Effects of anoxia and aglycemia on the adult and immature hippocampus. Brief anoxic episodes induce a loss of consciousness due to an interruption of synaptic responses and repeated on more sustained episodes induce a selective neuronal loss in vulnerable regions notably in the CA1-H1 region of the hippocampus. The use of brain slices kept in vitro has considerably improved our understanding of the mechanisms underlying anoxia and ischemia and the strategies of development of protective drugs or manipulations. This report reviews our present understanding of the effects of anoxia-ischemia on neuronal activity in particular concerning the abrupt and major changes in vulnerability which occur with maturation of the brain.


EEG sleep in Cushing's disease and Cushing's syndrome: comparison with patients with major depressive disorder. Because patients with Cushing' syndrome (CS) and Major depressive disorder (MDD) share features of hypercortisolism and the depressive syndrome, we compared electro-encephalographic (EEG) sleep in patients with pituitary-ACTH-dependent Cushing's syndrome (Cushing's disease, CD), patients with ACTH-independent Cushing's syndrome (AICS), patients with major depressive disorder (MDD), and normal subjects. There were substantial similarities in the abnormal polysomnography profiles of patients with CD, AICS, and MDD. All three patient groups demonstrated poorer sleep continuity, shortened rapid eye movement (REM) latency, and increased first REM period density compared with normal subjects. In addition, AICS patients and MDD patients had elevated REM activity and density. These findings are discussed in terms of models of pathophysiology that relate abnormalities in sleep, mood, and hypothalamic-pituitary-adrenal function.


Platelet imipramine binding and plasma cortisol levels in Israeli civilians during the Gulf War. [3H] Imipramine binding to platelets and plasma cortisol levels were measured in nine Israeli civilians before, during, and after repeated missile attacks. Hamilton Anxiety Rating Scale (HARS), and Beck Depression Inventory (BDI) were similar before and during the war and decreased significantly after the war. A trend toward increase in platelet imipramine binding values was observed during the war when compared with the postwar values (19%; p = 0.056), and/or when compared with prewar values (26%; p = 0.063). However, one-way analysis of variance with repeated measures did not reveal a significant statistical difference [F (2,6) = p = 2.07; NS] among the three time points. A significant correlation was found between HARS score, but not BDI, and imipramine binding values in the prewar and postwar time points (r = 0.87; 0.71, respectively). Plasma cortisol levels did not alter significantly during the study period.


Effect of cocaine injections on the neuroendocrine response to the serotonin agonist MK-212. This study was undertaken to examine whether several of the hormones that can be released by activation of serotonin receptors will be affected by long-term cocaine administration. Male rats received cocaine injections (15 mg/kg, IP) twice daily for 7 days. Forty-two hr after the last cocaine injection, the rats were challenged with increasing doses (0, 1, 5, 10 mg/kg, IP) of the 5-HT1/5-HT2 agonist MK-212 (6-chloro-2-[1-piper-azinyl]-pyrazine). The following observations were made: (1) cocaine reduced the rate of body weight gain; (2) cocaine inhibited the stimulatory effect of MK-212 on plasma vasopressin, oxytocin, and prolactin concentrations and on plasma renin activity and concentration; (3) cocaine did not inhibit the stimulatory effect of MK-212 on plasma ACTH or corticosterone concentrations. The data indicate that a wide-spectrum 5-HT (serotonin) agonist such as MK-212 can reveal differential neuroendocrine responses. This effect could be related to cocaine-induced changes in the different 5-HT receptor subtypes that regulate the secretion of these hormones.


Chronic parvovirus infection in a presumably immunologically healthy woman. Infection due to parvovirus B19 is common and usually resolves over several weeks. Prolonged infection has been reported primarily in immunodeficient hosts. The present report describes a chronic infection in an apparently immunologically healthy woman. The illness was characterized by recurrent episodes of paresthesia without anemia. Laboratory studies demonstrated persistence of parvovirus-specific DNA for nearly 4 years.


Ganciclovir treatment of cytomegalovirus ventriculitis in a patient infected with human immunodeficiency virus. Histopathologic evidence of central nervous system involvement with cytomegalovirus (CMV) has been well recognized in patients infected with human immunodeficiency virus (HIV). However, clinically symptomatic disease has been decidedly less common. In this report, we describe a patient infected with HIV who developed an acute change in neurological status. Gadolinium-enhanced magnetic resonance imaging and analysis of cerebrospinal fluid revealed CMV ventriculitis and meningoencephalitis. Treatment with ganciclovir resulted in radiological improvement of the ventriculitis and negative CMV cultures but little clinical neurological improvement.


Discrete gastrointestinal mass lesions caused by cytomegalovirus in patients with AIDS: report of three cases and review. We report the detailed clinical features of discrete mass lesions of the gastrointestinal tract caused by cytomegalovirus in three patients who had the acquired immunodeficiency virus syndrome. The disease occurred in the fundus of the stomach in one patient and in the cecum in the other two persons. The symptoms as well as radiographic and endoscopic findings in each case are described and are shown to be indistinguishable from those resulting from a neoplasm. The diagnosis was established by the presence of inflammation with cytomegalovirus-like inclusions and confirmed by immunoperoxidase staining. Cytomegalovirus infection should be considered, along with Kaposi's sarcoma and lymphoma, as a cause of focal mass lesions of the alimentary tract in persons infected with HIV.


Presence of cytomegalovirus and herpes simplex virus in middle ear fluids from children with acute otitis media. Twenty-seven (10%) of 271 infants and children with acute otitis media (AOM) were found to be infected with cytomegalovirus (CMV) or herpes simplex virus type 1 (HSV). CMV or HSV, alone or in combination with bacteria or other viruses, was isolated from the middle ear fluid (MEF) of 10 patients. In three cases, CMV alone was isolated from the MEF, and in one case, HSV alone was isolated. One of the CMV cases involved an acute primary or reactivation of CMV infection, with CMV-bacterial otitis and conjunctivitis as major manifestations. One patient with AOM and stomatitis had purulent otitis associated with the presence of HSV in MEF, with no other bacterial or viral pathogens noted in MEF or nasal wash specimens. While most patients with CMV infection were probably asymptomatic excreters at the time of development of AOM, CMV did enter the middle ear. The presence of CMV in MEF was prolonged, and the patients continued to have clinical signs of otitis despite negative bacterial cultures. Among patients with bacterial otitis, a higher proportion of those who had CMV found only in nasal wash specimens had persistent bacteria in MEF, compared with those who were concurrently infected with other viruses (57% vs. 19%; P less than .04). This report is the first to suggest an etiologic role for CMV and HSV in AOM.


New observations on middle term hydroxyapatite-coated titanium alloy hip prostheses. HA-coated hip prostheses were retrieved from elderly patients after death. Histological analysis, scanning electron microscopy and microanalysis by energy-dispersive X-ray spectrometry were performed on the same sections. These revealed good osseointegration of the implant material and evolution of bone and material.


A new model of human secretory diarrhoea using cholera toxin. Secretory diarrhoea is a major cause of morbidity and mortality worldwide. However, there is no biologically relevant test system in man for assessing new anti-diarrhoeal therapies prior to clinical trial. We have used highly purified cholera toxin in combination with the triple lumen jejunal perfusion technique to establish a subclinical model of cholera in man. Cholera toxin was administered either by mouth with sodium bicarbonate or directly into a 30 cm 'open' or 'closed' (isolated between two inflated balloons) jejunal segment in healthy adult volunteers. Both oral dosing and direct delivery into an 'open' jejunal segment failed to produce consistent secretion of water and electrolytes. In contrast 15 micrograms or 25 micrograms of cholera toxin elicited secretion of water and sodium 3 h after instillation into the balloon occluded 'closed' jejunal segment (P less than 0.05 vs. controls). The rate of secretion was constant over the maximal period studied (4.5 h) and was similar to that reported in human cholera. None of the subjects experienced troublesome diarrhoea. We believe this model offers a relevant test system for assessing anti-diarrhoeal therapy in man.


Increased responsiveness of the vascular bed to angiotensin I, angiotensin II and phenylephrine in acute and chronic ischemic hindlimbs in rats. Ischemia activates several compensatory mechanisms to restore blood supply. To investigate possible changes in the reactivity of blood vessels after acute and chronic ischemia of skeletal muscle, the response (resistance changes) of the vascular bed to angiotensin II (AII) and phenylephrine (PE) in a hindlimb perfusion model were studied in control, acutely ischemic (45 min) and chronically ischemic (4 weeks) spontaneously hypertensive rats. Furthermore, the effects of angiotensin I (AI) were studied to investigate the involvement of local angiotensin-I-converting enzyme (ACE) in adaptive responses. Ischemia was induced by partial occlusion of the left common iliac artery. Both in acute and chronic ischemia, the reactivity (maximal resistance change) of the vascular bed in the ischemic hindlimb to AI, AII and PE was increased only in severe ischemia (residual flow < 40%), whereas the sensitivity (ED50) was not influenced. The increase in reactivity was comparable for AI and AII, implying that local ACE seems not to be involved. These results suggest that severe ischemia of skeletal muscle results in nonselective hyperreactivity of the vascular bed, which may be due to alterations of receptor-linked mechanisms or ultrastructural changes of blood vessels.


Reconstituted collagen is not capable of activating factor XII but causes intrinsic coagulation by activating platelets. Using a rheological technique to measure the coagulation of plasma in collagen-coated tubes, we studied the intrinsic coagulant activities of different types (I, III, IV and V) and structures of reconstituted collagen. Recalcified, platelet-free plasma (PFP) in contact with the collagen surface did not clot, irrespective of the type and structure of collagen. Coagulation of platelet-rich plasma (PRP) did occur, although the time of onset of coagulation was highly dependent on the type and structure of the collagen used. Coagulation of PRP occurred rapidly on a collagen surface consisting of highly ordered fibrils (banded structure) to which large numbers of aggregated platelets adhered with shape change. In contrast, initiation of coagulation was delayed in PRP after incubation with 5 mM dibutyrylcyclic AMP (DB-cAMP) for 50 min. Coagulation of PRP was completely suppressed after 30 min incubation with colchicine (5.0 mg/ml). This suggested that disruption of platelet microtubules completely suppressed the stimulation of platelets associated with the initiation of intrinsic coagulation on the collagen surface. We conclude that reconstituted collagen is not capable of activating factor XII. Initiation of coagulation of recalcified PRP in contact with the reconstituted collagen surface is caused by the activation of platelets.


Effects of ATP and UTP on [Ca2+]i, membrane potential and force in isolated rat small arteries. We have investigated excitation-contraction coupling mechanisms associated with the activation of purinoceptors and putative pyrimidinoceptors by assessing the effects of ATP and UTP on cytoplasmic Ca2+ activity ([Ca2+]i), membrane potential (Em) and force in rat mesenteric small arteries. UTP induced a sustained concentration-dependent contractions, closely associated with concentration-dependent increases in [Ca2+]i. Superfusion with 0.1 mM UTP caused a sustained depolarisation of 12 +/- 1 mV (SE, n = 8). In Ca(2+)-free medium, the increase in [Ca2+]i and the contraction obtained with UTP (1 mM) were both transient and were inhibited by prior exposure to noradrenaline (NA). In vessels depolarised with KCl, UTP caused no change in Em, but a sustained increase in force and a transient increase in [Ca2+]i were induced, leading to an increased force/[Ca2+]i ratio. Similar effects on [Ca2+]i, Em and force were observed with ATP; but the effect of ATP on force was transient, whereas the effect on [Ca2+]i and Em declined only slowly. There was no crosstachyphylaxis between the responses to ATP and UTP: in the presence of 1 mM of either, the other drug induced contractions in low concentrations, as if they acted through distinct receptors. The results suggest that both UTP and probably ATP release intracellular Ca2+, possibly from the stores emptied by NA. The sustained response to UTP appears to be due to an influx of extracellular Ca2+. UTP but not ATP was found to enhance the force-generating effect of [Ca2+]i.


Characterization of human tissue-type plasminogen activator variants with amino acid mutations in the kringle 1 domain. Recombinant variants of tissue-type plasminogen activator (t-PA) were constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Five variants were designed to improve the function of t-PA by mutagenesis in the kringle 1 (K1) domain. The amino acids were replaced with the corresponding residues present in the kringle 2 (K2) domain of native t-PA. The t-PA mutants expressed were as follows: variant E94V.D95G with point mutations in Glu94----Val and Asp95----Gly; variant N115P.S119M, Asn115----Pro and Ser119----Met; variant P125A.R129Q.R13OS, Pro125----Ala, Arg129----Gln and Arg130----Ser; variant G161R.K162R.-S165W, Gly161----Arg, Lys162----Arg and Ser165----Trp; and variant N115P, Asn115----Pro, respectively. The half-life following intravenous bolus injection in rabbits was prolonged in all variants except P125A.R130S. This was particularly true for N115P.S119M. The kinetic parameters for plasminogen activation were improved in t-PA G161R.K162R.S165W which showed a 0.6-fold decrease in Km, and a 1.8-fold increase in Vmax, thus promoting a 2.7-fold increase in kcat/Km compared to native t-PA. For a similar degree of thrombolysis in the rabbit jugular vein thrombosis model, the thrombolytic activity of G161R.K162R.S165W, at the dose tested, was four-fold greater than that of native t-PA. Thus, the substitution of the amino acids in the K1 domain with those corresponding in the K2 domain significantly enhanced the enzymatic activity of t-PA and improved the plasma survival.


Prognostic significance of blood coagulation tests in carcinoma of the lung and colon. Blood coagulation test results were collected prospectively in patients with previously untreated, advanced lung or colon cancer who entered into a clinical trial. In patients with colon cancer, reduced survival was associated (in univariate analysis) with higher values obtained at entry to the study for fibrinogen, fibrin(ogen) split products, antiplasmin, and fibrinopeptide A and accelerated euglobulin lysis times. In patients with non-small cell lung cancer, reduced survival was associated (in univariate analysis) with higher fibrinogen and fibrin(ogen) split products, platelet counts and activated partial thromboplastin times. In patients with small cell carcinoma of the lung, only higher activated partial thromboplastin times were associated (in univariate analysis) with reduced survival in patients with disseminated disease. In multivariate analysis, higher activated partial thromboplastin times were a significant independent predictor of survival for patients with non-small cell lung cancer limited to one hemithorax and with disseminated small cell carcinoma of the lung. Fibrin(ogen) split product levels were an independent predictor of survival for patients with disseminated non-small cell lung cancer as were both the fibrinogen and fibrinopeptide A levels for patients with disseminated colon cancer. These results suggest that certain tests of blood coagulation may be indicative of prognosis in lung and colon cancer. The heterogeneity of these results suggests that the mechanism(s), intensity, and pathophysiological significance of coagulation activation in cancer may differ between tumour types.


Optimal administration of dual screening tests for detecting a characteristic with special reference to low prevalence diseases. We consider the problem of deciding optimally whether a characteristic exists based on one or two screening tests. We discuss the relative merits of giving either one or two tests, including the order in which they might be given, as well as their costs. Operating in the Bayesian mode, we derive posterior distributions for the accuracies of the tests and the prevalence of the characteristic. Applications to detecting rare conditions, such as the AIDS virus, are discussed.


Cadmium block of calcium current in frog sympathetic neurons. Cd2+ blocks whole-cell calcium currents in frog sympathetic neurons by 50% at approximately 300 nM. Strong depolarizations rapidly reverse that blockade (tau = 1.3 ms at +120 mV). Reblocking follows bimolecular kinetics (rate = 1.2 x 10(8) M-1 s-1) at voltages where channels are mostly open (0 to +30 mV). The unblocking rate is approximately 50 s-1, so the dissociation constant calculated from the rate constants is approximately 400 nM. Steady-state block is strong at -80 mV, so closed channels can also be blocked. However, reblocking is extremely slow (tau = 1-2 s) at voltages where the channels are mostly closed. The rates for Cd2+ entry and exit are greater than 100-fold lower for closed channels than for open channels, and closed channels appear to be closed at both ends.


Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2. Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.


Conditional probability analysis for a domain model of Ca(2+)-inactivation of Ca2+ channels. The domain model of Ca2+ inactivation of Ca2+ channels, which has been used to explain rapid inactivation of whole cell Ca2+ currents in pancreatic beta cells, is applied to single-time and conditional open probability measurements on guinea pig ventricular myocyte Ca2+ channels. These two measurements greatly constrain the choice of kinetic constants in the model. Calculations with the model provide a simple quantitative explanation of recent experimental results, including a slow increase in the inactivation rate.


Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling. Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.


Determination of fluid and gel domain sizes in two-component, two-phase lipid bilayers. An electron spin resonance spin label study. The average sizes of fluid and gel domains in the two-component, two-phase system formed from mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine were determined from an analysis of the electron spin resonance spectral lineshapes of a dimyristoyl phosphatidylcholine-nitroxide spin label as a function of spin label concentration. The ratio, R, of the intensities measured at two magnetic field strengths was found to be diagnostic of a statistical distribution of spin labels in disconnected domains. R is defined as V'/2Vpp, where Vpp is the maximum intensity and V' is the intensity at a position in the wings of a first derivative electron spin resonance line that is a constant multiple of the peak-to-peak linewidth. The intensity ratio for Gaussian or Voigt lineshapes is less than or equal to the value for a Lorentzian lineshape. The intensity ratio was found to be greater than the value for a Lorentzian line when spectra from disconnected domains containing a statistical distribution of spin labels undergoing spin-spin interactions were summed. The intensity ratio, R, calculated by spectral simulations as a function of the average number of labels per domain, N, was found to increase to a maximum with increasing N and then to decrease. The dependence on spin label concentration of the experimentally measured intensity ratios paralleled this predicted behavior. A method is presented to calculate the average number of lipids per fluid or gel domain based on a knowledge of R, and of the distribution of the spin label between the fluid and gel phases determined from the phase diagram. The results demonstrate that the number of lipids per domain increases linearly from a fixed number of nucleation sites, as the fraction of the phase that is disconnected increases. At any given mole fraction of the particular phase, the gel domains are bigger than the fluid domains because they have a lower nucleation density. The results also suggest that the disconnected domains are, in most cases, nonrandomly distributed in the plane of the bilayer.


Theory of excitation-contraction coupling in cardiac muscle. The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.


Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method. Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.


Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 3. Effects of phosphorylation by protein kinase C. The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5'-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha 1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 1991. Biophys. J. 60:890-901). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses. The observed Ca channels exhibited two open states (tau o1 = 5 ms and tau o2 = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -20.5 mV and K = 10.5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC. The inactivation time constant during the pulse was slightly increased(from 1.2 to 1.6 s), and the channel availability at the holding potential was decreased from 76 to 71%. Taken together, the results revealed that PKC increased Pmax largely through a shift in the voltage independent open-close equilibrium of the fully activated channels.This is in contrast with the effect of phosphorylation by PKA (Mundir'a-Weilenmann, C., J. Ma, E. Rios, and M. M. Hosey. 1991. Biophys.J. 60:902-909), which also increases Pmax but mostly by increasing the availability of channels and slowing inactivation during the pulse.


Na channels that remain open throughout the cardiac action potential plateau. In this paper we report the direct measurement of rare Na channel events that occur during the cardiac action potential, viz., channels that open at the upstroke and remain open throughout the plateau and early repolarization phase. The technique we use allows us to record channel activity and action potentials at the same time; thus, we are certain of when the Na channels open and when they finally close. The slow Na channels have the same voltage dependence, single-channel conductance, and TTX sensitivity as the fast Na channels, and they conduct Li. It therefore seems likely that the fast and the slow currents flow through the same channel. If this interpretation is correct, then the Na channel not only initiates the action potential but also helps to maintain its plateau. It is possible that the slow Na currents represent a separate collection of channels rather than a low-probability state of the fast Na channels. Regardless of which interpretation is correct, the present experiments allow us to assess the effect of the slow currents on action potential shape and on sustained Na entry.


Strong electrostatic loop-helix interactions in bundle motif protein structures. Based on CHARMM potential (Brooks et al., 1983) an energetic analysis has been carried out for four typical 4-alpha-helix bundle proteins, i.e., methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin. The bovine somatotropin possesses long loops, but all the other three proteins have short loops. It was found that in all these four 4-alpha-helix bundle motif structures the interaction between loops and helices was much stronger than the interaction among the four helices themselves. Particularly for the electrostatic interaction energy, the loop-helix interaction is overwhelmingly stronger than the interhelix interaction although the latter involves the favorable helix dipole interaction due to the antiparallel arrangement of neighboring alpha-helices. The present study indicates that such a conclusion holds true regardless of what loops, long or short, are in the 4-alpha-helix bundle protein, and also regardless of which empirical potential, ECEPP or CHARMM, is used for calculations although in CHARMM the electrostatic energy is much more heavily emphasized than in ECEPP. Therefore, no appropriate conclusion can be drawn in arguing whether the dipole interaction among the four alpha-helices play a stabilizing role or destabilizing role for a 4-alpha-helix bundle protein without taking into consideration the effect of interaction between helices and loops. The calculated results reported here provide, from a different point of view, insights that might be useful for revealing the essence of the driving forces during the folding of proteins.


Quantitation of liquid-crystalline ordering in F-actin solutions. Actin filaments (F-actin) are important determinants of cellular shape and motility. These functions depend on the collective organization of numerous filaments with respect to both position and orientation in the cytoplasm. Much of the orientational organization arises spontaneously through liquid crystal formation in concentrated F-actin solutions. In studying this phenomenon, we found that solutions of purified F-actin undergo a continuous phase transition, from the isotropic state to a liquid crystalline state, when either the mean filament length or the actin concentration is increased above its respective threshold value. The phase diagram representing the threshold filament lengths and concentrations at which the phase transition occurs is consistent with that predicted by Flory's theory on solutions of noninteracting, rigid cylinders (Flory, 1956b). However, in contrast to other predictions based on this model, we found no evidence for the coexistence of isotropic and anisotropic phases. Furthermore, the phase transition proved to be temperature dependent, which suggests the existence of orientation-dependent interfilament interactions or of a temperature-dependent filament flexibility. We developed a simple method for growing undistorted fluorescent acrylodan-labeled F-actin liquid crystals; and we derived a simple theoretical treatment by which polarization-of-fluorescence measurements could be used to quantitate, for the first time, the degree of spontaneous filament ordering (nematic order parameter) in these F-actin liquid crystals. This order parameter was found to increase monotonically with both filament length and concentration. Actin liquid crystals can readily become distorted by a process known as "texturing." Zigzaging and helicoidal liquid crystalline textures which persisted in the absence of ATP were observed through the polarizing microscope. Possible texturing mechanisms are discussed.


A method for estimation of drug affinity constants to the open conformational state of calcium channels. The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.


Interrupting autocrine ligand-receptor binding: comparison between receptor blockers and ligand decoys. Stimulation of cell behavioral functions by ligand/receptor binding can be accomplished in autocrine fashion, where cells secrete ligand capable of binding to receptors on their own surfaces. This proximal secretion of autocrine ligands near the surface receptors on the secreting cell suggests that control of these systems by inhibitors of receptor/ligand binding may be more difficult than for systems involving exogenous ligands. Hence, it is of interest to predict the conditions under which successful inhibition of cell receptor binding by the autocrine ligand can be expected. Previous theoretical work using a compartmentalized model for autocrine cells has elucidated the conditions under which addition of solution decoys for the autocrine ligand can interrupt cell receptor/ligand binding via competitive binding of the secreted molecules (Forsten, K. E., and D. A. Lauffenburger. 1992. Biophys. J. 61:1-12.) We now apply a similar modeling approach to examine the addition of solution blockers targeted against the cell receptor. Comparison of the two alternative inhibition strategies reveals that a significantly lower concentration of receptor blockers, compared to ligand decoys, will obtain a high degree of inhibition. The more direct interruption scheme characteristic of the receptor blockers may make them a preferred strategy when feasible.


A-type potassium channel clusters revealed using a new statistical analysis of loose patch data. The spatial distribution of ion channels over the surface of a neuron is an important determinant of its excitable properties. We introduce two measures of channel clustering for use in patch-clamp experiments: a normalized chi-squared statistic (eta) and the number of zero-channel patches in a data set (Z). These statistics were calculated for data sets describing the distribution of A-type potassium channels on neurons of the nudibranch Doriopsilla and measurements of Ca-dependent outward current channels on bullfrog hair cells, as well as simulated channel distributions. When channels are clustered, eta is approximately equal to the amount of current in a cluster. The analysis shows that somatic A-channels in the nudibranch are distributed in clusters of approximately 50 channels each. The clusters are < 2 microns wide and are separated, on average, by 3.2 microns. Outward current channels on hair cells occur in clusters of approximately 27 channels each, in agreement with the original analysis. Channel clustering may reflect properties of the insertion or regulation of channels in the membrane.


Effect of firing rate on the calcium permeability in adult neurons during spontaneous action potentials. Calcium channels in neurons mediate a wide variety of essential functions. In addition to contributing to action potential shape, they furnish a substrate that acts as an intracellular second messenger. This study shows that the shape of the neuronal action potential has characteristics that promote long openings of L-type (high threshold) calcium channels. We also present evidence that a change in the firing rate of isolated neurons modulates gating of single calcium channels. This mechanism could be important in modulating neuron excitability and providing a rise in intracellular Ca, when needed.


Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells. The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.


Orientational distribution of spin-labeled actin oriented by flow. Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.


Polarization sensitive coherent anti-Stokes Raman scattering spectroscopy of the amide I band of proteins in solutions. Polarization sensitive coherent anti-Stokes Raman scattering (PCARS) spectroscopy is a fruitful technique to study Raman vibrations of diluted molecules under off-electron resonant conditions. We apply PCARS as a direct spectroscopic method to investigate the broad amide I band of proteins in heavy water. In spontaneous Raman spectroscopy, this band is not well resolved. We fit a number of spectra taken of each protein under different polarization conditions, with a single set of parameters. It then appears that some substructure is observed in the amide I band. From this substructure, we determine the percentage of alpha-helix, beta-sheet, and random coil for the proteins lysozyme, albumin, ribonuclease A, and alpha-chymotrypsin.


Base pair selectivity in the binding of copper (II) tetrakis (4-N-methylpyridyl)porphine to polynucleotides under closely packed conditions. The base pair selectivity of the intercalative binding of the copper porphyrin, copper (II) tetrakis(4-N-methylpyridyl)porphine (Cu(II)TMpyP-4), to DNA has been investigated using a variety of DNA types and the synthetic polynucleotides poly(dG-dC)2 and poly(dA-dT)2. The studies utilize electron paramagnetic resonance of concentrated gels which are thought to mimic the closely packed state of nuclear DNA. The results indicate that intercalation of this porphyrin is preferred for sites containing two adjacent G-C base pairs, irrespective of sequence.


Effects of verapamil, gallopamil, diltiazem and nifedipine on sarcoplasmic reticulum function in rat heart. We investigated the effect of the calcium antagonists verapamil, gallopamil, diltiazem and nifedipine on cardiac sarcoplasmic reticulum function. In a cell-free homogenate from rat hearts, oxalate-supported Ca uptake was stimulated by verapamil, gallopamil and diltiazem at concentrations in the order of 10 nM to 100 nM, while higher concentrations were ineffective. Nifedipine was also ineffective. Peak stimulation of Ca uptake averaged 15-20% of control. Ca uptake is the difference between active Ca transport by Ca-ATPase and passive efflux through sarcoplasmic reticulum channels. In the presence of 300 microM ryanodine, which blocks sarcoplasmic reticulum channels, Ca uptake increased by 50%, but no further stimulation was produced by the addition of any calcium antagonist, at concentrations ranging from 1 nM to 100 microM. In a fraction enriched in sarcoplasmic reticulum, no drug affected the activity of Ca-ATPase at concentrations able to stimulate Ca uptake. We conclude that low concentrations of verapamil, gallopamil and diltiazem reduce Ca efflux through the Ca channels of the sarcoplasmic reticulum. Such an action might contribute to the clinical effect of these drugs.


Differential hydration of homopurine sequences relative to alternating purine/pyrimidine sequences. The minor groove ligand distamycin A has been used to probe the relative hydration of the minor groove of eight synthetic polynucleotides of known sequence and composition. A combination of densimetric, calorimetric, and temperature-dependent spectroscopic techniques have been used to obtain complete thermodynamic profiles (delta Gzero, delta Hzero, delta Szero, and delta Vzero) for the association of distamycin A to all polymer duplexes. In 10 mM phosphate buffer, pH 7, binding of the drug to each of the polymeric duplexes resulted in characteristic negative changes in both the volume and enthalpy. Although the binding constants were found to be identical for pairs of isomer polynucleotides having identical compositions but different sequences, the values of delta Hzero, delta Szero, and delta Vzero of each such pair were remarkably different. The entropy changes were found to roughly parallel the volume changes; no such trend was seen between delta Hzero and delta Vzero. The data support the hypothesis that the volume changes observed for these systems reflect the coulombic-hydration contribution to the entropy. The heteropolymer duplexes generated much larger exothermic contributions, less favorable entropies and larger volume contractions than did the corresponding homopolymer duplexes of identical composition, and strongly suggest that polynucleotides with homopurine sequences are more hydrated than polynucleotides with alternating purine/pyrimidine sequences. In addition, it was found that duplexes containing guanine sharply reduced the affinity for the drug, also lowering the exothermicity but raising the entropy. This may be explained by the presence of an amino group in the minor groove that prevents hydrogen bonding. Substitution of the guanine with inosine reversed this trend in the thermodynamic properties. Furthermore, substitution of poly(dA) for poly(rA) in a duplex produced a similar reduction in the affinity, while raising the exothermic contribution and greatly reducing the favorable entropy effect in agreement with an apparent increase in the hydration state.


Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH. In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.


Fine needle aspiration cytology of cutaneous vascular tumours. We have studied 12 cutaneous vascular tumours by means of fine-needle aspiration cytology (FNAC): six capillary haemangiomas, one cavernous haemangioma, one Masson's pseudo-angiosarcoma, two angiosarcomas, one benign haemangioendothelioma and one glomus tumour. We describe the main cytopathological findings and we discuss the differential diagnosis in each case. We consider that the cytopathological findings of the above lesions, evaluated in the context of the clinical findings, are sufficiently characteristic for us to be able to make a definitive diagnosis. We believe that FNAC can play an important part in the diagnosis and therapeutic planning of these tumours.


Acute hepatitis in childhood: virological, immunological and clinical aspects. Virological, immunological and clinical findings in 7 previously healthy children, aged 18 months to 11 years, with viral hepatitis are reported. Asymptomatic and fully recovering, although protracted, hepatitis B was diagnosed by chance in a 1 1/2 year-old boy. Anicteric and short-term hepatitis occurred in three children with Epstein-Barr virus infection, concomitantly with typical mononucleosis syndrome. On the contrary, cytomegalovirus (CMV)-associated hepatitis was severe and protracted in two children, and fatal in a 4-year-old girl, whose main autoptic finding was submassive hepatic necrosis. Therefore, our study showed that acute viral hepatitis in non-immunocompromised children is generally self-limited and that CMV hepatitis is more frequent and severe than commonly believed.


Inosine-containing primers in human papillomavirus detection by polymerase chain reaction. A PCR protocol has been developed using inosine-containing primers for human papilloma virus (HPV) detection. The HPV16 L1 region from CaSki-positive cells was efficiently amplified by a single reaction and directly analyzed by agarose gel electrophoresis. The method was found to be sensitive and reproducible, and it easy to use for HPV typing and screening.


Insulin-dependent release of 5'-nucleotidase and alkaline phosphatase from liver plasma membranes. Insulin treatment of isolated liver plasma membranes induced the release of 5'-nucleotidase and alkaline phosphatase. This effect was maximal at physiological hormone concentrations, being 36% and 17% for 5'-nucleotidase and alkaline phosphatase respectively, and was fully mimicked by the phosphatidylinositol specific phospholipase C (PI-PLC), thus confirming the presence of a glycosylphosphatidylinositol anchoring-system for these exofacial enzymatic proteins. The complete inhibition of insulin dependent enzyme release by neomycin is strongly supportive of an involvement of membrane-located PI-PLC activity. In addition, the insulin-like effect on enzyme release induced by the GTP non-hydrolysable analog, GTP-gamma-S, and its sensitivity to the pertussis toxin are in favour of a mediatory role exerted by the G proteins system, in the transduction of some actions of insulin.


Membrane vesicles from brown adipose tissue: a tool for the study of amino acid transport. The case of L-alanine. A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a linear (Kd = 0.498 min-1) and Na(+)-dependent saturable component (Km = 2.3 mM) and a Vmax = 19.9 pmol/micrograms protein.min). This pattern is similar to that shown by cells isolated from brown adipose tissue.


Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells. TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.


Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C. Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosyl phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.


On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts. Recombinant human interleukin-1 beta (IL-1 beta) and bradykinin (BK) synergistically stimulate prostaglandin E2 (PGE2) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1 beta per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1 beta, caused a rapid, transient rise of intracellular Ca2+ concentration ([Ca2+]i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1 beta did not change the effect of BK on [Ca2+]i. Ionomycin and A23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1 beta on PGE2 formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1 beta on PGE2 formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE2. In IL-1 beta treated cells, the enhancement of PGE2 formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1 beta. These data show that i) the synergistic interaction between IL-1 beta and BK on PGE2 formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca2+]i; ii) the signal transducing mechanism by which BK interacts with IL-1 beta, however, may be linked to a BK induced stimulation of [Ca2+]i and/or protein kinase C; iii) the mechanism involved in the action of IL-1 beta may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.


Mechanisms towards compensation of long-term haemopoietic injury in mice after 5 Gy irradiation: in vivo and in vitro enhancement of superoxide anion production by granulocytes. This paper analyzes the long-term (6 and 12 months) function of mouse granulocytes after total body irradiation with a single dose (5 Gy) of X-rays. Superoxide anion production has been investigated in granulocytes from peripheral blood, and also in those harvested from long term bone marrow cultures, with the aim of correlating the environmental damage induced by radiation with the functional properties of granulocytes. An in vivo and in vitro enhancement of superoxide anion production and protein levels in granulocytes from irradiated mice is described. The presence of some colony stimulating factor in the supernatant of cultures from irradiated mice could play an important role in the priming of granulocytes.


CCK modulates inhibitory synaptic transmission in the solitary complex through CCKB sites. The CCKB antagonists L-365, 260 and PD134308 and the CCKA antagonist L-364, 718 were applied to neurones of the rat solitary complex (SC) isolated in brainstem slices, while recording either intracellularly or by whole-cell patch-clamp. The CCKB antagonists increased the amplitude of spontaneous or solitary tract-evoked Cl(-)-dependent inhibitory synaptic events by 25 +/- 5% in 5/7 neurones. These events were identified as (1) reversed spontaneous potentials, (2) reversed multisynaptic potentials and (3) outward currents blocked by the GABAA antagonist bicuculline. The CCKB antagonists had no postsynaptic action and increased excitatory synaptic events by 16 +/- 5% in only 3/9 neurones. The CCKA antagonist depolarized neurones but had no effect on synaptic events. Results suggest that CCK, released from the SC tissue, modulates GABAergic interneurones through CCKB sites. This mechanism could contribute to panic attacks, probably mediated by CCKB receptors.


Metabotropic glutamate receptor agonists reduce epileptiform activity in the rat cortex. Slices of rat cingulate cortex, when incubated in magnesium-free medium, produce spontaneous epileptiform spikes. Here it is demonstrated that the metabotropic glutamate receptor (mGluR) agonists +/- trans-1-amino-cyclopentane-1,3-dicarboxylic acid (+/- Trans-ACPD), IS3R-ACPD and quisqualic acid (quis) can reduce the frequency of these bursts in a concentration-dependent manner. The IC50 values were 16, 12 and 26 microM, respectively. The low concentrations of +/- trans- and IS3R-ACPD used, plus the lack of NMDA antagonism shown by these compounds, suggest that the effect may be via a presynaptic reduction in glutamate release. The relative potency of the agonists IS3R-ACPD > Trans-ACPD > quis would seem to suggest that the mGluR1 receptor is not involved.


Pattern dependent form of synaptic modification and its quantal origins. Presynaptic mechanisms of post-activation changes of synaptic efficacy have been quantitatively reconstructed via computer simulations with the previously suggested double-barrier quantal model of a chemical synapse. Successful in predicting the global changes in synaptic efficacy during and after short trains of presynaptic impulses, computer reconstructions have revealed in the post-activation period the stimulus dependent form of synapse modification from short-term to longer-term plasticity. An analysis of the quantal origins of the phenomenon suggests semantic or S-pattern as the trigger of the phenomenon. The property of a semantic pattern to modify synaptic plasticity is presented in the form of the rule of S-pattern dependent synaptic modification or S-modification.


Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors. We have constructed recombinant baculoviruses containing cDNAs encoding either the alpha 1- or the beta 1-subunit of the bovine GABAA receptor. In Spodoptera frugiperda (IPLB-Sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional GABAA receptors were detected by whole-cell electrophysiological recordings. The threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 x 10(-6) M GABA, and for the co-infected cells was 8 x 10(-8) M GABA, suggesting that hetero-oligomeric channels formed in these cells. All GABA-induced currents were found to be inhibited by bicuculline and picrotoxin, potentiated by pentobarbital but were insensitive to benzodiazepines.


Ectopic granule cells in mouse cerebellum molecular layer express high affinity GABAA receptor. The antimitotic/mutagenic agent methylazoxymethanol when injected in mice pups at postnatal days 3 and 4 produces hypogranular adult cerebella with subpial cells clusters and with a supplementary, ectopic granule cells layer in the molecular layer. The internal granular layer of these treated mice displayed a much lower density of [3H]muscimol binding sites than in controls. At the same time these binding sites are expressed in the ectopic granule cells in the middle of the molecular layer, in spite of the unusual localization of the cells and of the alteration of their nerve inputs. The subpial cells do not express these sites. Our autoradiographic data confirm the suggestion that during ontogeny the GABAA receptors in the granule cells appear when these cells leave the subpial region and indicate that the expressed receptor subtype is the same whether granule cells are in the internal granular layer or in the middle of the molecular layer.


Redox modulation of NMDA receptor-mediated synaptic activity in the hippocampus. The sulfhydryl reducing agent dithiothreitol (DTT) and the oxidizing agent 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reversibly modulate the component of synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in slices of hippocampal area CA1. DTT (1 mM) reversibly potentiates NMDA receptor-mediated synaptic potentials while DTNB (1 mM) has the opposite effect. However, treatment of the slices with the irreversible sulfhydryl alkylating agent N-ethylmaleimide (300 microM) prevents DTNB from reversing the potentiation induced by DTT. These results further implicate the redox modulatory site as a regulator of the NMDA receptor-channel complex in vivo.


Localization of binding sites for oxytocin in the brain of the golden hamster. Using a radioiodinated ligand and autoradiography on film, specific binding sites for oxytocin were detected in the brain of the golden hamster. Sites were present in the endopiriform nucleus, the cingulate cortex, the islands of Calleja, the lateral septum, the dorsal hippocampus and the amygdala. The distribution of these binding sites was similar in males and females and was independent of the photoperiod. No binding sites were detected in the ventromedial hypothalamic nucleus or in the dorsal motor nucleus of the vagus nerve; areas which were labelled in the rat and where oxytocin is thought to participate in the control of sexual and autonomic functions. These data suggest that the role of oxytocin in the brain may differ among mammalian species.


Barbiturate sensitive components of visual ERPs in a reptile. The selective effects of methohexital anesthesia were used to differentiate components of visual event-related brain potentials (ERPs) in pond turtles (Pseudemys scripta). Tectal and forebrain omitted stimulus potentials (OSPs) were found to be particularly sensitive to the barbiturate; they are reversibly abolished while the large early wave of the forebrain flash visual evoked potential (VEP; 110-120 ms) is reduced by only 27 +/- 11% and that of the tectal VEP (55-65 ms) is increased by 40 +/- 12%. Concurrent with the decline of the OSP is the loss of late slow wave components (ca. greater than 125 ms) of forebrain and tectal VEPs and the appearance of irregularities in the responses of 5 Hz repetitive flashing. The barbiturate effects on the VEP and recovery cycles are remarkably similar to those reported in mammals.


Molecular characterization of type 1 serine/threonine phosphatases from Brassica oleracea. We describe the isolation of cDNA clones encoding type 1 serine/threonine protein phosphatase (PP1) from Brassica oleracea stigmas. We demonstrate that PP1 form a multigene family in Brassica. Within their most conserved domain, these phosphatases are 80-90% identical at the amino acid level. One cDNA (BoPP1) was found to encode a protein that shows 78-80% sequence identity to maize, rabbit, and yeast PP1. The accumulation of BoPP1 mRNA is developmentally regulated. Varying levels of BoPP1-homologous transcripts were detected in leaves, cotyledons, pistils, anthers and roots. In addition, distinct species of BoPP1 transcripts accumulated at different stages of Brassica microspore development, and mature trinucleate microspores contained a unique BoPP1 mRNA species not found at other stages of the plant's life cycle. Lastly, we show by genomic Southern blots that the Brassica genome might contain homologues of the mammalian PP1 inhibitor-1.


Phospholipids in the nucleus--metabolism and possible functions. Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca2+ homoeostasis.


[Role of limbic structures in the mechanisms of post-traumatic epilepsy]. It was shown in the experiments on rats that intracerebroventricular administration of kainic acid (0.01, 0.05 microgram) after brain trauma, resulted in the occurrence of behavioral and electrographic convulsive disturbances; maximal expression of epileptic activity was obtained in entorhinal cortex and ventral hippocampus. Kainic acid induced epileptic reactions in nontraumatized rats only if injected in dose 0.1 microgram. Brain trauma did not lead to changes in seizures intensity induced by systemic picrotoxin administration. It is concluded that the formation of generator of pathologically enhanced excitation in limbic structures via increase of excitor glutamatergic neurotransmission is the important mechanism of traumatic epilepsy.


[Adrenergic mechanisms in the formation of adaptive response of different tissues]. The proliferative tissue activity of the small intestine, kidney, liver and submandibular salivary gland was studied in 10-h immobilization after blocking peripheral beta-adrenoreceptors by obsidan. It is shown that the proliferation increased in all the tissues examined, except the submandibular gland. The injection of obsidan before and during immobilization led to a decrease and delay of DNA synthesis (the intestine, liver), short-term proliferative inhibition (kidney), to stimulation of stress-inhibited proliferation (submandibular gland). It is suggested that the pattern of tissue adaptive response is defined by beta-adrenergic mechanisms.


[Effects of multipotent hematopoietic stem cell inhibitory factor on formation of immune response to viral antigen]. An immunostimulating effect of PIF was studied. The augmentation of antibody production to Coxsackie A13 virus as well as protective effect during influenza infection have been found out in mice after PIF injections. An immunostimulating effect of PIF after SRBC immunization of mice has been also revealed. Possible mechanisms of immunostimulating effect of PIF are discussed.


[Morphofunctional characteristics of wound inflicted by an ultrasonic destroyer]. A surgical model of EstoRex ultrasound destroyer operating at a frequency of 60 KHz, power of 6 W, and vibration amplitude at the tip of the tool of 15 microns was used to make incisions on rat liver. 5 to 7 s or 24 hr after surgery the specimens of the wound wall were fixed and further processed for electron microscopy and histochemical visualization of glucose-6-phosphatase at the ultrastructural level. In a separate series 2 mm-thick strips of the tissue were excised from the liver, processed by the destroyer for 45 s, and then exposed to a digestion treatment with mixture of trypsin and chymotrypsin for 24 hr at 37 degrees C or in solution of cathepsin L for 60 hr at 25 degrees C. The results showed that ultrasound caused not only thermal but also nonthermal ultrastructural and histochemical alterations, due probably to cavitation and viscous stresses. The ultrasound wound did not contain any proteolytically resistant material. Since ultrasound-processed tissue turned out to be highly susceptible to proteolytic digestion we suggest that the ultrasound destroyer, unlike Nd:YAG laser surgical instrument, would be a promising surgical tool with respect to wound cleaning and healing.


[Use of spectral characteristics of ryodipine in the study of dihydropyridine receptor complex of neuronal membranes]. A fluorescent dihydropyridine (DHP) derivative, ryodipine, was used to study structural characteristics of the DHP-sensitive Ca-channels in nerve terminals (synaptosomes) isolated from the rat cerebral cortex. It was found that an inductive resonance energy transfer from membrane proteins to ryodipine occurred in synaptosomal membranes. Two groups of membrane proteins differentially accessible to ryodipine were found by quenching of their own fluorescence. The percentage of group I proteins (20%) whose fluorescence was quenched by up to 1 microM ryodipine, was increased by 50% upon K(+)-depolarization and remained unchanged upon the addition of 100 microM Ni2+, whereas the addition of 100 microM Cd2+ prevented the increase induced by K(+)-depolarization. Nifedipine and nicardipine competed with ryodipine for the DHP receptor as evidenced by the change in percentage of group I proteins. The percentage of group II proteins (50% at 10 microM ryodipine) remained unchanged during various functional alterations of the synaptosomal membranes. Model experiments on proteoliposomes demonstrated that binding of ryodipine to synaptosomal membranes was due mainly to the hydrophobicity of DHP but not the ligand-receptor interaction. Nonetheless we that the membrane proteins-ryodipine system could be a qualitative test for the functional state of DHP-sensitive Ca-channels.


Anisodamine antagonizes acetylcholine-induced inhibition of adrenergic neurotransmission in the canine saphenous vein. The effect of anisodamine, an alkaloid structurally related to atropine and isolated from a Chinese herb, on adrenergic neurotransmission was studied using isolated canine saphenous veins. Helical strips of vein were incubated in modified physiological salt solution containing L-(7- 3H)-norepinephrine (3 x 10(-7) mol/L) for two hours and then mounted for isometric tension recording and superfusion. Increases in isometric tension and efflux of total tritiated compounds and 3H-norepinephrine evoked by electrical stimulation were inhibited by acetylcholine (10(-6) mol/L). This effect of acetylcholine was antagonized to comparable levels by anisodamine (10(-6) mol/L) or atropine (10(-6) mol/L). The results demonstrate that anisodamine blocks prejunctional muscarinic receptors.


Retinoic acid counteracts both the downregulation of thrombomodulin and the induction of tissue factor in cultured human endothelial cells exposed to tumor necrosis factor. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) shift the hemostatic balance of endothelial cell surfaces in favor of prothrombotic properties by downregulating thrombomodulin (TM) and inducing tissue factor (TF) expression. We investigated the effects of retinoic acid (RA) on the prothrombotic properties of cultured umbilical vein endothelial cells exposed to TNF-alpha. The approximate 50% downregulation of TM antigen and cofactor activity induced by TNF-alpha (10 U/mL for 24 hours) was completely prevented when the cells were coincubated with both TNF-alpha and 10 mumol/L RA. In accordance with changes in cell surface TM antigen levels, the 70% decrease in TM messenger RNA (mRNA) induced by TNF-alpha was also prevented by 10 mumol/L RA. TNF-alpha induced TF activity of lysed cells (100-fold greater than untreated controls), an effect prevented when the cells were coincubated with both the TNF-alpha and 10 mumol/L RA. The 34-fold increase in TF mRNA levels induced by TNF-alpha (10 U/mL for 3 hours) was only two-fold in the presence of both TNF-alpha and RA. The effects of RA on the regulation of TM and TF expression in the cells exposed to TNF-alpha was dose-dependent from 0.01 to 10 mumol/L RA. The present results suggest that RA may affect on the mRNA level to alter TM and TF expression, effectively counteracting expression of prothrombotic properties of endothelial cells induced by inflammatory cytokines such as TNF-alpha.


Unexpected consistent involvement of V beta gene segments in inappropriate T-cell receptor beta gene rearrangements occurring in B-lineage acute lymphoblastic leukemias. T-cell receptor beta (TCR beta) gene rearrangements occur in a third of early B-cell acute lymphoblastic leukemias (ALLs). V, D, and J segments involved in these inappropriate rearrangements remain unknown and are of interest, both because partial D beta J beta and complete V beta D beta J beta recombinations occur at distinct stages of thymic maturation and because these rearrangements are regulated differently. We have therefore studied in detail seven cases of B-lineage ALL that show inappropriate clonal TCR beta gene rearrangements. Analysis of genomic DNA by Southern hybridization with C beta, J beta 1, V beta 8, and V beta 11 probes suggested the involvement of V beta segment in tumor cell rearrangements. A complete genomic library constructed from one case was screened with a C beta probe, and the TCR beta gene rearrangement was cloned and fully sequenced to show an out of frame V beta 2.2-J beta 2.6 recombination. TCR beta gene rearrangements occurring in other cases were further analyzed by polymerase chain reaction (PCR) using J beta and V beta primers and the resulting specific PCR products were sequenced. Evidence of clonal V beta rearrangements was obtained in all cases. These unexpected findings represent the first definitive demonstration that complete V beta(D beta)J beta rearrangements can occur in B-lineage cells and contrast with the previously reported lack of V beta(D beta)J beta rearrangement in B cells from V beta-J beta-C beta-E mu transgenic mice. In the context of increasing evidence that rearrangements are linked to transcription of unrearranged gene segments, these data prompt a search in B-lineage ALL cells for the presence of germline V beta transcripts whose deregulated expression may be linked to early transforming events.


Roles of heme insertion and the mannose-6-phosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase. Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme-free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO-related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)


Circulating cytomegalovirus (CMV) neutralizing activity in bone marrow transplant recipients: comparison of passive immunity in a randomized study of four intravenous IgG products administered to CMV-seronegative patients. Forty-two cytomegalovirus (CMV)-seronegative bone marrow transplant (BMT) recipients were randomized in a double-blind fashion to receive one of four commercially available intravenous Ig (IVIgG) products (Gamimmune N, Immune Globulin Intravenous, Gammagard, or Sandoglobulin) at a dose of 500 mg/kg every other week. The four treatment groups were similar in distribution of patient ages, weights, autologous versus allogeneic donor type, and underlying diseases. Every other week administration of IVIgG provided total serum IgG levels within the physiologic range for age. CMV titers by latex agglutination were stable (average geometric mean titer of 18.4 after the second IVIgG dose), with no statistically significant differences among the four product groups. CMV neutralizing activity (CMVNA) and CMV enzyme-linked immunosorbent assay (ELISA) titers were determined on a subset of sera from 27 study patients representing the four product groups. Patient serum samples obtained before IVIgG infusions and 2 weeks after the second IVIgG dose (ie, 3 weeks post-BMT) were assayed for CMVNA and CMV ELISA titers. Geometric mean titers of CMVNA and CMV ELISA varied among the product groups. The highest mean 50% CMVNA was 1:43 for product B, whereas the lowest mean 50% CMVNA was 1:14 for product A; two of the IVIgG product groups showed intermediate 50% mean titers of 1:27 (product C) and 1:26 (product D) for an overall P = .02. CMV ELISA titers (expressed as Paul Ehrlich International units [PEI U]) also showed the highest mean of 2.95 PEI U/mL for product B and the lowest mean of 1.34 PEI U/mL for product A. Intermediate mean values of 2.27 PEI U/mL and 2.03 PEI U/mL were obtained with products C and D, respectively (overall P = .003). The CMV ELISA titers show a minimal correlation (r = .566) to the observed CMVNA titers. We conclude that commercially available IVIgG products provide passive CMVNA, and that the level of circulating CMVNA is affected by the IVIgG product used.


DNA topoisomerase-targeting antitumor agents and drug resistance. A review of the chemotherapeutic agents which have been developed by targeting DNA topoisomerase I and II is presented. Camptothecins as topoisomerase I-targeting agents and newly developed topoisomerase II-targeting agents with unique properties are expected to be promising anticancer agents in the near future. An important issue is how cellular sensitivity to these agents is controlled. One approach is to establish and characterize drug-resistant human cancer cell lines, which would provide powerful tools to understand their intracellular target sites and also the mechanisms for acquirement of drug resistance to topoisomerase inhibitors. Drug resistance to topoisomerase-targeting agents appears to be closely correlated with two events, namely decreased expression and point mutation of topoisomerase genes.


A phase I clinical and pharmacokinetic study of the topoisomerase I inhibitor topotecan (SK&F 104864) given as an intravenous bolus every 21 days. Topotecan (SK&F 104864) is a novel antitumor agent whose mechanism of action is inhibition of the DNA unwinding protein topoisomerase I. An analog of camptothecin, topotecan was designed to be more water soluble in an effort to decrease the severe and sporadic toxicities experienced during phase I/II trials of the parent compound. In this phase I clinical and pharmacological trial, topotecan was given as a bolus intravenous (i.v.) infusion over 30 min every 21 days. A total of 42 patients entered the study, receiving doses ranging from 2.5 to 22.5 mg/m2. The maximum tolerated dose (MTD) of topotecan given in this schedule was 22.5 mg/m2. Myelosuppression, primarily neutropenia, was dose-limiting. The extent of prior therapy did not predict for more severe neutropenia. Non-hematologic toxicities were mild and included low-grade to moderate fever, nausea, vomiting, alopecia, diarrhea and skin rashes. There were no objective partial or complete responses, although there was a suggestion of antitumor activity in three patients. Topotecan undergoes pH-dependent hydrolysis of the lactone ring; only the closed, lactone form is active. The lactone form predominated during infusion, with hydrolysis occurring rapidly following the end of infusion. There were linear relationships between dose administered and peak plasma lactone concentrations as well as AUC lactone to AUC total. The lactone was rapidly cleared from plasma with a total body clearance of 25.7 (+/- 6.7) l/h/m2. The plasma lactone concentration declined rapidly with a harmonic mean terminal half-life of 3.4 (+/- 1.1)h. Lactone hydrolysis and renal excretion were the major routes of elimination.(ABSTRACT TRUNCATED AT 250 WORDS)


Multidrug resistance in a small cell lung cancer line: rapid selection with etoposide and differential chemosensitization with cyclosporin A. We developed a multidrug resistant small cell lung cancer line, VPR-2, by exposing H69 parent cells to etoposide (20 microM) for 1 h daily for 3 days every 21-28 days, a schedule similar to that used in the clinic. Resistance (20-fold) to the cytostatic and DNA cleavage activities of etoposide emerged after the third treatment, and this phenotype was stable in the absence of drug exposure for 2.5 years. VPR-2 cells exhibited cross resistance to intercalating agents and vinca alkaloids, but remained sensitive to X-radiation, cisplatin and 5-fluorouracil. The human mdr1 gene was overexpressed in the resistant line, but steady-state concentrations of etoposide were reduced only 1.5-fold. Topoisomerase II catalytic and etoposide stimulated DNA cleavage activity in nuclear extracts from both lines were identical despite retention of a 3-fold level of resistance to etoposide-induced strand breaks in isolated nuclei from VPR-2 cells. Cyclosporin A and verapamil, both of which bind to P-glycoprotein, enhanced accumulation of etoposide in VPR-2 cells, and H69 cells to a lesser extent. Yet only cyclosporin A was effective in differentially enhancing etoposide cytostasis in VPR-2 relative to H69. In VPR-2 whole cells, cyclosporin A enhanced etoposide-induced DNA single-strand break frequency 9-fold but had no effect in isolated nuclei. Rapid selection of this line with a clinically relevant drug exposure schema and stability of the resistant phenotype suggest these cells may have been a steady subpopulation of the parent line through years of serial passage in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)


Efficacy of rifaximin on symptoms of uncomplicated diverticular disease of the colon. A pilot multicentre open trial. Diverticular Disease Study Group. Diverticular disease of the colon is a common health problem in western societies. Most patients with colonic diverticula are asymptomatic; it has been estimated that only 20% of individuals harboring diverticula will develop symptoms and signs of illness and a minority will develop major complications. Although the efficacy of a high fiber diet in the management of symptomatic uncomplicated diverticular disease is still controversial, bran and bulking agents are commonly used. Antibiotics are used to treat major inflammatory complications of diverticular disease but apparently there is no rationale for the use of antibiotics in uncomplicated disease where an inflammatory component is by definition excluded. In a multicenter open trial, 217 patients with symptomatic uncomplicated diverticular disease were treated with glucomannan (110 pts) or with glucomannan plus a poorly absorbable antibiotic (rifaximin 400 mg bid for 7 days each month) (107 pts). Clinical evaluation was performed bimonthly for 12 months using a global score system for 8 clinical variables. After 12 months, patients treated with glucomannan plus rifaximin showed a 63.9% reduction of the score as compared to 47.6% in patients treated with glucomannan only (p < 0.001). Cyclic administration of rifaximin appears to be of some advantage in obtaining symptomatic relief in uncomplicated diverticular disease.


Spontaneous rupture of hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is the most frequent malignant tumour of the liver. HCC has an incidence that changes with geographic areas (1.2-2.5% in western countries and 13-53% in Asia and Africa) as the risk of tumour bleeding. The patient arrives to the surgeon in emergency with no possibility of radical resection because of the patient's general conditions, the tumour's stage and the cirrhosis. Palliative treatments are: resection, direct suture of the bleeding tumour, artery embolization and selective binding of the hepatic artery. The Authors describe two cases of spontaneous rupture of HCC observed in the surgical department of Venice Hospital. A review of the literature is also reported.


Circadian and photoperiodic time measurement in male Syrian hamsters following lesions of the melatonin-binding sites of the paraventricular thalamus. Autoradiographic studies using [125I]iodomelatonin in several species, including the Syrian hamster, have revealed that the rostral region of the anterior paraventricular nucleus of the thalamus (aPVT) contains a very high density of binding sites for melatonin. In two studies, small or large bilateral electrolytic lesions of the aPVT were made in adult male hamsters maintained on long days (LD 16:8). The hamsters were then transferred to short days (LD 8:16) to test whether testicular regression could occur in response to a decrease in photoperiod. Serum prolactin concentrations were measured as a second photoperiodic response. All unoperated control hamsters showed the typical short-day photoperiodic response: A decrease in serum luteinizing hormone (LH) and prolactin concentrations and testicular regression all occurred within 6 weeks in short days, followed by the development of scotorefractoriness. Lesions of the aPVT did not significantly affect the rate or the degree of the short-day-induced decline in serum levels of LH or prolactin, nor the pattern of testicular regression and the subsequent expression of refractoriness. To enable us to determine whether the aPVT might be involved in the entrainment or the expression of circadian rhythms, locomotor activity was monitored continuously in lesioned and control groups in Experiment 2, prior to and following the switch to short days. The reduction in photoperiod (involving an 8-hr advance in the time of lights-off and an 8-hr extension of the dark phase) caused a decompression of the nocturnal activity bout of control animals, so that after 2 weeks in short days, activity onset had also advanced to regain its phase relationship to the timing of lights-off. A similar pattern of reentrainment was observed in lesioned animals, and no differences were observed between treatment groups in the rate of entrainment and decompression. In addition, both intact controls and animals bearing large bilateral lesions of the aPVT exhibited robust free-running circadian rhythms of locomotor activity when held under constant dim red light. In summary, the integrity of the aPVT is not necessary for the seasonal response of the reproductive axis and prolactin secretion to photoperiod, nor for photic entrainment of activity rhythms, in the Syrian hamster.


Enzymes in saliva from four parasitic arthropods. Enzyme assays and SDS polyacrylamide gel electrophoresis were carried out on saliva and in some cases homogenates of salivary gland and gut from four parasitic arthropods (the cattle tick, Boophilus microplus (Canestrini); the mosquito, Aedes aegypti (L.); non-parasitic adult and parasitic larval blowfly of sheep, Lucilia cuprina (Wiedemann); the buffalo fly, Haematobia irritans exigua de Meijere). Saliva from all species showed large differences in the number and molecular weight of components, as judged by electrophoresis. Enzyme profiles, however, showed similar enzyme activities (phosphatase, esterase/lipase) in saliva from species with dissimilar feeding behaviours. There were obvious differences in the enzyme profiles of saliva and gut tissues from the different species that reflected feeding strategies. These differences were mainly in the type and levels of glycosidase and protease activities. It was concluded that many of the components of saliva from different species had similar functions, although a small number of them may be specifically adapted to the mode of feeding for each parasite.


Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods. Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non-viraemic guinea-pig. This phenomenon has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea-pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea-pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.


Quantitation by fast-atom bombardment mass spectrometry: assay of cytidine 3',5'-cyclic monophosphate-responsive protein kinase. A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.


The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells. We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.


Genetic mapping of symbiotic loci on the Rhizobium meliloti chromosome. To facilitate genetic analyses of Rhizobium meliloti genes that are involved in symbiosis, we determined the map positions of 11 symbiotic loci on the R. meliloti chromosome by using a combination of the Tn5-Mob conjugational transfer method described by Klein et al. (S. Klein, K. Lohmann, G. C. Walker, and E. R. Signer. J. Bacteriol. 174:324-326, 1992) and co-transduction of genetic markers by bacteriophage phi M12. Loci involved in effective nodule formation (fix-379, fix-382, fix-383, fix-385, and fix-388), polysaccharide synthesis (exoR, exoS, exoC, and ndvB), nodule invasion (exoD), and nitrogen regulation (ntrA) were ordered with respect to previously mapped markers and each other. The positions of two other loci, degP and pho-1, were also determined.