Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes.

The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein-Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1-6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV-positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.


Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34.

Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.


Band 3 Tuscaloosa: Pro327----Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2.

Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56----Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic phenotype.


Nucleotide sequence of a 2 kb plasmid from Pseudomonas cepacia implicated in the degradation of phenylcarbamate herbicides.

The complete nucleotide sequence of a very small plasmid whose presence and level in Pseudomonas cepacia have been linked to herbicide resistance is presented. The structural features of the plasmid are discussed.


Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.


Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.


Alternative secondary structures in the phage G4 origin of the complementary DNA strand synthesis: effects of NaCl concentration on the bleomycin-DNA interaction.

The effects of NaCl concentration on bleomycin-induced cleavages of single-strand and double-strand DNA fragments containing the phage G4 origin of complementary DNA strand synthesis were investigated. It was found that bleomycin could be used as a reagent to analyze secondary and tertiary structures and subtle changes of DNA structures. The effects of NaCl concentration on cleavages of single-stranded DNA were distinct at every target site, indicating that the diversity of topolotical properties of DNA might change the selectivity of the bleomycin-induced DNA cleavage. These results showed alternative secondary structures within and close to the G4 origin of complementary DNA strand synthesis.


[Experimental research on the effective mechanism of jianweiling].

The purpose of this study is to find out the effective mechanism of Jianweiling (JWL) in treating some gastrointestinal (GI) diseases. The functions of GI movement, bile and pancreatic secretion and intestinal absorption were measured after giving JWL to the experimental rats. The results showed that JWL could adjust GI movement once it was in abnormal conditions. When the gastrointestine was in paralysis under the influence of abdominal operation, JWL could make GI myoelectric activity return to normal; and JWL could relax it when the gastrointestine was in a cramp state resulted from Neostigmini Methylsulfurici injection. In addition, the pancreatic secretion, the amylase activity in pancreatic juice and the intestinal absorption for D-xylose in JWL group were obviously better than those of the control groups. These results suggested that the effective mechanism of JWL on some GI diseases can be realized by adjusting and promoting GI functions in various ways.


Defects in thymocyte differentiation and thymocyte-stromal interactions in the trisomy 16 mouse.

We have examined fetal thymic development in the trisomy 16 (Ts16) mouse, which is considered to be a model for human trisomy 21, or Down Syndrome. The Ts16 thymus contains 10 to 20% of the number of lymphocytes found in a normal thymus at a comparable stage. Expression of thymocyte differentiation markers (Thy-1, CD5, CD8, CD4, CD3, and HSA) is severely affected in Ts16 fetuses aged 14-18 gestational days. When thymuses from 14-day Ts16 mice were cultured in vitro, these markers eventually reached levels of expression comparable to those seen in normal thymuses in culture. On the other hand, expression of CD44 appears to be unaffected in Ts16 thymuses in vivo, but declines in vitro relative to normal thymuses. Reconstitution of depleted thymic stroma with thymocytes showed evidence of defects in both developmental compartments.


Comparison of immunocytochemical and Holzer's methods for detection of acute and chronic gliosis in human postmortem material.

Immunocytochemical staining for glial fibrillary acidic protein (GFAP) allows more specific identification of astrocytes and their processes than classical histochemical techniques and has therefore recently been used by some investigators to quantify gliosis. However, although the immunocytochemical method is superior for delineation of reactive astrocytes, the examples presented here and previous work by others demonstrate that chronic fibrillary gliosis may be best detected by Holzer's method and not by GFAP immunocytochemistry. The authors' studies indicate that if, as in a recent study of gliosis in schizophrenia, computer-assisted densitometry is to be used to measure gliosis, the immunoperoxidase method may not be a sensitive technique to demonstrate glial changes in human postmortem material.


Indications for aspirin as a palliative for tinnitus caused by SOAEs: a case study.

This paper explores the effect of aspirin on the tinnitus of one patient for whom two contralateral spontaneous otoacoustic emissions (SOAEs) caused binaural tinnitus. The relation between the SOAEs and tinnitus was explored during a preliminary testing session, after which the SOAEs were measured for 7 days. During days 1, 2, 5, 6 and 7 of the trial, a placebo (two 50-mg tablets of ascorbic acid) was administered four times per day. During days 3 and 4, a drug (two 300-mg tablets of aspirin) was administered four times per day. During day 2, the right ear's SOAE was low level and labile, sometimes disappearing into the noise floor. The effect of aspirin on an emission which is not consistently observed, cannot be assessed so this report focuses primarily on the left ear. During days 1 and 2, the SOAE in the left ear was present and the tinnitus was audible. By day 3 (after 2.4 g of aspirin), the SOAE in the left ear had been abolished, and the tinnitus was not audible. On the fifth day (24 hours after the last aspirin), both the SOAE and the tinnitus in the left ear had returned. There were no reported side-effects of the aspirin. Thus, aspirin seemed to provide an acceptable palliative for this patient's SOAE-caused tinnitus.


The non-peptide tachykinin antagonist, CP-96,345, is a potent inhibitor of neurogenic inflammation.

1. Release of the tachykinin, substance P, from the peripheral terminals of polymodal afferent C-fibres is thought to be largely responsible for the vasodilatation and plasma protein extravasation described as neurogenic inflammation. The effects of CP-96,345, a non-peptide antagonist at the substance P (NK1) receptor, on these vascular reactions were investigated in the rat. 2. Intravenously (i.v.) injected CP-96,345 (0.4-3.0 mumol kg-1) prevented the drop in blood pressure, a measure of the peripheral vasodilatation, evoked by substance P and neurokinin A in a dose- and time-dependent manner, but did not affect that elicited by the non-tachykinin peptides calcitonin gene-related peptide and vasoactive intestinal polypeptide. 3. Plasma protein extravasation evoked by i.a. infusion of substance P, antidromic stimulation of the saphenous or the vagus nerve, and stimulation of cutaneous afferent nerves with mustard oil, were each significantly inhibited by CP-96,345 (3.0-9.0 mumol kg-1, i.v.). Furthermore, CP-96,345 was orally active in blocking mustard oil-induced plasma extravasation with an ED50 of 10 mumol kg-1. 4. The inhibition of substance P-induced vasodilatation and of neurogenic plasma extravasation by CP-96,345 was stereospecific as the inactive isomer CP-96,344 (2R, 3R enantiomer of CP-96,345) had no effect. 5. Thus CP-96,345 is a specific, highly potent, long-acting and orally active inhibitor of tachykinin-mediated neurogenic inflammation.


Induction and potential biological relevance of a Ca(2+)-independent nitric oxide synthase in the myocardium.

1. We have investigated whether the myocardium and isolated cardiac myocytes can express a Ca(2+)-independent NO synthase after treatment with endotoxin or cytokines. Nitric oxide synthesis was measured in cytosols from the left ventricular wall from rats treated with endotoxin, or from freshly isolated myocytes from adult rats treated in vitro with cytokines. 2. Cytosols from the ventricle of saline-treated control animals showed only Ca(2+)-dependent NO synthesis. After treatment with endotoxin, the expression of an inducible, Ca(2+)-independent NO synthase was observed. The activity of this enzyme was maximal at 6 h and returned towards control levels by 18 h; no alterations occurred in the Ca(2+)-dependent NO synthase activity. Parallel to this enzyme induction there was an increase in myocardial guanosine 3':5'-cyclic monophosphate (cyclic GMP) and plasma nitrite and nitrate (NOx-). All these changes were prevented by pretreatment of the rats with dexamethasone. 3. Myocytes possessed Ca(2+)-dependent NO synthase activity and expressed, after treatment with tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), a Ca(2+)-independent NO synthase, the induction of which was prevented by dexamethasone and cycloheximide. 4. Since increases in cyclic GMP levels in the heart are associated with reduced myocardial contractility, it is possible that the enhanced production of NO by a Ca(2+)-independent enzyme accounts, at least in part, for the depression of myocardial contractility seen in septic shock, cardiomyopathies, allograft rejection, burn trauma, as well as during anti-tumour therapy with cytokines.


Mechanism of action of MY-1250, an active metabolite of Repirinast, in inhibiting histamine release from rat mast cells.

1. When MY-1250 (3.6 x 10(-5) M) was added to mast cells, it caused a rapid increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP) and decrease in adenosine 5'-triphosphate (ATP), both of which recovered to their original levels within 2 min. The accumulation of cyclic AMP was maximal at 20 s after challenge with MY-1250. The minimum level of ATP was observed at 20 s after addition of MY-1250. 2. The initial rise in [Ca2+]i and the histamine release induced by DNP-AS antigen (40 micrograms ml-1) was most strongly inhibited at 20 s after incubation of the mast cells with MY-1250. 3. MY-1250 strongly and dose-dependently inhibited the histamine release from rat mast cells induced by antigen. Moreover, MY-125 strongly inhibited calcium ion mobilization from the intracellular Ca(2+)-store. 4. These results suggested that the inhibitory mechanism of MY-1250 on the initial rise in [Ca2+]i and histamine release induced by antigen was due to the inhibition of ATP-dependent Ca(2+)-release from the intracellular Ca(2+)-stores.


The role of cyclic AMP in non-adrenergic non-cholinergic contraction in guinea-pig bronchi.

1. We investigated the role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in non-adrenergic non-cholinergic (NANC) contraction in guinea-pig bronchial strips. 2. Forskolin (3 nM to 1 microM) reduced NANC contraction induced by electrical field stimulation (EFS) in a concentration-dependent fashion (-log EC50 was 7.22 +/- 0.12 M and maximum inhibition was 100 +/- 0.01%). However, forskolin (less than 1 microM) did not alter the contraction induced by substance P (SP, 1 microM). 3. Dibutyryl cyclic AMP (1 mM) also reduced NANC contractions induced by EFS (100 +/- 0.01%) without significant effect on SP (1 microM)-induced contractions. In contrast, dibutyryl cyclic GMP (1 mM) was without effect against either NANC or SP-induced contractions. 4. Both the beta 2-adrenoceptor agonist, procaterol (0.1 nM to 3 nM) and theophylline (100 nM to 1 mM) concentration-dependently reduced EFS-induced NANC contractions without significant effect on SP (1 microM)-induced contractions. 5. In contrast to forskolin, procaterol and theophylline, both sodium nitroprusside and cromakalim inhibited the EFS-induced contractions only at those concentrations that similarly reduced the contractions induced by SP (1 microM). 6. These results suggest that cyclic AMP may mediate pre-junctional inhibition of NANC contractions in guinea-pig bronchi.


Pharmacological and histological examinations of regional differences of guinea-pig lung: a role of pleural surface smooth muscle in lung strip contraction.

1. Parenchymal lung strip preparations have been widely used as an in vitro model of peripheral airway smooth muscle. The present study examined functional responses of 4 consecutive guinea-pig lung parenchymal strips isolated from the central region (segment 1) to the distal edge (segment 4) of the lower lung lobe. The middle two segments were designated as segments 2 and 3. 2. Lung segments 1 and 4 exhibited significantly greater contraction than the other 2 segments to KCl when responses were expressed as mg force per mg tissue weight. Contractile responses to bronchospastic agents including histamine, carbachol, endothelin-1, leukotrienes (LT) B4 and D4, and the thromboxane A2-mimetic U46619 demonstrated no significant difference in EC50 values among the 4 lung segments. 3. Contractile responses of segments 1 and 4 to antigen-challenge (ovalbumin), ionophore A23187 and substance P were significantly greater than the other 2 segments with respect to either sensitivity or maximum responsiveness. 4. U46619-induced contractions of the 4 lung segments were relaxed in similar manner by papaverine and theophylline up to 100%, salbutamol up to 80%, and sodium nitroprusside by only 20%. In contrast, sodium nitroprusside markedly reversed U46619-induced contraction of pulmonary arterial rings and bronchial rings. 5. Histological studies identified 2-4 layers of smooth muscle cells underlying the lung pleural surface. Mast cells were prominent in this area. Moreover, morphometric studies showed that segment 4 possessed the least amount of smooth muscle structures from bronchial/bronchiolar wall and vasculatures as compared to the other 3 segments, and a significant difference in this respect was evident between segment 1 and segment 4.6. Since lung segments 1 and 4 are covered with larger surface area of lung pleura, the present results suggest that the significantly greater intrinsic contractile responses of segments 1 and 4 are associated with the presence of increased lung pleural surface possibly together with more mast cells. Thus, a primary contribution to the net contraction of the lung parenchymal strips may be smooth muscle from the lung pleura, alveolar ducts and interstitial contractile cells rather than from bronchi/bronchioles and microvasculatures.


Positive inotropic and negative chronotropic effects of (-)-cis-diltiazem in rat isolated atria.

1. The cardiovascular effects of (-)-cis-diltiazem, an optical isomer of diltiazem, were studied in the isolated atrium and aortic strip. (-)-cis-Diltiazem (30 microM or more) increased the developed tension of the rat left atrium, while (+)-cis-diltiazem (1 microM or more) decreased it. 2. (-)-cis-Diltiazem (1 to 100 microM) decreased the rate of spontaneous beating in the right atrium as did (+)-cis-diltiazem. 3. The potency of the positive inotropic action of (-)-cis-diltiazem was almost the same as that of ouabain in the rat left atrium, but in the guinea-pig left atrium it was considerably weaker than that of ouabain. 4. In both endothelium-intact and endothelium-denuded aortic strips, (-)-cis-diltiazem relaxed the Ca(2+)-induced contraction. In the endothelium-intact rat aortic strip depolarized by 15 mM KCl, Bay K 8644, a calcium channel agonist, increased the contractile force, whereas (-)-cis-diltiazem did not. 5. These results indicate that (-)-cis-diltiazem has a positive inotropic action in isolated atria in rats and guinea-pigs, but the mode of positive inotropic action of (-)-cis-diltiazem is different from that of ouabain or Bay K 8644.


Alpha 4-2 beta 2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs.

1. Xenopus oocytes were injected with various muscle and neuronal nicotinic acetylcholine receptor (ACh receptor, cholinoceptor) subunit RNA combinations and their pharmacological properties studied using two-electrode voltage clamp. The functional expression of one of these combinations, rat alpha 4-2 beta 2, has not been previously described. The alpha 4-2 mRNA is a splicing variant transcribed from the alpha 4 gene. In the experiments reported here, the alpha 4-2 beta 2 subtype was functionally indistinguishable from the alpha 4-1 beta 2 subtype. 2. For each subtype, the relative potency of nicotine compared with acetylcholine was obtained by estimating the relative concentration of nicotine which would elicit the same current response as 0.1 microM Ach. The ratios of these concentrations (nicotine: ACh) for the mouse muscle ACh receptor-(alpha 1 beta 1 gamma delta) was 96.1:1. In contrast, the ratios for the rat neuronal subtypes were: alpha 2 beta 2, 1.01:1; alpha 3 beta 2, 2.01:1; alpha 4 beta 2, 0.76:1 and alpha 4-2 beta 2, 0.76:1. The much greater relative nicotine sensitivity of the neuronal subtypes as compared with muscle receptors illustrates their potential to mediate the psychoactive and addictive effects of nicotine. However, it does not appear that the differences in relative nicotinic sensitivity among the neuronal receptors themselves can be used as a simple discriminative tool in neuronal tissue. 3. The slopes of the log dose-log response curves at low ACh concentrations were all greater than 1 but less than 2, suggesting that at least two agonist binding sites mediate the functional response of each hetero-oligomer. 4. The response of all the neuronal subtypes to ACh could be inhibited by the psychoactive drugs mecamylamine, amitriptyline, phencyclidine, trifluoperazine and promethazine. With the exception of the very potent antagonist, mecamylamine, the degree of block of the peak current to ACh produced by 10 microM concentrations of these drugs was remarkably similar (around 50%). 5. The degree of inhibition produced when the antipsychotic drug, trifluoperazine, was co-applied with ACh increased as the duration of application increased. Such an effect was not observed with promethazine, a related phenothiazine derivative which does not have antipsychotic actions.


Effect of K+ channel-modulating drugs on the vasoconstrictor responses of leukotrienes C4, D4 and angiotensin II in the guinea-pig isolated perfused heart.

1. The vascular actions of leukotrienes C4 (LTC4) and LTD4 in the guinea-pig isolated perfused heart were studied in the presence of potassium (K+) channel modulatory compounds. 2. Cromakalim (0.35-10 microM), a K+ channel activator, inhibited the vasoconstrictor responses of LTC4 (30 pmol), LTD4 (30 pmol) and angiotensin II (AII) (100 pmol) in a concentration-dependent manner. 3. Glyceryl trinitrate (10 mgl-1) and vasoactive intestinal peptide (10 nM) induced a similar vasodilator action to cromakalim in the isolated heart but had no effect on responses to LTC4 and LTD4. 4. The inhibitory action by cromakalim (10 microM) on the LTC4 (30 pmol) response could be reversed in the presence of an equimolar concentration of glibenclamide. However, glibenclamide (10 microM) only partially restored the LTD4 (30 pmol) actions. 5. Galanin (10 nM) and charybdotoxin (60 nM) had no effect on the vascular responses to LTC4 and LTD4 (30 pmol). 6. Inhibition by cromakalim of coronary vasospasm induced by vascular LTC4, LTD4 and AII appears to be separate from its vasodilator action and it is postulated that a cromakalim-sensitive mechanism in the coronary vasculature is important in the vasoconstrictor responses to LTC4, LTD4 and AII.


Multiphasic effect of morphine on the release of substance P from rat trigeminal nucleus slices.

It is generally accepted that morphine acts presynaptically to inhibit substance P (SP) release from afferent terminals in the trigeminal nucleus. Recent studies, however, provide evidence that opioids produce both inhibitory and excitatory effects on SP release which are concentration- and receptor subtype-dependent. In the present study, we have examined a wide range of morphine concentrations on K(+)-evoked SP release from rat trigeminal nucleus caudalis slices. Immunoreactive SP was measured in perfusates. Morphine produced multiphasic effects on K(+)-evoked SP release without affecting basal release. A very low nanomolar concentration (1 nM) suppressed release, higher nanomolar concentrations (100-300 nM) facilitated release, a low micromolar concentration (3 microM) suppressed release, and a higher micromolar concentration (30 microM) facilitated release. These effects were abolished by opioid receptor blockade with naloxone (30 nM). Thus, morphine produces a complex bi-directional modulation of SP release from TNC which is concentration- and possibly receptor subtype-dependent.


Morphine produces a multiphasic effect on the release of substance P from rat trigeminal nucleus slices by activating different opioid receptor subtypes.

Morphine (MOR) produces a concentration-dependent multiphasic effect (inhibitions and facilitations) on K(+)-evoked substance P (SP) release from rat trigeminal nucleus slices. In this study, we tested the action of selective opioid receptor antagonists on this multiphasic effect of MOR. 1 nM MOR produced an inhibition of K(+)-evoked release of SP that was affected only by the selective mu 1-opioid receptor antagonist naloxonazine (1 nM). MOR at 100 nM elicited an increase in SP release which was abolished selectively by the mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA; 20 nM) and attenuated by the delta-opioid receptor antagonist, ICI 174,864 (0.3 microM). 3 microM MOR produced an inhibition of SP release that was reversed only by ICI 174,864 (0.3 microM). MOR at even higher concentrations (30 microM) produced an enhancement of SP release that was reversed selectively by 3 nM n-binaltorphimine (n-BNI; 3 nM), a kappa-opioid receptor antagonist. In slices pretreated with 20 nM beta-FNA and in the presence of 0.3 microM ICI 174,864 (mu- and delta-opioid receptor blockade), both 100 nM and 3 microM MOR elicited a strong facilitation of K(+)-evoked SP release which was sensitive to 3 nM n-BNI. Thus, the increase in SP release produced by 100 nM may be mediated by the simultaneous stimulation of beta-FNA-sensitive mu- and excitatory delta-opioid receptors whereas the facilitation of SP release induced by 30 microM MOR could be due to the activation of kappa-opioid receptors. 1 nM and 3 microM MOR may inhibit SP release by stimulating naloxonazine-sensitive mu 1- and inhibitory delta-opioid receptors, respectively.


Effect of peptidase inhibition on the pattern of intraspinally released immunoreactive substance P detected with antibody microprobes.

Antibody microprobes bearing antibodies to the C-terminus of substance P (SP) were used to measure release of immunoreactive (ir) SP in the dorsal horn of barbiturate anaesthetized spinal cats. Electrical stimulation of unmyelinated primary afferents of the ipsilateral tibial nerve produced a relatively localised release of ir SP in the superficial dorsal horn. Prior microinjection of the peptidase inhibitors kelatorphan and enalaprilat in the dorsal horn resulted in ir SP being detected over the whole of the dorsal horn and the overlying dorsal column. This pattern had previously been observed with evoked release of ir neurokinin A and supports the proposal that a slow degradation results in a neuropeptide accessing many sites remote from sites of release.


The medial dorsal nucleus is one of the thalamic relays of the cerebellocerebral responses to the frontal association cortex in the monkey: horseradish peroxidase and fluorescent dye double staining study.

To reveal the thalamic relay nucleus of the cerebellocerebral responses in the frontal association cortex, simultaneous labeling of the cerebellothalamic (C-T) terminals and the thalamocortical (T-Cx) neurons was performed in three monkeys. Horseradish peroxidase (HRP) was injected into the deep cerebellar nuclei and small doses of HRP or fluorescent dye were injected into the prefrontal cortex. The distribution of anterogradely labeled C-T terminals and retrogradely labeled T-Cx neurons was examined in the same sections. In addition to being distributed in the ventral thalamic nuclei and nucleus X, as previously reported, anterogradely labeled terminals were distributed in the ventrolateral part of the medial dorsal (MD) nucleus where retrogradely labeled thalamo-frontal projection neurons were localized. This study revealed that the ventrolateral parts of the MD together (MDmf, MDpc and MDdc) form one of the thalamic relays of the cerebelloprefrontal responses.


Distribution of clusterin in Alzheimer brain tissue.

The immunohistochemical distribution of clusterin (SP40,40, SGP-2) was determined in Alzheimer disease (AD) and normal human brain tissue and compared with the distributions of vitronectin, protectin and the complement membrane attack complex (MAC). Antibodies to all four proteins showed staining of dystrophic neurites and neuropil threads in AD tissue, and residual serum in normal tissue, but only antibodies to clusterin and vitronectin strongly stained amyloid deposits in senile plaques. The clusterin antibody also showed punctate staining of some normal appearing AD pyramidal neurons, and very scattered staining of intracellular neurofibrillary tangles. Clusterin, vitronectin and protectin are all believed to inhibit membrane insertion by the MAC, and these data are consistent with upregulation of all three proteins in response to MAC formation in AD, and with a neuronal origin of clusterin.


Carcinoid tumor localized in the liver--two cases report: immunohistochemical and ultrastructural studies.

Two cases of carcinoid tumors, considered to be probably hepatic in origin, occurring in a 53-year-old man and a 69-year-old woman are reported. In both cases no endocrine syndrome appeared, and an alternative primary source of the tumor was not found in case two, despite an intensive search at operation. The neoplasms in both cases were soft, firm, brown-pink and well-encapsulated. They were composed of small uniform cells, that had distinct borders and grew in insular, nests, trabeculae and strands that were separated by a delicate fibrous stroma. Stains of argentaffin and argyrophil showed strong positivity in both cases. The immunohistochemical and ultrastructural studies all demonstrated characteristics of a carcinoid. The postoperative recovery was good. They have remained well 5 years later in case 1 and 3 years in case 2 after surgical treatment. Literature concerning this rare condition is also reviewed.


Plasticity in expression of calcitonin gene-related peptide and substance P immunoreactivity in ganglia and fibres following guanethidine and/or capsaicin denervation.

This study was designed to investigate the effects of multiple denervation procedures on calcitonin gene-related peptide- and substance P-immunoreactive neurons in sympathetic and sensory cranial ganglia and in selected targets. Sympathectomy by long-term guanethidine treatment induced a pronounced increase in calcitonin gene-related peptide-immunoreactive and substance P-immunoreactive nerve fibres in all the tissues investigated, in contrast to a significant reduction of immunoreactive cell bodies. Neonatal capsaicin treatment abolished substance P immunoreactivity in many targets and caused a dramatic reduction of substance P-immunoreactive sensory nerve cell bodies; calcitonin gene-related peptide-immunoreactive nerve density was decreased, but the number of immunoreactive nerve cell bodies was unchanged. Guanethidine treatment of capsaicin-injected rats reversed the loss of calcitonin gene-related peptide-immunoreactive nerves, but not that of substance P-immunoreactive neurons. In the iris, capsaicin treatment had little effect on calcitonin gene-related peptide- and substance P-immunoreactive nerves, suggesting that in rats the majority of these fibres originate from capsaicin-insensitive neurons. The results suggest that the denervation procedures used in this study alter the synthesis and transport of neuropeptides in sensory neurons in conjunction with changes in the number of nerve fibres.


Inhibition of production of monocyte/macrophage-derived angiogenic activity by oxygen free-radical scavengers.

We showed previously that thiol-containing compounds inhibited the production of macrophage-mediated angiogenic activity. Since thiol-containing compounds may act on macrophages by affecting activation and inhibiting the production of oxygen free-radicals, we studied the effects of oxygen free-radical scavengers on production of angiogenic activity by elicited mouse peritoneal macrophages and lipopolysaccharide stimulated normal human monocytes. Monocyte/macrophage conditioned media were potently angiogenic when assayed in rat corneas, while conditioned media, from oxygen free-radical scavenger-treated cells were not. The inhibitory effect of oxygen free-radical scavengers was due to a direct effect on monocyte/macrophage production of angiogenic activity but was not due solely to a decrease in the production of the macrophage-derived angiogenic cytokine tumor necrosis factor-alpha. We conclude that oxygen free-radical scavengers are potent inhibitors of the production of macrophage-mediated angiogenic activity.


Gap junctional channels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology.

The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9 +/- 97.1 M omega (mean +/- SEM, n = 10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16 +/- 0.32 G omega, n = 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243-257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electronic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.


Acidic and basic fibroblast growth factors in adult rat heart myocytes. Localization, regulation in culture, and effects on DNA synthesis.

Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) are involved in the induction of embryonic mesoderm, angiogenesis, neuronal differentiation, and proliferation and survival of many cell types. In cardiac myocytes their roles are not well understood. Effects of fibroblast growth factors on reexpression of fetal actin genes have been reported. In freshly isolated adult rat cardiac myocytes, bFGF mRNA was not detectable by in situ hybridization, although the cells contained significant amounts of bFGF and aFGF as quantified by radioimmunoassays, mitogen assays with immunoneutralization, and Western blotting. After culturing, bFGF mRNA was detected (aFGF mRNA was not studied), and the cells contained 2.5-fold more bFGF and 60% more aFGF than freshly isolated cells. The FGFs were not found in conditioned medium. They were localized, especially in cultured cells, to the nucleus. Cultured myocytes bound fourfold more 125I-FGF than freshly isolated cells and expressed the fibroblast growth factor R-1 (flg) gene. The addition of bFGF or aFGF in serum-free medium to pure populations of myocytes (after 10 days in culture, at which time they are spread, beating, and multinucleated) led to increased thymidine incorporation. Expression of fibroblast growth factors and fibroblast growth factor receptors by adult cardiac myocytes that survive the shock and "dedifferentiation" of culturing may contribute to DNA synthesis and, by analogy, to other cell types, to regulation of ribosomal and actin genes, and to cell survival. These possibilities and their in vivo relevance will require further study.


Balloon injury and interleukin-1 beta induce nitric oxide synthase activity in rat carotid arteries.

Experiments were performed to investigate whether balloon injury induces nitric oxide synthase activity in the blood vessel wall. Contractions to phenylephrine were compared in left carotid arteries of the rat, previously injured by balloon catheterization and excised either immediately (t = 0), 6, or 24 hours after the procedure, with those in control right carotid arteries (with and without endothelium). Phenylephrine evoked comparable concentration-dependent contractions in balloon-injured (t = 0) and control carotid arteries without endothelium, whereas those in control arteries with endothelium were depressed. In the balloon-injured carotid arteries (6 and 24 hours), the concentration-contraction curves to phenylephrine were shifted to the right compared with those observed in balloon-injured arteries (t = 0). In balloon-injured carotid arteries (6 hours), the hyporeactivity to phenylephrine was enhanced by superoxide dismutase. In balloon-injured carotid arteries (24 hours), nitro-L-arginine and methylene blue restored full contractions, whereas superoxide dismutase potentiated the hyporesponsiveness to phenylephrine. The depressed contractions were associated with a concomitant increase in the basal level of cGMP; this production was abolished by nitro-L-arginine. The depression of the concentration-contraction curves to phenylephrine and the increase of the tissue level of cGMP induced by interleukin-1 beta (4 hours) were more pronounced in balloon-injured arteries (24 hours) than in control arteries without endothelium. The effects of interleukin-1 beta were inhibited by nitro-L-arginine. These observations indicate that in vivo endothelial injury of the rat carotid arteries induces the production of nitric oxide from L-arginine in the blood vessel wall, an effect which is potentiated by interleukin-1 beta.


Effects of norepinephrine on the oxidative pentose phosphate pathway in the rat heart.

To examine whether stimulation of alpha-adrenergic receptors may affect the oxidative pentose phosphate pathway (PPP) in the rat heart, norepinephrine (NE) and the alpha-adrenergic agonist norfenephrine were used. NE was administered as a continuous intravenous infusion in awake rats for 3 days. It stimulated the activity of cardiac glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of the oxidative PPP, in a dose-dependent manner. With the highest dose (0.2 mg.kg-1.hr-1), there was also a time-dependent enhancement. The increase observed after 48 hours was attenuated partially by the beta-receptor blocker metoprolol and the alpha-receptor blocker prazosin. It was entirely abolished when both drugs were administered. Carvedilol, a beta-adrenergic blocker and vasodilator with alpha 1-blocking activity (0.5 mg.kg-1.hr-1), prevented the NE-induced increase in cardiac G-6-PD activity, in functional parameters (heart rate, left ventricular systolic pressure, and left ventricular dP/dtmax), and in the heart weight/body weight ratio. The alpha-adrenergic stimulator norfenephrine increased myocardial G-6-PD activity; prazosin prevented this stimulation. NE and norfenephrine also elevated the available pool of cardiac 5-phosphoribosyl-1-pyrophosphate. G-6-PD activity was enhanced in cardiac myocytes freshly isolated from the left ventricle of rats that had received NE infusion for 3 days (12.3 +/- 1.4 units/g protein) compared with control rats (1.5 +/- 0.4 units/g protein). The activity of 6-phosphogluconate dehydrogenase, one of the enzymes in the oxidative PPP, was elevated only moderately from 12.7 +/- 0.7 to 19.1 +/- 1.4 units/g protein. Combined alpha- and beta-receptor blockade with carvedilol attenuated these effects.(ABSTRACT TRUNCATED AT 250 WORDS)


Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning.

The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC16); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.


CD3+ CD57+ lymphocytes are not likely to be involved in antigen-specific rejection processes in long-term allograft recipients.

Cytofluorometric investigation of peripheral blood lymphocytes in 380 long-term (greater than 1 year posttransplantation) allograft recipients showed a significant increase in the proportion of CD3+57+ lymphocytes (greater than 20%) in 20% of patients with renal allografts, 66% of patients with cardiac allografts and 44% of patients with liver allografts. Most of these CD3+57+ cells expressed the CD8 antigen and a variable proportion the HLA-DR antigen. A retrospective analysis showed a poorer prognosis for the clinical outcome in those patients with elevated numbers of CD3+57+ cells in peripheral blood. However, CD57+ lymphocytes could rarely be detected in renal infiltrates by immunohistology. Using the Southern blot technique to analyse the T cell receptor rearrangement of separated CD57+ cells, no clonal or oligoclonal expansion of T cell clones could be detected. Nevertheless, there might be a bias towards the use of particular TCR-V beta gene families in at least some patients, as shown by analysis with monoclonal antibodies. In summary, CD57+ T cells are not likely to be directly involved in the rejection process. The data support the idea of a polyclonal and/or superantigen-driven expansion, but not of an antigen-driven expansion of these cells.


IgG antibodies from patients with primary Sjögren's syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.

Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (less than or equal to 7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21-41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21-41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.


Adhesion molecule expression in Graves' thyroid glands; potential relevance of granule membrane protein (GMP-140) and intercellular adhesion molecule-1 (ICAM-1) in the homing and antigen presentation processes.

To assess the potential role of adhesion molecules in the pathogenesis of Graves' disease, we examined the expression of several of these adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and granule membrane protein-140 (GMP-140), in sections of Graves' thyroid glands and control thyroids, using immunohistochemical techniques. Up-regulated expression of GMP-140 was frequently observed on endothelial cells (EC) of post-capilliary venules in all Graves' thyroids examined, compared with an occasional weak staining on EC control glands. Some capillary EC around thyroid follicles (perifollicular EC) were strongly positive for GMP-140 in the Graves' thyroids in contrast to a negative staining on the same structures in the control glands. In addition, there was a correlation between the reactivity and frequency of GMP-140 expression on EC and the severity of mononuclear cell (MNC) infiltration in the Graves' thyroids. The expression of ICAM-1 was up-regulated on perifollicular EC and EC of small venules in some thyroids of both Graves' and control groups. Conversely, no significant expression was observed on any type of EC for both endothelial-leucocyte adhesion molecule-1 (ELAM-1) and VCAM-1. However, dendritic-like cells, present within lymphocytic infiltrates, were positive for VCAM-1 in most of the Graves' thyroids examined, especially in those with a severe lymphocytic infiltration. Thyrocytes were constantly negative for the expression of all four adhesion molecules investigated. These data suggest that GMP-140, as well as ICAM-1, could play an important role in the initiation of MNC infiltration in Graves' disease. ELAM-1 and VCAM-1 appear not to be relevant for the migration of MNC from the blood vessels into the target gland, although VCAM-1 expression on dendritic-like cells might play an additively tissue-selective role in autoantigen presentation and subsequent elicitation of autoimmune phenomena.


[CSF monoamine metabolites in patients with progressive supranuclear palsy].

Neurochemical abnormalities in the patients with progressive supranuclear palsy (PSP) are not well understood. We investigated CSF levels of the monoamine metabolites HVA, 5HIAA and MHPG in 5 patients with PSP in order to investigate, especially dopaminergic, serotonergic and noradrenergic metabolism. Results were as follows (value; mena +/- S.D.). [table: see text] The levels of HVA were significantly lower than those in controls. These findings suggest that there is a hypofunction of dopaminergic neurons in PSP patients.


Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.


Comparative efficacy of antidepressants.

Selective serotonin reuptake inhibitors (SSRIs) are a recently developed class of drugs with significantly greater antidepressant efficacy than placebo. Generally, in double-blind comparative trials, all SSRIs demonstrated antidepressant efficacy similar to that of the 'standard' tricyclic antidepressants amitriptyline and imipramine; a meta-analysis of controlled trials found the efficacy of the SSRIs to be equivalent to that of the 2 tricyclics. Nevertheless, because of small patient numbers included in most studies that compare SSRIs with other antidepressants, no definitive statements about relative efficacy can be made. In these studies it is simply possible to state that no statistically significant differences were identified between SSRIs and the comparative antidepressants. Importantly, differences in clinical characteristics exist between the SSRIs-differences in elimination half-life (t1/2 beta) between fluoxetine and/or its metabolite (total t1/2 beta = 330 hours) and other SSRIs (t1/2 beta range = 15 to 30 hours), for example. This has implications in terms of potential drug interactions and must be considered when patients have to be switched to treatment with monoamine oxidase inhibitors. Studies with fluvoxamine have been conducted in both in- and outpatients, whereas trials with other SSRIs have been confined largely to outpatient populations. Fluvoxamine has been associated with a high incidence of nausea (37%), although this may have resulted from high initial dosages (rather than upward dose titration protocols) used in early trials. Of further interest, fluoxetine doses of 20mg may be sufficient to produce a satisfactory antidepressant response, and this SSRI may be particularly useful in patients with chronic retarded depression. More clinical data are required before the efficacy of sertraline and citalopram relative to standard antidepressants can be clearly defined. Preliminary data indicate that SSRIs are effective in the treatment of panic disorder, obsessive-compulsive disorder (OCD), eating (e.g. anorexia and bulimia) and personality disorders (e.g. anger, impulsiveness) and substance abuse (e.g. alcoholism); early results with fluvoxamine in the treatment of panic disorder and OCD, and with fluoxetine in the treatment of bulimia, personality disorders and alcohol abuse, have been encouraging. SSRIs have a more favourable tolerability profile than tricyclic antidepressants and, unlike the tricyclics, are not associated with anticholinergic adverse effects, sedation, cardiotoxicity or weight gain. SSRIs are associated with a relatively high incidence of nausea, particularly if high doses are used at the start of treatment. However, the incidence of nausea appears to decrease as treatment is continued.(ABSTRACT TRUNCATED AT 400 WORDS)


Suicide and antidepressants.

Depression is associated with a risk of suicide 13 to 30 times greater than in the general population, and antidepressants are among the drugs most frequently implicated in fatal overdose, resulting in a dilemma for the clinician. Suicidal thoughts are a core symptom of depression, and there is evidence that the selective serotonin (5-hydroxytryptamine) reuptake inhibitors (SSRIs), including fluvoxamine, are of particular benefit in reducing their occurrence. Data from controlled studies indicate that the SSRIs do not exacerbate suicidal ideation; on the contrary, SSRIs protect against the emergence of suicidal thoughts, which reflect the natural history of the disease. The SSRIs also appear to be particularly effective in patients who are highly suicidal at treatment initiation, and to be more effective than the tricyclic antidepressants in treating severe depression and improving anxiety symptoms associated with depression. Thus, fluvoxamine, in common with other SSRIs, is of particular benefit in treating depression in patients with prominent suicidal thoughts or who are at increased risk of suicide.


Pharmacological differences of serotonin reuptake inhibitors and possible clinical relevance.

Depression is a heterogeneous disease state characterised by complex alterations in several CNS neurotransmitter and receptor systems. All antidepressants are thought to act by causing postsynaptic adaptive changes (e.g. in transducers or second messengers) within these systems. Thus, the mechanism of action of selective serotonin reuptake inhibitors (SSRIs) cannot simply be explained in terms of inhibition of serotonin (5-hydroxytryptamine) [5-HT] reuptake. Fluvoxamine, sertraline and fluoxetine downregulate central beta-adrenoceptors, and all SSRIs are believed to normalise central 5-HT1A- and 5-HT2-receptor density and function in patients with depression. SSRIs are as effective as tricyclic antidepressants in the treatment of depression, but have distinct tolerability advantages--they are not associated with anticholinergic adverse effects, cardiotoxicity, sedation or weight gain. However, gastrointestinal reactions (e.g. nausea, diarrhoea/loose stools, constipation) are relatively common during SSRI therapy. Additionally, in contrast to tricyclic antidepressants, SSRI dosage adjustments appear to be unnecessary in elderly depressed patients. Fluvoxamine has a much shorter elimination half-life than fluoxetine and its active metabolite, norfluoxetine, and therefore a reduced potential for drug interactions. Only small amounts of fluvoxamine and fluoxetine, but large quantities of paroxetine, are secreted in breast milk. Furthermore, genetic polymorphism has not been documented for fluvoxamine metabolism, whereas slow and fast metabolisers of paroxetine, and fast metabolisers of fluoxetine have been identified. SSRIs have a better tolerability profile than tricyclic antidepressants, as indicated by lower mean rank scores for behavioural toxicity. Moreover, SSRIs are associated with a much lower incidence of fatal toxicity than tricyclics, and appear to be relatively safe in overdosage.(ABSTRACT TRUNCATED AT 250 WORDS)


Efficacy of fluvoxamine in severe depression.

The therapeutic efficacy of fluvoxamine has been demonstrated in large numbers of patients participating in comparative double-blind placebo-controlled studies using imipramine as the active reference compound. Overall, patients treated with fluvoxamine for 4 to 6 weeks had significant amelioration of their depression compared with placebo-treated patients. More importantly, the response rate in patients with severe depression was greater than in patients with mild or moderate depression. However, several investigators have observed high placebo response rates, and in some studies there has been a similar response rate to imipramine and placebo treatment. While the high placebo response rate may have resulted from methodological problems, the issue does raise some questions that can only be resolved by further investigation.


The safety of antidepressants.

In this article, a distinction is proposed between safe and less safe antidepressants. The safety of 18 antidepressants is discussed in relation to 3 principal issues: the safety of the drug in the event of an overdose; the seriousness of its side effects; and the existence of dangerous interactions. On the basis of present information, it can be said with reasonable confidence that fluoxetine, fluvoxamine and paroxetine are safe antidepressants, and with some reservation (mainly because of hypnosedation) the same can be said of mianserin and trazodone.


Review of fluvoxamine safety database.

A review of the safety and tolerability of fluvoxamine in worldwide marketing studies involving 24,624 patients, predominantly receiving fluvoxamine treatment in uncontrolled studies in depression, has been conducted. There was a marked preponderance of female patients and patients aged between 30 and 50 years. The majority of patients were treated for 6 weeks, with the most frequent modal total daily dose being 100mg. The greatest proportion of adverse experiences occurring, by COSTART body system, affected the digestive system (24.1%), the nervous system (23.7%), and the body as a whole (15.3%). The only adverse experience with an incidence greater than 10% was nausea (15.7%), with somnolence (6.9%) and asthenia (6.2%) as the next most frequent experiences. Notably, the rates of agitation and anxiety were only 1.4 and 1.3%, respectively. The incidences of adverse experiences increased with age, and were slightly higher in females than males. 15.1% of patients discontinued treatment prematurely as a result of adverse experiences, principally nausea, dizziness, vomiting, somnolence, abdominal pain, and headache. The overall incidence of serious adverse events associated with fluvoxamine treatment was 2.5%, and the incidence of overall suicidality, including suicidal ideation, overdose, and intentional overdose as well as attempted and completed acts of suicide, was remarkably low at 0.8%.


Ictal spikes: a marker of specific hippocampal cell loss.

Spontaneous seizures recorded from mesial temporal depth electrodes in the human are commonly manifested by one of two onset patterns: a high frequency discharge or a periodic spike discharge morphologically similar but clearly distinguished from ongoing interictal activity. We categorized medial temporal lobe seizure onset for the presence of periodic ictal spikes at a frequency of less than 2 Hz lasting for more than 5 sec to investigate the relationship of this ictal pattern to anatomical changes in the resected temporal lobe tissue. Fifty-one patients had hippocampal depth electrode recordings of spontaneous seizures, subsequent hippocampal resection, and quantitative cell counts of hippocampal subfields. Thirty-two of these patients had ictal spikes lasting at least 5 sec in more than 50% of their seizures. The presence of ictal spikes was significantly correlated with decreased cells in CA1 only (P = 0.015). The correlation of a common ictal pattern with focal cell loss in the hippocampus suggests that electrophysiological manifestations of seizures provide a clue to the underlying pathological substrate. Ictal spikes may be a cause or result of the cell loss. These observations should be correlated with independent investigations in humans and animal models that reflect the CA1 cell loss associated with temporal lobe epilepsy.


Comparative coherence studies in healthy volunteers and Down's syndrome patients from childhood to adult age.

Within the scope of the Munich Pediatric Longitudinal Study, EEG coherence was studied in 212 Down's syndrome patients and 342 healthy controls aged from 6 months up to 30 years. The digitalized EEG records were subjected to spectral analysis. Frequency band-related coherences were calculated to reveal age-specific differences in the functional relationship between two brain areas in Down's syndrome patients and controls. The results show that in the "eyes-open" state the intra-hemispheric coherence in the alpha band was significantly lower (P less than 0.05) in the Down's syndrome patients than in the controls whereas that in the delta bands it was generally higher. The intra-hemispheric coherence in the "eyes-closed" state was generally higher in the Down's syndrome groups than in the controls; however, significant differences could be detected only in some age groups. The age-specific development of coherence in the inter-hemispheric parieto-occipital region was almost identical in Down's syndrome children as in controls, both with open and closed eyes. The most distinct differences were found in the fronto-central inter-hemispheric coherence (P less than 0.01), while the coherence deficiencies in the Down's syndrome group became more prominent with increasing age from school age onwards. These electrophysiological results are compared with the results of neuropathological and neurophysiological studies of other authors. It can be suggested that there are correlations with a significantly small number of dendritic spines in Down's syndrome patients, which was determined in neuropathological examinations. A neuronal model of interpretation is presented which explains the increasing developmental deficit with age in Down's syndrome children.


The topographical features of EEGs in patients with affective disorders.

EEG data were obtained in the basic state from 16 scalp sites of 44 patients with affective disorder, diagnosed by DSM-III criteria, and 44 normal controls. The EEG power spectra were computed and the t statistic significance probability mapping (SPM) was applied to visualize regions where the patient group showed differences in the EEG topogram from the controls. The results show: (1) left occipital predominance (P3, O1) of alpha activities in the patients with affective disorders, (2) decreased alpha activities in Fp2 and F8 areas in patients with major depression without melancholia, (3) decreased alpha activities in F7 area in patients with bipolar disorder, manic, and (4) increased beta 2 activity in F4 and C4 areas in patients with major depression with melancholia. These results suggest that inter-hemispheric and intra-hemispheric relationships may be disturbed in patients with affective disorder.


Periodic activity in cerebral arousal mechanisms--the relationship to sleep and brain damage.

Periodic activity during light sleep is well recognised in many physiological systems, particularly respiration. In damaged brains this activity can become exaggerated. It involves the autonomic nervous system, the muscles, the cerebrospinal fluid (CSF) pressure, the cerebral blood flow and the electroencephalogram (EEG). It is related to the level of arousal. The EEGs of 52 subjects were studied. In stage 0-1 sleep, periods of alpha activity alternated with periods of theta activity related to the level of arousal. The intervals between the alpha onsets were measured and the data pooled. There was a dominant interval of about 16 sec. It is suggested that this is a physiological cerebral rhythm involving the cortex and the brain-stem activating mechanisms, responsive to outside stimuli but essentially endogenous. It is related to the controls of the autonomic, motor, and some cerebral auto-regulatory mechanisms. It may be severely disturbed in brain damage.


The different patterns of the photoparoxysmal response--a genetic study.

In order to investigate the significance of different patterns of the photoparoxysmal response (PPR), an electroencephalographic family study on 135 probands and 371 relatives was carried out. The PPR was subclassified in 4 phenotypically different types. Considering all types of PPR, the incidence was equal in siblings of probands with epilepsy and in siblings of probands without epilepsy. Type 4 (generalized spikes and waves) occurred more often both in probands with epilepsy and their siblings than the respective controls. Thus, the coincidence of photosensitivity will appear as higher if only a PPR with generalized spikes and waves is considered to be indicative of photosensitivity. A striking age dependency of the phenotypical expression of the PPR was found. These findings suggest that the phenotypical expression of the PPR is age-related and modified by other factors predisposing to generalized epilepsy, the varied patterns of the PPR only representing different levels of expression of the same genetically determined trait. The importance of an adequate technique of intermittent light stimulation is discussed.


Late magnetic fields and positive evoked potentials following infrequent and unpredictable omissions of visual stimuli.

Randomized and infrequent omissions during presentation of a steady train of visual stimulation produced distinctive wave forms of both the magnetic fields and electrical potentials. Electrical potentials at Pz showed a positive peak in response to the omitted stimuli which occurred on the average 445 msec after the time when a stimulus was anticipated. Analyses of the magnetic wave forms indicated that at least two separate sources appear to be active coincident with the electrical positive peak. One source localized in the occipital lobes in the vicinity of the visual cortex while the other source was located in the medial aspects of the temporal lobe or even deeper in the lateral thalamus. Judging from the calculated direction of current flow it appeared that the deep source would contribute greater potentials in the frontal areas of the scalp while the source in the occipital area would contribute to more posterior placement of electrodes, especially at Pz.


Contralateral effects of cerebello-pontine angle exposure on human auditory brain-stem evoked potentials.

Auditory brain-stem evoked potentials (ABEPs) were recorded during surgical procedures which exposed the cerebello-pontine angle (CPA) in humans. Recordings made with the CPA contralateral to stimulus exposed were compared with those obtained with the skin sutured at the end of surgery. Single-channel as well as 3-channel Lissajous' trajectory (3-CLT) analyses were used to evaluate the effect of the surgical exposure on ABEP. The results suggest that exposure of the CPA contralateral to the stimulated ear did not affect dipole equivalent orientation nor magnitude, but did affect timing of the recorded activity being more pronounced for segments 'd'-'e' (corresponding to waves IV-V) than for 'a'-'b' (waves I-II). The results imply that the effects of disrupting the volume conductor may have been overwhelmed by other effects, such as local temperature changes. These changes, although not associated with clinical sequella, should be accounted for when analyzing subtle quantitative changes involving surgical exposures.


An electroencephalographic comparison of effects of propofol and methohexital.

Thirty-eight patients were randomly allocated to receive propofol 1 mg/kg (group A, N = 10), methohexital 0.7 mg/kg (group B, N = 9), propofol 2 mg/kg (group C, N = 10), methohexital 1.5 mg/kg (group D, N = 9). They were all male with a mean age of 65.8 years (range, 46-85) and a mean weight of 76.2 kg (range, 50-109). Patients received no premedication. All drugs were administered as a single i.v. bolus. After baseline EEG recordings were obtained, i.v. bolus doses were given and the recording continued until the patients became fully responsive to verbal commands. The EEGs were visually analyzed and classified into 4 phases: phase 0, the wake physiologic pattern; phase 1, initial changes after i.v. bolus doses; phase 2, state of deep anesthesia; and phase 3, stage of recovery. The main change during phase 1 was increase in the amplitude of the background rhythms. Phase 2 was characterized by theta and delta activity and burst suppression in some patients. During phase 3 beta activity was seen following methohexital. Propofol produced a much deeper level of anesthesia compared to methohexital. The stage of deep anesthesia was prolonged following propofol. The clinical and EEG recoveries were prolonged after induction doses of propofol. The quality of recovery, however, was far superior with propofol. Methohexital produces a "hang over" effect which delays full recovery.


Primary and secondary bilateral synchrony in epilepsy: differentiation by estimation of interhemispheric small time differences during short spike-wave activity.

Estimation of interhemispheric small time differences (TDs) during spike-wave bursts in the EEG by coherence and phase analysis is useful for differentiation between primary bilateral synchrony (PBS) and secondary bilateral synchrony (SBS) in epilepsy. Because the previous method via Fast Fourier Transform needed long bursts for reliable analysis, a method using a 2-dimensional autoregressive model was newly developed to enable estimation of TDs even in 1.2 sec bursts, and applied to 19 epileptic patients with apparently bilaterally synchronous spike-wave bursts. At the onsets of bursts, estimated maximal TDs were 5.8 msec or less and inconsistent in leading hemispheres in 10 patients with a clinical diagnosis of idiopathic, cryptogenic or symptomatic generalized epilepsy indicating PBS, while the maximal TDs were 9.3-41.5 msec and consistent in leading in 7 patients with clinically symptomatic partial epilepsy and also in two with idiopathic and symptomatic generalized epilepsy suggesting SBS. Among 8 patients with bursts which suggested SBS and long enough for evaluation of intra-burst TD variation, TDs tended to disappear in the middle to end parts of the bursts in 5 cases, but not in the other 3, suggesting 2 different pathophysiological mechanisms in SBS.


Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.


Reliable phenotyping of alpha-1-antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient.

Genetically determined phenotypes of the highly polymorphic human alpha 1-antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.


Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum.

For the identification of apolipoprotein E isomorphic phenotypes, fresh or thawed serum was analyzed without prior delipidation or other pretreatment. Using 5% polyacrylamide gels with a 40 mm interelectrode distance, the isoforms were separated by isoelectric focusing in immobilized pH gradients ranging from pH 5 to 6.5, and transferred onto polyvinylidene difluoride membranes by contact blotting for 1 h. The apolipoprotein E isoforms were identified following immunostaining. The electrophoresis required less than 2 h and the entire procedure could be completed within 6 h.


Monoclonal antibody (Mab) markers for subpopulations of rat tracheal epithelial (RTE) cells.

We sought monoclonal antibodies (Mabs) that would recognize distinct subsets of rat tracheal epithelial (RTE) cells. Mice were immunized with pronase-dissociated RTE cells and hybridomas whose supernatants immunocytochemically stained subpopulations of tracheal cells were selected. We report the immunohistochemical staining properties of the antibodies and give the results of preliminary biochemical characterization of the antigens. Four different types of antibodies were produced. Antibody RTE 1 stained most RTE cells. Three antibodies (RTE 2, 7, and 13) recognized a subpopulation of nonciliated cells, both columnar and basal cells. Antibody RTE 3 intensely labeled the surface of ciliated cells. Three antibodies reacted with granule components of secretory cells; antibodies RTE 9 and 11 reacted with mucous-type secretory cells and antibody RTE 12 stained all tracheal surface secretory cells. As described in detail, some antibodies were RTE cell specific while others also reacted with cells and secretions in other organs; the antibodies did not cross react with guinea pig or rabbit tissues. Periodate sensitivity of the antigens suggested that some antibodies recognized carbohydrate moieties while others detected peptide epitopes. In some cases, Western blotting revealed the molecular weights of the antigens, but some antigens were denatured by sodium dodecyl sulfate (SDS) and heat treatment. These antibody probes provide a useful means to immunochemically study changes in cell type distribution and/or epitope expression during development, injury, and regeneration.


The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.


Different types of K+ channel current are generated by different levels of a single mRNA.

A cloned human voltage-sensitive K+ channel HLK3 which is present in T-lymphocytes and in the brain was expressed in Xenopus oocytes and after permanent transfection of a human B-lymphocyte cell line (IM9). Injections of low cRNA concentrations into Xenopus oocytes led to the expression of a transient K+ current, with saturating current-voltage (I-V) relationship, which was abolished by repetitive stimulations due to a slow recovery from inactivation. This transient K+ channel current was fully inhibited by 10 nM charybdotoxin. Injection of high concentrations of the same RNA led to a non-inactivating K+ current, with linear I-V curve, which did not undergo use-dependent inactivation and was hardly sensitive to 10 nM charybdotoxin. Intermediate behaviour due to changing proportions of these two types of K+ channel expression were observed at intermediate RNA concentrations. Transient and non-inactivating K+ currents were also observed by both whole-cell and single channel patch-clamp recording from HLK3 transfected IM9 cells. The main conductance of the channel in the two different modes (inactivating and charybdotoxin-sensitive or non-inactivating and charybdotoxin-resistant) is the same (12-14 pS). Destruction of the cytoskeletal elements with cytochalasin D, colchicine or botulinum C2 toxin in oocyte experiments prevented expression of the sustained mode of the K+ channel. The results suggest that the sustained mode obtained at high RNA concentrations corresponds to channel clustering involving cytoskeletal elements. This differential functional expression of K+ channels associated with different levels of mRNA appears as a new important factor to explain the biophysical and pharmacological diversity of voltage-sensitive K+ channels.


Characterization of a Shaw-related potassium channel family in rat brain.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.


Regulation of CFTR expression and function during differentiation of intestinal epithelial cells.

CFTR, the protein defective in cystic fibrosis is regulated during differentiation of intestinal epithelial cells. The undifferentiated cells (Caco-2 and HT-29) show a lower level of CFTR mRNA, while a 10-fold increase is seen in differentiated cells. These differences correlate well with those of other intestinal-specific genes, including sucrase-isomaltase, villin and alpha 1-antitrypsin, indicating that the regulation is cell specific. In Caco-2 cells the increase in CFTR mRNA cannot be accounted for by increased transcription of the gene. These data indicate that CFTR mRNA stabilizing factor(s) might be present in differentiated cells. The higher levels of CFTR mRNA in differentiated cells are accompanied by decreased protein levels, indicating, as well, involvement of translational control in the regulation of CFTR in these cells. Finally, fully differentiated cells show lowered levels of cyclic AMP-activated C1- transport, the characteristic function of CFTR. Thus, CFTR function in differentiated cells is modulated by a complex interaction of regulatory elements. Caco-2 and HT-29 cells provide a suitable in vitro system in which to study the mechanism of regulation of CFTR.


Suppression of beta-1,3-glucanase transgene expression in homozygous plants.

A chimeric construct containing the Nicotiana plumbaginifolia beta-1,3-glucanase gn1 gene was introduced into Nicotiana tabacum SR1 to produce high levels of the enzyme constitutively. We determined that the GN1 protein represents a basic beta-1,3-glucanase isoform which accumulates into the vacuoles of the transgenic plants. Analysis of the progeny of the transgenic plant with the highest levels of gn1 expression revealed an unexpected phenomenon of gene suppression. Plants hemizygous for the T-DNA locus contained high levels of gn1 mRNA and exhibited a 14-fold higher beta-1,3-glucanase activity than untransformed plants. However, the expression of gn1 was completely suppressed in the homozygous plants: no corresponding mRNA or protein could be detected. This suppression mechanism occurs at a post-transcriptional level and is under developmental control. In addition, by generating haploid plants we found that this silencing phenomenon is not dependent on allelic interaction between T-DNA copies present at the same locus of homologous chromosomes, but rather is correlated with the transgene dose in the plant genome. We postulate that high doses of GN1 protein relative to the level(s) of other still unknown plant products could trigger the cellular processes directed to suppress gn1 expression.


DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen.

Singlet oxygen (1O2), generated by exciting an eosin-Tris complex with a high intensity beam of radiation at 532 nm, was used to chemically modify bases in fragments of DNA containing the lac UV5 promoter in the presence of the DNA binding proteins, RNA polymerase and CRP (cAMP receptor protein). Subsequent treatment with piperidine selectively cleaved the DNA at specific modified bases in the sequence. Using this technique we show first that the reactivity of DNA bound by CRP differs in the presence and absence of RNA polymerase. Hence the local conformation of CRP-bound DNA must change during the transition to the open complex. However, no reactivity is observed at the sites of the 40 degrees kinks described in the cocrystal structure (Steitz, 1990). Secondly we show that there is unique CRP-dependent reactivity at a specific site (position -46 on the upper strand) in the open complex. Finally, in the open complex, 1O2 also reacts with sites 90 bp upstream from the transcription start point. This reactivity is qualitatively CRP-independent. We infer that 1O2 reacts at sites where the promoter DNA is significantly distorted, and suggest that the pattern observed reflects the functional orientation of an active transcriptional complex in which the DNA is bent to form an extended loop.


The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain. This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein. We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.


Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.


DNase-I hypersensitivity analysis of the L-type pyruvate kinase gene in rats and transgenic mice.

The rat L-type pyruvate kinase gene possesses two promoters located 500 bp apart. The L' promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue-specific and hormone-regulated gene expression are located within 3.2 kbp of 5' flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu-Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19,904-19,910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase-I-hypersensitivity analysis of the endogenous rat L-type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS-1 in adult liver and HSS-A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L' promoter. Two other distal groups were detected; HSS-2 at -3 kbp (with respect to the liver-specific cap site) in adult liver and HSS-B around -4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS-2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS-1 appears to be a transcription-associated site, being greatly weakened in fasted rats, while HSS-2 is transcription independent. The pattern of DNase-I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver-specific HSS-1 and HSS-2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS-1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS-2 deletion, while this transgene is very weakly (but nevertheless tissue-specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS-2, the DNA is in a potentially active configuration.


Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation.

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)


An improved procedure for reconstitution of the uncoupling protein and in-depth analysis of H+/OH- transport.

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H+ uptake in K(+)-driven exchange diffusion quantitatively analyzed. UCP is isolated and reconstituted with medium-length linear-chain alkyl polyoxyethylene. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 microliters/mg phospholipid with a vesicle diameter of 100 nm. One vesicle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H(+)-transport activity by UCP. Asolectin completely inactivates UCP. K(+)-gradient-driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H+ transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane-bound H+ pools. Total H+ uptake after addition of carbonylcyanine m-chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H(+)-transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K+ electrode and 86Rb measurements. Recording delta psi using 3,3'-diispropylthiadicarbocyanine shows a rapid generation of delta psi on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin. The delta psi collapses only after addition of external K+. By demonstrating that valinomycin-induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first-order rate, VH+/CH+, in which the rate, VH+, is related to H(+)-uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on delta psi of H+ transport is measured by varying external K+ concentration. A virtually linear relation of the rate to the K(+)-diffusion potential is observed, although the capacity is only slightly changed. The linear VH+/delta psi relationship resembles an open-channel type of transport, but is discussed in terms of a low-activation-barrier type of carrier mechanism, in contrast to the log (VH+/delta psi) relation found for the ADP/ATP carrier with high activation barriers.


The role of calcium and protein kinase C in the IgE-dependent activation of phosphatidylcholine-specific phospholipase D in a rat mast (RBL 2H3) cell line.

Our previous studies have suggested that phosphatidylcholine-specific phospholipase D (PtdCho-PLD) plays a role in IgE-dependent diacylglycerol production, protein kinase C activation and mediator release in the RBL 2H3 mast cell line. We have extended these studies to examine the mechanisms by which PtdCho-PLD may be regulated in these cells. RBL 2H3 cellular lipids were labeled with [14C]arachidonic acid or [3H]myristic acid, then PtdCho-PLD activity was monitored by the formation of radiolabeled phosphatidylethanol when ethanol was included in the incubation medium. Trinitrophenol-ovalbumin conjugate (10 ng/ml), when added to cells previously sensitized with anti-(trinitrophenelated mouse IgE) (0.5 microgram/ml), ionomycin (1 microM) and thapsigargin (0.1 microM), stimulated PtdCho-PLD activation and mediator release in cells incubated in buffer containing 1.8 mM calcium, but not in cells incubated in calcium-free, buffer. Phorbol 12-myristate 13-acetate (0.1 microM) activated PtdCho-PLD in both buffers, but on its own did not trigger mediator release. When intracellular calcium was chelated with 5,5'-dimethyl-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, trinitrophenol-ovalbumin conjugate failed to activate PtdCho-PLD and histamine release. Similarly, down-regulation of protein kinase C activity by long-term exposure to the phorbol ester (0.1 microM) and preincubation of the cells with protein kinase inhibitors resulted in the loss of the trinitrophenol-ovalbumin response on PtdCho-PLD activity and histamine release. Taken together, the above results suggest that IgE-dependent PtdCho-PLD activation is dependent on both activation of protein kinase C and a rise in the intracellular free calcium concentration.


Differential regulation of the transcript levels of some nuclear-encoded and mitochondrial-encoded respiratory-chain components in response to growth activation.

Biogenesis of mammalian mitochondria requires the participation of both nuclear and mitochondrial genes. In order to study the expression and coordination of these two sets of genes, serum-deprived, quiescent NIH 3T3 cells were activated by serum addition. The steady-state levels of the transcripts for two growth-response genes (the mitochondrial adenine-nucleotide translocator and non-mitochondrial beta-actin), one nuclear-encoded respiratory-chain component (F1-ATPase beta-subunit) and the mitochondrial-encoded subunit I of cytochrome oxidase decreased significantly in quiescent cells and were rapidly restored with similar kinetics after addition of serum. The transcripts for two additional nuclear-encoded mitochondrial genes (cytochrome c1 and cytochrome oxidase subunit IV) did not respond to serum deprivation or growth activation. These results imply that mitochondrial biogenesis is at least partially regulated through growth-dependent mechanisms. Furthermore, the expression of nuclear genes encoding mitochondrial respiratory-chain components does not appear to be tightly coordinated, suggesting the existence of multiple control circuits.