecutively at the Memorial Sloan-Kettering Cancer Center between 1988 and 1990. Biopsies from testicular and extragonadal primary and metastatic lesions studied included all histological subtypes of germ cell tumors and cases of malignant transformation. Nonrandom numerical and structural chromosomal abnormalities including i(12p), the previously described characteristic marker of these tumors, were determined, and their frequency was compared between histological subtypes, between gonadal and extragonadal lesions, and between primary and transformed lesions. The frequency and copy number of i(12p) were found to be higher in nonseminomas compared with seminomas. Nonrandom sites of chromosome rearrangements associated with specific histologies comprised 1p32-36 and 7q11.2 in teratomas and 1p22 in yolk sac tumors. Some tumors that underwent malignant differentiation exhibited chromosome changes previously described to be nonrandomly associated with de novo tumors with the same histological characteristics. Cytological evidence of gene amplification in the form of homogeneously staining regions and/or double minutes was detected in 24% of extragonadal lesions, mainly metastatic tumors, suggesting amplification of a gene(s) associated with metastatic progression of these tumors. While a number of previous small cytogenetic series or individual case reports of germ cell tumors identified several of the features of these tumors reported here, this series comprises analysis of the largest group of tumors ascertained consecutively at a single institution, defines the incidence of nonrandom abnormalities in tumor subsets, and addresses their biological significance.


First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids SM4s-Gal and SM4g, from a patient with lung cancer. Several human monoclonal antibodies directed to tumor-associated glycolipid antigens have been established, but more than one-half of them react with gangliosides and the others react with neutral glycolipids. We report here the first establishment of a human IgM monoclonal antibody directed to the sulfated glycolipid. This monoclonal antibody, M14-376, did not react with SM3 and SB1a which have a terminal HSO3----3Gal beta 1----R1, but with the simple sulfolipids SM4s-Gal and SM4g which contain a terminal HSO3----3Gal beta 1----O----CH2----R2; however, lyso-SM4s-Gal and lyso-SM4g did not bind M14-376. These results suggest that terminal HSO3----3Gal and part of the hydrophobic region of the glycolipid are recognized by M14-376. Directly biotinylated M14-376 was used for immunohistochemical staining of 140 formalin-fixed, paraffin-embedded lung cancer tissue sections to study the distribution of the antigen. A high incidence of positive staining was found in adenocarcinoma (39.5%, 17 of 43), followed by large cell carcinoma (20.0%, 5 of 25), while this antigen was rarely detected in small cell carcinoma (4.7%, 1 of 21) and squamous cell carcinoma (3.9%, 2 of 51). Thin layer chromatography immunostaining of glycolipids extracted from lung cancer tissues showed the presence of only SM4s-Gal in adenocarcinoma, but SM4g was not found in any subtype of lung cancer. Immunohistochemical staining revealed that this antigen was expressed in normal kidney, testis, and brain, but erythrocytes, granulocytes, and lymphocytes were negative in cytofluorometric analysis.


Prognostic influence of HSP-27 expression in malignant fibrous histiocytoma: a clinicopathological and immunohistochemical study. Malignant fibrous histiocytoma is a very aggressive sarcoma. After the tumor has disseminated, chemotherapy is of little influence on the course of the disease because of the resistance to most chemotherapy regimens. We evaluated by immunohistochemistry the prognostic influence of the expression of a member of the stress polypeptides family, the heat-shock protein of 27 KDa (HSP-27). HSP-27 was found to be associated with an aggressive behavior in breast carcinoma and was related to chemoresistance in cell cultures. Forty-three malignant fibrous histiocytomas with no evidence of metastases at the time of diagnosis and resected between 1974 and 1985 were retrieved from the files of the Pathology Department of L'Hôtel-Dieu de Québec hospital. The immunostaining was performed on Bouin-fixed, paraffin-embedded tissue. Regardless of the percentage of positive cells, HSP-27 was expressed in the cytoplasm of 25 (58.1%) cases. HSP-27 expression was associated with a more favorable prognosis, and a significant correlation was observed with overall survival (P less than 0.025) and metastasis-free survival (P less than 0.05). HSP-27 expression was found to be the strongest prognostic factor, and multivariate analysis revealed that it was independent of tumor size, necrosis, and histological subtype. However, in the 13 patients with recurrent disease who underwent chemotherapy, the antigenic expression did not help to predict the treatment response. HSP-27 expression is one of the rare prognostic markers in this tumor type.


Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment. The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the "critical" cysteine residue, were added at 30 microM concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier; control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.


Role of the phosphoprotein (P) in the encapsidation of presynthesized and de novo synthesized vesicular stomatitis virus RNA by the nucleocapsid protein (N) in vitro. Encapsidation of presynthesized and nascent (synthesized de novo) vesicular stomatitis virus (VSV) leader RNA in vitro by the nucleocapsid protein (N) and the role of the phosphoprotein (P, previously known as NS) in this process were examined. Presynthesized VSV leader RNAs were derived from the SP6 transcription vectors containing both (+) and (-) leader genes while the nascent RNA was derived from transcription of viral ribonucleoprotein (RNP) complex. The N and the P proteins were made by transcription from SP6 vectors containing the genes, followed by translation of the mRNAs in rabbit reticulocyte lysate. Here, we demonstrate that the N protein alone encapsidated presynthesized VSV leader RNA; however, prior formation of N-P complex totally abolished the encapsidation property of N. On the other hand, encapsidation of nascent RNA by the N protein was stimulated by the N-P complex. These results suggest that encapsidation by the N protein of presynthesized and nascent VSV RNA are separate biochemical processes which can be distinguished by the differential role of the phosphoprotein P in the two reactions.


Sumatriptan in the treatment of acute migraine with aura. The efficacy of the selective 5HT1-like agonist sumatriptan in acute treatment of classical migraine (i.e. migraine with aura) was assessed in a double-blind, placebo-controlled, parallel group randomized trial. An oral dose of 200 mg was chosen on the basis of the efficacy rates achieved (70-85%) with 70-280 mg in open studies (1, 2). The dose of 200 mg was also chosen for the study because preliminary data from an oral pilot study indicated that efficacy increased with increasing dose up to 200 mg. Each patient was treated for a maximum of three separate attacks of migraine with aura within a three months' period. Three attacks were treated so that we could examine consistency of response across more than one attack. For attack 1, 200 mg sumatriptan was significantly more effective, safe and well tolerated than placebo at relieving headache 2 h after treatment was given (p = 0.023). In subsequent attacks, i.e. in attacks 2 and 3, there was no such significant effect of sumatriptan compared with placebo in relieving headache. This reduced efficacy of sumatriptan in the second and third attacks may be due to a high incidence of vomiting induced by the high dose of dispersible formulation and also by the bitter taste of the tablets. In addition, there was an increase in placebo response in attacks 2 and 3 compared to the first attack.


The collagen shield as a collagenase inhibitor and clinical indicator of collagenase activity on the ocular surface. Collagen shields applied to the corneas of patients with bacterial keratitis degrade rapidly, often within a few hours. Once treatment brings the infection under control, subsequently applied collagen shields degrade more slowly. In vitro models were established to evaluate the significance of these observations. Twenty-four and 72-hour collagen shields were incubated with collagenase from Clostridium histolyticum. The in vitro rate of digestion of the shields was directly proportional to the concentration of collagenase, with the rate of digestion of the 24-hour shields being greater than that of the 72-hour shields. Therefore, the rate of collagen shield degradation may be a clinically useful index of collagenase activity on the ocular surface. Ultrastructural studies of collagen shields from patients with acute bacterial keratitis revealed irregular degradation of shield matrix with no evidence of adherence of microorganisms or inflammatory cells. Co-incubation of deepithelialized rabbit corneas and collagen shields resulted in inhibition of the digestion of the rabbit corneas when the weight:weight ratio of collagen shield:rabbit cornea was increased to greater than or equal to 2:1. Collagen shields may inhibit corneal collagen degradation in infectious ulceration and melting disorders by effectively competing for collagenase on the ocular surface.


Thyrotoxicosis and hyperemesis gravidarum associated with a serum activity which stimulates human thyroid cells in vitro. To investigate in-vitro thyroid stimulatory activity in the serum of patients with hyperemesis gravidarum and thyrotoxicosis. Serum from hyperthyroid patients was incubated with cultures of human thyroid cells. Attempts were made to neutralize stimulatory activity with antisera to hCG. Five patients presenting in early pregnancy with hyperemesis and thyrotoxicosis. Serum concentrations of thyroid hormones (total and free), TSH and hCG. Accumulation of extracellular cAMP in response to serum. All five patients had biochemical hyperthyroidism with no evidence of an underlying autoimmune disease. The mean cAMP accumulation over 4 hours with sera from 12 non-pregnant controls was 130.6 (121.8-142.8), from 12 pregnant controls 132.4 (118.1-143.8), compared with values of 144.7, 159.1, 166.2, 178.9 and 320.5 for the thyrotoxic patients. The stimulatory activity could not be neutralized by addition of anti-hCG or by depleting the sera of hCG. Thyrotoxicosis may present with hyperemesis in early pregnancy. Clinical and biochemical features may be masked by the pregnancy or by the intercurrent illness. The hypothesis that hCG is a thyroid stimulator in patients with hyperemesis gravidarum is not supported by the immuno-neutralization data in this study.


The radioprotective effects of WR-2721 on radiation-induced goblet cell loss. We assessed the efficacy of the radioprotective effects of WR-2721 in radiation-induced goblet cell loss. Fourteen female New Zealand white rabbits were divided into radioprotected, radiounprotected, and control groups. Ten rabbits received 5,000 rad of beta irradiation to the temporal conjunctiva of both eyes. The right eyes were pretreated with topical applications of WR-2721 30 minutes and 1 minute before irradiation. The left eyes received irradiation only. Four additional rabbits received neither irradiation nor pretreatment with WR-2721. After a 2-month waiting period, each animal was killed and goblet cell densities per high-power field (GCD/HPF) were determined over the areas in question and compared using paired sample t tests. The mean GCD/HPF for the radioprotected, unprotected, and control groups were 10.93, 4.68, 23.15 (right eye), and 26.25 (left eye). Paired sample t tests showed a significant radioprotective effect when comparing WR-2721-treated animals with those receiving radiation only (p less than 0.003). Independent sample t tests showed significant goblet cell loss in both the radioprotected (p less than 0.018) and radiounprotected eyes (p less than 0) when compared with control groups. These data suggest a radioprotective effect of WR-2721 in radiation-induced goblet cell loss.


The evaluation of therapeutic responses in experimental keratomycosis. Two different measures of response to therapy were evaluated in a model of keratitis caused by Aspergillus fumigatus in Dutch-belted rabbits. Combined pre and post-inoculation treatment with oral fluconazole 37.5 mg/kg bid or itraconazole 40 mg/kg bid was compared to post-inoculation treatment only and untreated controls using a standardized clinical disease severity score and quantitative isolate recovery techniques. For both drugs, there was no difference in isolate recovery rates among all three groups. However, a significant improvement in clinical disease was noted in the pre and post-inoculation treatment group compared to controls (p less than .01) and to the post-inoculation group (p less than .05) for fluconazole. A similar trend, though not statistically significant, was apparent with itraconazole treatment. This disparity highlights the difficulties associated with measuring responses to therapy in keratomycosis and emphasizes the need for more sensitive and specific measures.


Protection from retinal necrosis by passive transfer of monoclonal antibody specific for herpes simplex virus glycoprotein D. Passive administration of antibody against herpes simplex virus type 1 (HSV-1) has been shown to protect against stromal keratitis and death from encephalitis. Although the exact mechanism by which passively-transferred antibody protects is not known, one of the features of protection by passively-transferred antibody is interference with the ability of the virus to spread within the nervous system. In the experiments reported herein, studies were performed to determine if 8D2, a monoclonal antibody against a type-common epitope of glycoprotein D, could protect mice from retinal necrosis following uniocular anterior chamber inoculation of HSV-1. Mice were protected from retinal necrosis when the antibody was administered 2 hours before virus inoculation or 24 hours after virus inoculation. When antibody was injected 2 hours before virus inoculation, the titer of virus at day 1 p.i. in the injected eyes of antibody-treated and control mice was the same, but by 3 days p.i., the titer of virus in the antibody-treated mice was significantly lower than that recovered from control mice. The titers of virus in the brains and in the uninoculated eyes of antibody-treated mice were also significantly lower than in control mice. The results of these studies suggest that passively-transferred antibody protects against retinal necrosis by limiting spread of virus to the CNS or replication of virus within the CNS.


Effects of cyclic nucleotide analogs on intraocular pressure and trauma-induced inflammation in the rabbit eye. In this study the effects of cell-permeable 8-bromo-cAMP and 8-bromo-cGMP on intraocular pressure (IOP) and puncture-induced inflammatory response were investigated. Both 8-bromo-cAMP and 8-Bromo-cGMP reduced IOP when given subconjunctivally, but not topically. Subconjunctival administration of 8-bromo-cAMP induced a moderate disruption of the blood-aqueous barrier (BAB); in addition, subconjunctival 8-bromo-cAMP, but not topical 8-bromo-cAMP or subconjunctival 8-bromo-cGMP, reduced the disruption of the BAB and elevation of the aqueous PGE2 level after puncture trauma. It is concluded that the effects of 8-bromo-cAMP depend on the mode of administration, since this determines the concentration of 8-bromo-cAMP reached in the aqueous humor. It is suggested that 8-bromo-cAMP can partially suppress a slight inflammatory response by interference with the release of arachidonic acid from the tissues surrounding the aqueous humor.


Protective effects of anti-glycoprotein D monoclonal antibodies in murine herpetic keratitis. The protective effects of passive immunization with two kinds of anti-glycoprotein D (anti-gD) monoclonal antibodies, having different antiviral activities, were investigated in murine herpetic keratitis. One monoclonal antibody, designated M1, had high virus-neutralizing antibody titers, along with undetectable levels of complement-dependent cytolysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC); the other, designated M12, exhibited extremely low titers of virus-neutralization with high level of CDC and ADCC. When systemically administered 24 hours prior to virus inoculation to the cornea, both M1 and M12 almost completely prevented the development of stromal keratitis. The protective efficacy of both was observed to be dose-dependent. Pepsin-treated M1 retained its efficacy in suppressing stromal keratitis, whereas pepsin-treated M12 did not. When the administration of M1 and M12 were delayed, both provided significant (but less complete) protection, up to 24 hours after virus inoculation. These results suggest that both virus neutralization and CDC/ADCC play an important role in preventing virus growth in the corneal stroma during the early stage of corneal infection.


[Flow cytometric analysis in non-small-cell bronchial carcinoma and its prognostic significance]. Tumor and lymph node infiltration, and the DNA-ploidy status of a tumor contain prognostic information in addition to the information obtained by histological examination of surgical samples. Specimens from 112 patients with non-small-cell lung carcinoma obtained immediately after surgery were investigated by means of flow cytometry. DNA-aneuploidy was found in 43% of the primary tumors. Independent from tumor stage, patients with DNA-euploid tumors lived significantly longer (p less than 0.01) than with DNA-aneuploid carcinomas. In 29 cases the DNA-ploidy status of the primary tumor (PTU) could be compared with that of the N2 lymph node metastases (LM). 7 samples revealed a change from DNA aneuploidy in the PTU to DNA-euploidy in the LM. Patients with DNA-euploid PTU and DNA-euploid LM lived significantly longer than patients with DNA-aneuploid PTU/DNA-euploid LM, and patients with DNA-aneuploid PTU/DNA-aneuploid LM. In case of lymph node infiltration only the simultaneous measurement of DNA ploidy of PTU and LM offers an accurate prognostic evaluation. Local tumor recurrence exhibited stability of DNA ploidy, showing DNA euploidy in 12 out of 13 PTU and their corresponding recurrent tumor. Thus, the DNA-ploidy status offers additional prognostic informations which is useful for an extended tumor classification.


[The ileoanal reservoirs as a rectal replacement. The late complications and long-term functional results]. Late complications and functional results in 44 consecutive patients (17 women, 27 men; mean age 36 years) were collected prospectively after continence-preserving proctocolectomy for ulcerative colitis. In all patients a J-pouch was placed by direct end-to-side machine-stapled anastomosis without rectal cuff. 5 years postoperatively 12 of 13 (92%) were totally continent during the day, 11 of 13 (85%) at night. Stool frequency at that time was 6.3 +/- 2 per 24 hours; 4 patients (31%) had on average one motion during the night. Lasting sexual dysfunction occurred in three men. Fistulae, as a result of previously undiagnosed Crohn's disease, developed postoperatively in three patients: between pouch and vagina in one, between pouch and bladder in another. The pouch had to be removed in both instances. In the third patient an enterocutaneous fistula was closed. If strict indications are followed, the clinical and functional results of a pelvic ileoanal reservoir meet the daily demands of life at both a professional and social level.


Streptokinase is a flexible multi-domain protein. The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.


Variation in dietary fibre, beta-glucan, starch, protein, fat and hull content of oats grown in Sweden 1987-1989. Samples of oat groat from cultivars grown in Sweden during 3 years (1987-1989, 50 samples/year) were investigated. On an average, the kernels contained 15.9% protein, 7.0% fat, 9.7% dietary fibre and 63.2% starch (% of dry matter). The coefficient of variation was highest for fat (26%) and lowest for starch (7%). The thousand kernel weight was on average 34.7 g. The content of dietary fibre and fat was significantly negatively correlated and the starch positively correlated with the thousand kernel weight. The samples contained mean (and range) 9.7% (5.0-13.4) total dietary fibre, 3.5% (2.0-5.0) soluble dietary fibre, and 4.6% (3.5-5.7) beta-glucan. The beta-glucan content was significantly positively correlated with insoluble and total dietary fibre as well as with the fat content, and significantly negatively correlated with starch.


The Scottish Heart Health Study. Dietary intake by food frequency questionnaire and odds ratios for coronary heart disease risk. II. The antioxidant vitamins and fibre. High serum antioxidant vitamins are increasingly being associated with reduced risk of coronary heart disease (CHD). Previous studies have not addressed the relationship between dietary antioxidant vitamins and risk of CHD although diet is a key factor which modifies blood antioxidant vitamin levels. In prospective studies, high-fibre diets have also been associated with reduced CHD incidence. In this analysis CHD-diagnosed, -undiagnosed and non-CHD controls were selected from 10,359 men and women aged 40-59 who participated in a cross-sectional study of CHD risk factors. Diet was assessed by food frequency questionnaire, odds ratios were adjusted for the classical CHD risk factors (+/- social class) and calculated relative to the first quintile for each vitamin and total fibre. The antioxidant vitamins were further combined in a principal component analysis and the odds ratios for undiagnosed and diagnosed CHD were again calculated. For undiagnosed CHD, risk was significantly lower in the highest quintiles of beta-carotene, fibre and vitamin C, E and A for men, but only lower for fibre in women. Opposite trends were observed in the odds ratios for vitamin C and E and fibre for male-diagnosed CHD which possibly indicates changes in diet as a result of diagnosis. Principal component analysis showed significantly reduced risk of undiagnosed CHD in the top three quintiles for men (odds ratios 0.66, 0.67 and 0.64; P less than 0.05 in each case). A similar trend occurred for women but was non-significant. The results suggest that high dietary intake of the antioxidant vitamins may reduce risk of CHD, particularly in men, and that fibre may be equally cardio-protective in both sexes.


An eight-month controlled study of a low-fat high-fibre diet: effects on blood lipids and blood pressure in healthy young subjects. The effects on blood lipids and blood pressure of a diet corresponding to present Nordic Nutrition Recommendations, i.e. less than 30% of energy from fat and with a fibre content exceeding 3 g/MJ, were studied in 18 men and 12 women (mean age, 24 years) under strict dietary control over 8 months. Blood sampling, blood pressure and body weight measurement were performed at four occasions on their habitual diet and once a month during the intervention period. An age-matched control group (17 men, 8 women) was followed with monthly measurements parallel to the intervention group. The habitual diets, assessed by 7-day records, showed an average fat content corresponding to 36% of energy. Initial levels of total cholesterol and HDL cholesterol (X +/- SD) were 4.21 +/- 0.61 and 1.23 +/- 0.23 mmol/l for the men in the intervention group; 4.35 +/- 0.79 and 1.21 +/- 0.26 mmol/l for the male controls; 4.61 +/- 0.59 and 1.46 +/- 0.31 mmol/l for the women in the intervention group and 4.48 +/- 0.64 and 1.48 +/- 0.29 mmol/l for the female controls. Significantly decreased levels of total cholesterol and HDL cholesterol throughout the experimental period were seen for both sexes in the intervention group. Total cholesterol fell 0.49 mmol/l (95% CI: 0.41-0.56) in the male subjects and 0.49 mmol/l (95% CI: 0.39-0.59) in the female subjects. The fall in HDL cholesterol was 0.16 mmol/l (95% C: 0.13-0.18) and 0.18 mmol/l (95% CI: 0.12-0.23), respectively. Total cholesterol changes were independent of initial values. All subjects were normotensive at the start of the study with an average blood pressure of 122/68 mmHg for men and 112/68 mmHg for the women. Systolic blood pressure dropped gradually and significantly in the male subjects of the intervention group. A minimum of 6 mmHg below initial values was noted after six months of dietary intervention. No significant changes in dietary intake and blood lipids were observed in the control group. Thus, changes of present dietary habits of young healthy Danish subjects to an intake in accordance with the Nordic Nutrition Recommendations 1989 will favourably affect suggested risk factors for disease.


The influence of rice bran on plasma lipids and lipoproteins in human volunteers. Eighteen healthy male normocholesterolaemic men consumed diets supplemented with 15 g wheat bran, 15 g and 30 g rice bran for 3-week treatment periods. Plasma lipoprotein concentrations were determined on the last 3 days of each treatment period. Nutrient intakes estimated by analysis of duplicate diets were similar during all treatments. Plasma triglyceride concentrations were statistically significantly (P less than 0.05) lowered by 15 g/day rice bran compared with 15 g/day wheat bran. Plasma total cholesterol, low density lipoprotein and high density lipoprotein cholesterol, apoprotein AI and B concentrations and body weight were unaffected by the supplements. It is concluded that the consumption of realistic amounts of rice bran does not contribute to a reduction in plasma cholesterol concentrations in man.


The effects of sugar-beet fibre supplementation for five weeks on zinc, iron and copper status in human subjects. The present study was conducted to evaluate the effects of fibre supplementation on zinc, iron and copper status in human subjects. Ten males (53 +/- 8 years of age) participated in this study which consisted of three phases: baseline-1 period (2 weeks) in which subjects were on their normal, habitual dietary intake, followed by a period of fibre supplementation (5 weeks) in which subjects were supplemented with 26 g dietary fibre/d, and baseline-2 period (4 weeks) in which fibre supplement was withdrawn. Parametric measurements of zinc, iron and copper status were conducted on weeks 1,2 (zero-time), 7 and 11. Results showed that fibre supplementation for 5 weeks did not cause any significant change in the status of zinc (measured by concentration of zinc in plasma and urine and alkaline phosphatase activity), iron (measured by packed cell volume (PCV%), HB, transferrin saturation % and ferritin), or copper (measured by plasma copper concentration and erythrocyte superoxide dismutase activity). We conclude that consumption of sugar-beet fibre added to the daily diet does not constitute any risk with respect to zinc, iron and copper nutriture.


Beriberi cardiomyopathy.

Beriberi cardiomyopathy. In Indonesia beriberi is still endemic, but subclinical cases are not uncommon. Three patients suffering from beriberi presented with different clinical manifestations. One had the classical features of Shoshin beriberi and the other two had the non-alcoholic cardiac beriberi (chronic type). The cardiac symptoms of all three patients responded dramatically to thiamine tetrahydrofurfuryl disulfide; there was also some improvement of their polyneuropathy, consistent with the neurophysiologic findings and somatosensory evoked potentials (SSEPs). We conclude that SSEPs provide additional clinical information on beriberi polyneuropathy. The mortality of untreated cardiovascular beriberi is high. In view of the harmless nature of the treatment, a good case could be made for routine administration of thiamine to all patients in whom heart failure is present without clear evidence of the cause.


Effect of endothelin-1 in man: pretreatment with nifedipine, with indomethacin and with cyclosporine A. The rise in blood pressure following the intravenous administration of endothelin-1 remained unchanged in healthy male volunteers pretreated with either the calcium-channel antagonist nifedipine (10 mg orally), the cyclo-oxygenase inhibitor indomethacin (150 mg day-1 for three days) or the immunosuppressive agent cyclosporine (5 mg kg-1 body weight for five days). Following administration of nifedipine the rise in plasma concentrations of endothelin-1 during the infusion of the peptide was markedly higher (P less than 0.01) than during control experiments without nifedipine. It is concluded that, in healthy men, nifedipine, indomethacin and cyclosporine do not exert a major influence on the pressor action of endothelin-1. However, nifedipine apparently influences the elimination of endothelin-1 from the circulation in healthy men.


Mast cells in the anterior uvea of the rabbit. Intraocular effects of compound 48/80 in the rabbit. In the present study, the presence and localization of mast cells and the intraocular effects of compound 48/80 have been studied in detail in the rabbit eye using histochemical and physiological methods. In histochemical studies mast cells were localized in the anterior uvea, especially in the ciliary and iridial processes. Intracamerally injected, compound 48/80 caused an increase in the intraocular pressure, disruption of the blood-aqueous barrier and an increase in the cAMP content in the aqueous humour. Miosis was observed only after higher doses of compound 48/80 (greater than 100 micrograms) and even then only one-half of the eyes responded. The intraocular effects, excluding miosis, of compound 48/80 resembled an on/off-type of response, where 20 micrograms caused only minor changes, if any, and 50 micrograms gave a maximal response. The ocular hypertensive reaction developed a tachyphylaxis so that the second and third consecutive dose of compound 48/80 (100 micrograms) produced no significant change in IOP. The results indicate that mast cells, which are present in the anterior uvea in an extent not known previously, might be involved in certain inflammatory reactions in the rabbit eye. The inconsistent and slight miosis after the intracameral application of compound 48/80 indicates that the mechanism is different from that caused by sensory nerve stimulation. The rapid development of tachyphylaxis after consecutive application of compound 48/80 suggests that mast cells are easily depleted which might be useful for further studies to evaluate the functional role of mast cells in different pathophysiological conditions in the rabbit eye.


Effects of estrogen and fimbria/fornix transection on p75NGFR and ChAT expression in the medial septum and diagonal band of Broca. NGF receptor-expressing cells located in the basal forebrain have recently been shown to contain estrogen (E) receptors (Toran-Allerand and MacLusky. 1989. Soc. Neurosci. Abstr. 15: 954). In the present study, we have examined the effects of E-treatment on p75NGFR and choline acetyltransferase (ChAT) expression by neurons in the medial septum (MS) and the vertical (VDB) and horizontal (HDB) limbs of the diagonal band of Broca using immunocytochemical and in situ hybridization techniques. First, since E-treatment has been shown to affect neuronal survival and to stimulate synaptic reorganization and growth within various regions of the brain, we hypothesized that E-treatment might attenuate the loss of p75NGFR immunoreactivity (IR) which occurs in the MS and VDB following transection of the fimbria/fornix. Contrary to our hypothesis, E-treatment did not attenuate the effects of fimbria/fornix transection. In fact, E-treatment alone produced a significant decrease in the number of p75NGFR-IR cells detected in the MS. Subsequent experiments confirmed that chronic E-treatment produces a down-regulation of both p75NGFR-IR and p75NGFR mRNA in the MS and VDB. In the MS, estrogen appeared to affect a subpopulation of p75NGFR-expressing neurons which were also affected by fimbria/fornix transection since the effects of these two treatments were not additive. In addition, effects of E-treatment on p75NGFR-IR were sex-specific (observed in females but not in males) and were reversible in the MS after 2 weeks, but not after 4 weeks (allowing 2 weeks recovery), of E-treatment. A time-course analysis revealed that effects of E-treatment on p75NGFR-IR were not observed until after 16 days (MS) or 30 days (VDB) of E-treatment and were preceded by a significant and transient increase in ChAT expression in both the MS and VDB. The data are consistent with the possibility that continuous, long-term exposure to gonadal steroids may contribute to a loss of p75NGFR-expressing neurons with age. In addition, the data suggest that p75NGFR expression may play a role in regulating the functioning of specific basal forebrain cholinergic neurons. Different mechanisms by which E-treatment might influence ChAT and p75NGFR expression in brain are discussed.


PML/RAR-alpha rearrangement in acute promyelocytic leukaemias apparently lacking the t(15;17) translocation. Recent investigations have clarified some of the molecular mechanisms underlying the t(15;17) translocation specific for acute promyelocytic leukaemia (APL). Together with providing new insights into the pathogenesis of the disease, the identification of breakpoints within the RAR-alpha and PML loci on chromosomes 17 and 15 has allowed a new relevant diagnostic tool for the recognition of this leukaemic form. We report the molecular characterization of 6 cases of acute myelogenous leukaemia (AML) in which a diagnosis of typical M3 by conventional morphocytochemistry (FAB criteria) was not accompanied by cytogenetic evidence of the specific t(15;17) aberration. DNA rearrangements were documented in all cases at the PML and RAR-alpha loci. Moreover, in 4 cases also analysed by Northern blot hybridization, we could detect aberrant RAR-alpha transcripts. These findings highlight the specificity of PML/RAR-alpha rearrangements in APL, whereas the lack of t(15;17) may be attributed to sub-microscopic translocations as well as to the presence of non-neoplastic cells undergoing mitosis in the samples examined for karyotype.


Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4) contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.


Direct induction of G1-specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis. In Saccharomyces cerevisiae, the genes encoding the HO endonuclease, G1-specific cyclins CLN1 and CLN2, as well as most proteins involved in DNA synthesis, are periodically transcribed with maximal levels reached in late G1. For HO and the DNA replication genes, cell cycle stage-specific expression has been shown to be dependent on the Cdc28 kinase and passage through START. Here, we show that cells released from cdc28ts arrest in the presence of cycloheximide show wild-type levels of induction for HO, CLN1, and CDC9 (DNA ligase). Induction is gradual with a significant lag not seen in untreated cells where transcript levels fluctuate coordinately with the cell cycle. This lag may be due, at least in part, to association of the Cdc28 peptide with G1 cyclins to form an active kinase complex because overexpression of CLN2 prior to release in cycloheximide increases the rate of induction for CDC9 and HO. Consistent with this, release from pheromone arrest (where CLN1 and CLN2 are not expressed) in cycloheximide shows no induction at all. Transcriptional activation of CDC9 is likely to be mediated through a conserved promoter element also present in genes for other DNA synthesis enzymes similarly cell cycle regulated. The element contains an intact MluI restriction enzyme recognition site (consensus approximately 5'-A/TPuACGCGTNA/T-3'). Insertion of a 20-bp fragment from the CDC9 promoter (containing a MluI element) upstream of LacZ confers both periodic expression and transcriptional induction in cycloheximide following release from cdc28ts arrest. High levels of induction depended on both the MluI element and CDC28. These results suggest that the activity of trans-acting factors that operate through the MluI element may be governed by phosphorylation by the Cdc28 kinase.


A mouse cdc25 homolog is differentially and developmentally expressed. The timing and activation of the p34cdc2 kinase in mammals is associated with dephosphorylation of phosphotyrosine and phosphothreonine residues on the p34cdc2 kinase. For fission yeast, the timing of mitosis is regulated by cyclic accumulation of cdc25, which promotes dephosphorylation of p34cdc2 and concomitant protein kinase activation. We report the identification and characterization of a structural and functional mouse homolog, Cdc25M2, of the cdc25 phosphatase. Cdc25M2 shows high sequence identity to the previously reported human homolog cdc25Hu2. Cdc25M2 can functionally complement for a Schizosaccharomyces pombe cdc25ts mutation, and when expressed in Escherichia coli and purified, Cdc25M2 is an active phosphatase. cdc25M2 mRNA shows variation in expression in different tissues in the mouse embryo and is expressed in a developmental and cell-cycle-dependent fashion. We suggest that the expression and accumulation of the cdc25 mitotic inducer may play a critical role in the regulation of mouse development.


Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression. Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.


Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry. We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.


Reduced expression of manganese superoxide dismutase in cells resistant to cytolysis by tumor necrosis factor. Tumor necrosis factor (TNF) induces synthesis of manganese superoxide dismutase (MnSOD). It was previously shown that overexpression of MnSOD protected some mammalian cells from TNF cytotoxicity. The purpose of this study was to establish whether MnSOD was increased in cells selected for resistance to cytolysis by TNF in combination with cycloheximide. Melanoma SK-MEL-109 and HeLa cell-resistant variants were selected by repeated treatments with TNF and cycloheximide. The SK-MEL-109 variants had relatively low levels of MnSOD that were inducible by TNF. Surprisingly, the HeLa variants had very low levels of MnSOD that were poorly inducible by either TNF or interleukin-1 alpha. Therefore, an elevated level of MnSOD was not required to protect these cells from TNF-mediated cytolysis. The HeLa variants were more sensitive than parental cells to superoxide radical (O2-) generating compounds, such as paraquat or xanthine/xanthine oxidase. Pretreatment of these variants with TNF did not provide protection against damage by superoxide radicals.


Stabilities of hydroxyl radical spin adducts of PBN-type spin traps. The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.


[Signal transduction of prostaglandin E2 and thromboxane A2]. The signal transduction of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), cyclooxygenase products of arachidonic acid, was investigated in smooth muscle preparations and 1321N1 human astrocytoma cells. While PGE2 has been known to stimulate (via EP2 receptor) or inhibit (via EP3 receptor) adenylate cyclase, PGE2 activated phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLase C) in non-vascular smooth muscles (via EP1 receptor), resulting in accumulations of inositol trisphosphate (IP3) and diacylglycerol to elicit intracellular Ca2+ mobilization. On the other hand, STA2, a TXA2 receptor analogue, also accumulated IP3 in human astrocytoma cells. [3H]SQ 29548, a TXA2 receptor antagonist, specifically bound to astrocytoma membranes. TXA2-receptor antagonists (ONO NT-126, S-145, SQ29548 and ONO3708) concentration-dependently inhibited PIP2-specific PLase C activation by STA2, and they also inhibited [3H]SQ 29548 binding in human astrocytoma cells. The Ki value of each antagonist in PIP2-specific PLase C inhibition was similar to that in [3H]SQ29548 binding inhibition. In membrane preparations, STA2 activated PIP2-specific PLase C in the presence of GTP gamma S. Pertussis toxin (IAP) did not affect STA2-induced PLase C activation. The results suggest that stimulation of TXA2 receptors activates PIP2-specific PLase C via an IAP-insensitive G-protein.


[Central stimulating effect of the combination of the new quinolone group of antimicrobials and nonsteroidal anti-inflammatory drugs in mice]. Six new quinolones: enoxacin, norfloxacin, ofloxacin, ciproflosacin, lomefloxacin, and tosufloxacin and eight nonsteroidal anti-inflammatory drugs: fenbufen, flurbiprofen, ketoprofen, pranoprofen, ibuprofen, indomethacin, mefenamic acid and aspirin were tested for their ability to produce a central stimulating effect in mice. At 5 min after the oral administration of one of the nonsteroidal anti-inflammatory drugs, a new quinolone was administered orally. The combination of drugs induced convulsions in a dose-dependent manner, and some mice died as a result of the convulsions. The survival time was used as an index to measure the intensity of convulsions induced by the drug combination. The new quinolones in combination with fenbufen at 100 mg/kg produced convulsions in the following order of potencies: enoxacin greater than lomefloxacin greater than norfloxacin. In contrast, administration of fenbufen together with ofloxacin, ciprofloxacin, or tosufloxacin up to a dose of 1000 mg/kg caused no convulsions. Four nonsteroidal anti-inflammatory drugs combined with enoxacin at 100 mg/kg also caused convulsion dose-dependently. The order of potency in producing convulsion was as follows: fenbufen greater than flurbiprofen greater than ketoprofen = pranoprofen. However, no convulsions were produced by treatment of ibuprofen, indomethacin, mefenamic acid or aspirin together with enoxacin. From these results, the important chemical structures of the new quinolones particularly concerned with the appearance of convulsion were discussed.


[Effects of the combination of new quinolones and a nonsteroidal anti-inflammatory drug, fenbufen, on the EEG of rabbits]. A combination of fenbufen, a non-steroidal anti-inflammatory drug and the new quinolone produces a central stimulating action. To confirm the action, we used 6 kinds of new quinolones: enoxacin, norfloxacin, ofloxacin, ciprofloxacin, lomefloxacin and tosufloxacin in this experiment. The convulsive effects of these drugs were tested on the EEG recorded from the neocortex and subcortical regions of the rabbits. Animals treated with fenbufen (50-200 mg/kg, p.o.) tended to have a high amplitude slow wave in their EEG. Rabbits treated with the new quinolones at the dose of 100 mg/kg, p.o., with the exception of tosufloxacin, also tended to show a high amplitude slow wave in their EEG. Each new quinolone given 30 min after fenbufen (50 mg/kg, p.o.) elicited characteristic spikes on the EEG. Then, high-frequency-spikes and epileptiform seizure waves appeared for a long experimental period with this combination. The combination of fenbufen and tosufloxacin (100-400 mg/kg, p.o.) caused no changes in EEG and behavior. The spike and epileptiform wave could be suppressed only temporarily with diazepam (1-4 mg/kg, i.v.). These results suggest that combined use of fenbufen and one of the new quinolones, except for tosufloxacin, produces the seizure. Not only GABA but also several other mechanisms in the central nervous system may be involved in the convulsion.


[Mechanism of the anti-osteoarthritic action of ulinastatin in comparison with those of indomethacin and triamcinolone]. We investigated the effect of ulinastatin, a candidate anti-osteoarthritic drug, in comparison with indomethacin and triamcinolone, two well-known drugs for osteoarthritis, on IL-1 production by monocytes, proteoglycan synthesis by chondrocytes and superoxide generation by leukocytes. Ulinastatin, a glycoprotein purified from human urine, suppressed both the IL-1 production and the IL-1 induced reduction of proteoglycan synthesis. In addition, ulinastatin inhibited superoxide generation. These actions of ulinastatin seemed to be related to its inhibitory actions against serine proteases such as trypsin, alpha-chymotrypsin, plasmin, leukocyte elastase and leukocyte cathepsin G. Triamcinolone suppressed the IL-1 production more potently than ulinastatin and it also suppressed the IL-1 induced reduction of proteoglycan synthesis. Triamcinolone alone, however, reduced the proteoglycan synthesis, and it did not affect the superoxide generation. In contrast, indomethacin had no effect on proteoglycan synthesis and superoxide generation, although it accelerated the IL-1 production. These results indicate that these three drugs have different mechanisms of action on the factors involved in the pathogenesis of osteoarthritis. Since ulinastatin has broad actions, which are considered to be beneficial for preventing some process of osteoarthritic pathogenesis, ulinastatin is expected to be an useful drug for the treatment of osteoarthritis.


[Physiological analyses of secretory kinetics of adrenocorticotropic hormone (ACTH) from anterior pituitary cells: development and application of a microperfusion system]. A microperfusion system was developed to study detailed kinetics of adrenocorticotropic hormone (ACTH) secretion by dispersed rat anterior pituitary cells responding to various ACTH secretagogues. The system approaches hydrodynamics to square-wave stimuli and enables kinetic analysis of ACTH secretion with intervals as short as 5 sec. ACTH secretion initiated within 5 sec of exposure of the cells to corticotropin-releasing factor (CRF), arginine vasopressin (AVP), oxytocin (OT) or angiotensin II (A-II) and reached a maximum within 20-40 sec. CRF induced a plateau-shaped secretion of ACTH which remained constant as long as CRF was perifused. In contrast, the ACTH secretion responding to AVP, OT and A-II rose rapidly to a peak and fell to the baseline despite continued perifusion of these agents. There were two components of ACTH secretory response to AVP and OT. AVP had synergistic effect with CRF only if it was perifused simultaneously with CRF or immediately after CRF was stopped. The ACTH secretory response to A-II was greatly diminished when cells were exposed to AVP or OT before A-II perifusion. Prior exposure to A-II had no effect on the magnitude of the ACTH secretory response to either AVP or OT. Epinephrine, nor-epinephrine, gastrin-releasing peptide, atrial natriuretic factor and cholecystokinin stimulated no significant ACTH secretion in the microperfusion system, although some of them induced ACTH secretion by same cell preparation in static culture systems.(ABSTRACT TRUNCATED AT 250 WORDS)


The nature of breast dense core granules: chromogranin reactivity. Ultrastructural immunoreactivity for chromogranin with the LK2H10 antibody was examined in nine cases of breast carcinoma and one example of normal resting breast. These tissues were selected for study on the basis of argyrophilia or LK2H10 immunostaining by light microscopy. In two cases, positive reactivity with the chromogranin antibody was seen in breast neuroendocrine-like dense core granules. In a further six cases chromogranin reactivity was not seen in similar granules that showed the neuroendocrine properties of ultrastructural argyrophilia and uranaffinity. Inability to exhibit the full complement of neuroendocrine characteristics in breast carcinomas contrasts with the facility to demonstrate them in tissues of usual neuroendocrine differentiation. Furthermore, in two mucoid carcinomas and one example of normal resting breast ultrastructural cytoplasmic LK2H10 reactivity was evident, which was not localized to dense core granules, although these were present in two of the cases. Our findings demonstrate the heterogeneity of breast dense core granules and discourage acceptance of such characteristics as evidence of histogenesis of these carcinomas from a neuroendocrine cell type in breast tissue.


Malignant rhabdoid tumour of the liver. A case report. A case of primary malignant rhabdoid tumour of the liver occurring in a 5-month-old girl is reported. Histologically the neoplasm presented the 'typical' features as described in malignant rhabdoid tumour arising in the kidney. In addition, some areas of the tumour showed a 'pseudoglandular' growth pattern. Immunohistochemistry revealed the neoplastic cells to be intensively positive for vimentin and for cytokeratin 19. Variable immunoreactivity for cytokeratin polypeptides 7, 8 and 19 was also detected. On electronmicroscopy the neoplastic cells contained bundles of intermediate filaments, tonofilaments and intercellular junctions. These findings are suggestive of a primitive epithelial differentiation.


Micro-invasive breast carcinoma with granulomatous stromal response. Two cases of micro-invasive breast carcinoma with granuloma formation associated with invasive foci are presented. This is an unusual feature which may aid in the recognition of stromal micro-invasion. Micro-invasive carcinomas are defined as predominantly non-invasive tumours in which there are one or more foci of infiltration, none measuring more than 1 mm in maximum diameter.


Assignment of the beta-subunit of rod photoreceptor cGMP phosphodiesterase gene PDEB (homolog of the mouse rd gene) to human chromosome 4p16. The gene encoding the beta-subunit of rod photoreceptor cGMP phosphodiesterase (gene symbol PDEB, homolog of the mouse rd gene) is mapped to human chromosome 4 using somatic cell hybrids and further localized to the chromosome band 4p16 using in situ hybridization. A mutation in the mouse gene underlies the recessive trait of retinal degeneration in the rd mouse. Thus, the human homolog is a candidate for lesions causing retinal degeneration.


Use of an enzyme-linked immunosorbent assay for detection of infection with pseudorabies virus on a herd basis. The use of an ELISA that can differentiate between swine infected with pseudorabies virus (PRV) and swine vaccinated with a specific PRV vaccine was evaluated on an individual and herd basis, and a system for interpreting ELISA results on a herd basis was developed. In 17 herds, recently introduced replacement gilts, seronegative for PRV, were vaccinated with a thymidine kinase- and glycoprotein X (gpX)-deleted vaccine. After vaccination, blood samples were collected from these gilts approximately every 1 to 2 months for up to 19 months. Serum samples were analyzed for antibodies to gpX antigen, using a commercially available ELISA kit according to the manufacturer's protocol. Herd status was determined as positive, suspect, or negative, according to the serum sample:negative control (S:N) values of the samples collected from the herd. From the 17 herds, 130 evaluations were performed. On 49 (38%) of the 130 herd evaluations, 1 or more gilts had suspect test results. Additional testing was required in 19 (39%) of these 49 herd evaluations to determine the PRV infection status of the herd. Status of herds having gilts with suspect results and no positive results was usually negative after retesting. Herds having gilts with positive results were unlikely to have negative status after retesting.


Compounds that increase cAMP prevent ischemia-reperfusion pulmonary capillary injury. This study evaluated the physiological effects of compounds that increase adenosine 3',5'-cyclic monophosphate (cAMP) on changes in pulmonary capillary permeability and vascular resistance induced by ischemia-reperfusion (I-R) in isolated blood-perfused rabbit lungs. cAMP was elevated by 1) beta-adrenergic stimulation with isoproterenol (ISO, 10(-5) M), 2) post-beta-receptor stimulation of adenylate cyclase with forskolin (FSK, 10(-5) M), 3) and dibutyryl cAMP (DBcAMP, 1 mM), a cAMP analogue. Vascular permeability was assessed by determining the capillary filtration coefficient (Kf,c), and capillary pressure was measured using the double occlusion technique. The total, arterial, and venous vascular resistances were calculated from measured pulmonary arterial, venous, and capillary pressures and blood flow. Reperfusion after 2 h of ischemia significantly (P less than 0.05) increased Kf,c (from 0.115 +/- 0.028 to 0.224 +/- 0.040 ml.min-1.cmH2O-1.100 g-1). These I-R-induced changes in capillary permeability were prevented when ISO, FSK, or DBcAMP was added to the perfusate at reperfusion (0.110 +/- 0.022 and 0.103 +/- 0.021, 0.123 +/- 0.029 and 0.164 +/- 0.024, and 0.153 +/- 0.030 and 0.170 +/- 0.027 ml.min-1.cmH2O-1.100 g-1, respectively). I-R significantly increased total, arterial, and venous vascular resistances. These increases in vascular resistance were also blocked by ISO, FSK, and DBcAMP. These data suggest that beta-adrenergic stimulation, post-beta-receptor activation of adenylate cyclase, and DBcAMP prevent the changes in pulmonary vascular permeability and vascular resistances caused by I-R in isolated rabbit lungs through a mechanism involving an increase in intracellular levels of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)


Follistatin is a developmentally regulated cytokine in neural differentiation. Activin acts mitogenically on P19 cells as well as being inhibitory of the differentiation of retinoic acid-treated P19 cells and some neuroblastoma cell lines. Here, we show some lines of evidence that follistatin, an activin-binding protein, is also involved in neural differentiation. Counteracting the activity of activin, addition of follistatin suppresses the anchorage-independent growth of P19 cells in soft agar and stimulates neurite outgrowth of a neuroblastoma cell line, IMR-32 cells. While activin does not seem to be expressed significantly, follistatin is demonstrated in the conditioned medium of these cells. Furthermore, the expression of follistatin in P19 cells is subject to dynamic fluctuations in response to retinoic acid treatment. These neural cells may produce follistatin in a cell stage-specific manner in order to interact with exogenously derived activin.


Phosphatidic acid is a specific activator of phosphatidylinositol-4-phosphate kinase. The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.


Truncation of the cytoplasmic tail of the lutropin/choriogonadotropin receptor prevents agonist-induced uncoupling. An agonist-induced change in the functional properties of a constant number of receptors seems to be a ubiquitous phenomenon involved in the regulation of cell surface receptors. Although the mechanisms responsible for this phenomenon (called uncoupling or desensitization) have been studied in detail using beta 2-adrenergic receptors it is unclear if the models derived from these studies are applicable to other members of the family of G protein-coupled receptors. Since it has been shown previously that truncation of the C-terminal cytoplasmic tail of the beta 2-adrenergic receptor results in a delay in the onset of agonist-induced uncoupling (Bouvier, M., Hausdorff, W.P., De Blasi, A., O'Dowd, B.F., Kobilka, B.K., Caron , M.G., and Lefkowitz, R.J. (1988) Nature 333, 370-373), we now present experiments designed to test the effects of a similar truncation of the lutropin/choriogonadotropin (LH/CG) receptor on its functional properties. The results presented herein show that (i) clonal lines of human embryonic kidney cells stably transfected with cDNAs encoding for the wild-type (rLHR-wt) or a mutant receptor truncated at amino acid residue 631 (rLHR-t631) express functional LH/CG receptors as judged by their ability to bind hCG and to respond to it with increased cAMP accumulation; (ii) a preincubation of the cells expressing rLHR-wt with hCG leads to a reduction in the ability of hCG to activate adenylylcyclase; and (iii) this reduction is severely blunted in cells expressing rLHR-t631. These results demonstrate that the C-terminal cytoplasmic tail of the LH/CG receptor is necessary for agonist-induced uncoupling.


Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin. Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.


The nickel site in active Desulfovibrio baculatus [NiFeSe] hydrogenase is diamagnetic. Multifield saturation magnetization measurement of the spin state of Ni(II). The magnetic properties of the nickel(II) site in active Desulfovibrio baculatus (DSM 1743) [NiFeSe] hydrogenase have been measured using the multifield saturation magnetization technique. The periplasmic [NiFeSe] hydrogenase was isolated from bacteria grown in excess selenium in the presence of 57Fe. Saturation magnetization data were collected at three fixed fields (1.375, 2.75, 5.5 tesla) over the temperature range from 2 to 100 K. Mössbauer and EPR spectroscopies were used to characterize the magnetic state of the two [4Fe-4S] clusters of the enzyme and to quantitate the small amounts of iron impurities present in the sample. The nickel(II) site was found to be diamagnetic (low spin, S = 0). In combination with recent results from extended x-ray absorption fine structure studies, this magnetic state indicates that the nickel(II) site of active D. baculatus [NiFeSe] hydrogenase is five-coordinate.


Liver-specific expression of the gene coding for human factor X, a blood coagulation factor. Factor X is a vitamin K-dependent glycoprotein that plays an essential role in both the intrinsic and extrinsic pathways of blood coagulation. Studies on a recombinant lambda phage containing the 5'-flanking region of the human factor X gene showed that the factor X gene was linked to and was located at the 3' end of the factor VII gene: the initiation codon of the factor X gene was 2823 base pairs (bp) downstream from the polyadenylation site of the factor VII gene. This 2.8-kilobase intergenic region, and progressively deleted fragments of it, was fused to the chloramphenicol acetyltransferase gene, and transient expressions in HepG2 cells, human fibroblasts, and Chinese hamster ovary cells were measured. A liver-specific promoter element, FXP1-binding site, essential for hepatocyte-specific transcription was identified. This promoter sequence, further localized to -63 to -42 bp in DNase I footprint studies, was homologous to LF-A1 or hepatic nuclear factor-4 recognition sequence and was equally functional in the normal and inverse orientations. FXP1 site bound to nuclear protein(s) from HepG2 cells and complex formation was partially abolished by the presence of duplex oligonucleotides containing liver factor-A1 or hepatic nuclear factor-4-binding sequences. Two additional positive elements located upstream of the promoter region, spanning from -215 to -149 bp (FXP2 site), and -457 to -351 bp (FXP3 site), were also established by reporter gene assays.


Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.


Residues in the longitudinal, hydrophobic strip-of-helix relate to terminations and crossings of alpha-helices. An alpha-helix terminates when the virtual extension of its most hydrophobic, longitudinal strip containing Leu, Ile, Val, Phe, and Met lacks those residues. In each of 247 helices a template was fitted to maximize the mean hydrophobicity of  positions forming a longitudinal strip-of-helix. The template was then extended into sequences beyond the ends of the helices. Leu, Ile, Val, Phe, and Met occurred in  positions in the longitudinal strip-of-helix at an increased frequency (p less than 0.001), but in the first and second  positions beyond either end of each true helix, they occurred at the same frequency as for their empirical distribution over all the proteins. Excesses of Asp and Glu were found in the N-terminal loop, and of Arg, His, and Lys in specific positions about the C terminus of helices. The longitudinal hydrophobic strip, the smallest amino acid in that strip, and charged amino acids in that strip, related to rotational and longitudinal orientation of alpha-helices in 15 proteins. Adjacent helices generally crossed through their longitudinal hydrophobic strips. They usually crossed through the smallest residue in the strip. Charged residues, when they occurred in the strips, were excluded from the crossing regions.


Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex. The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.


Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction. Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.


Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue. Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.


Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor. Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.


Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme. Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.