The role of lipopolysaccharide in the exposure of protective antigenic sites on the major outer membrane protein of Chlamydia trachomatis.

A species-specific monoclonal IgM antibody (mAb) 9BF8 directed against the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized several chlamydial serovars in a complement-independent manner. The presence of Mg2+ ions negated the neutralization in serovars F, L1 and L2, but not in serovars A, B, E, D and K. The ability of monovalent Fab-fragments of this mAb to neutralize chlamydial infectivity in a Mg-independent manner suggested that conformational alterations on the chlamydial surface induced by the cation hindered the IgM but allowed the smaller Fab fragment access to its epitope. In order to determine the chlamydial component that binds Mg, elementary bodies (EB) of serovars E and L1 were treated with EDTA at pHs 8 and 9. The infectivity of the treated EB and the amount of released LPS were determined. Only after EDTA treatment at pH 9, as the LPS release increased, did the binding of the mAb on the chlamydial surface become Mg-independent. The infectivity of the EB was almost completely lost after such a treatment. These results suggest that the chlamydial LPS has the potential to modulate the exposure of antigenic sites on the MOMP, when it is cross-linked by Mg2+. They further imply that serovars protected by Mg and those that are not differ in the surface topology of one particular MOMP epitope, but are antigenically very similar. This difference might be of considerable importance in vivo.


Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.


The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA.

Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.


Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.


Antitumor activity of some metal complexes: effect on hepatic DNA, RNA, and protein synthesis in rats bearing transplanted tumors by Dalton's lymphoma cells.

In order to ascertain the antitumor properties of metal complexes, we have investigated their effect on hepatic DNA, RNA pools, and protein levels in rats bearing transplanted tumors by Dalton's Lymphoma cells because antitumor agents are directed against DNA, RNA transcription, and synthesis. These coordination complexes have been synthesized by the condensation of 2,6-diacetylpyridine with FaHh and DAP-Th2, thereafter they were complexed with nickel, cobalt, and iron. The structure of these metal complexes have been elucidated on the basis of IR, UV, NMR, Mass, EPR, and magnetic studies. The tumor-transplanted rats were treated with these metal complexes and ligands (2 ml of 1 mM sol/Kg body weight, s.c.) for two weeks to study their effect on DNA, RNA pools, and protein levels. The metal complexes have altered the hepatic DNA, RNA pools, and protein levels in the liver, kidney, and spleen of rats.


Membrane currents of horizontal cells isolated from turtle retina.

1. Membrane currents of horizontal cells isolated from the retina of the turtle, Pseudemys, were characterized by the whole-cell patch-clamp technique. 2. Four membrane currents were identified: an anomalous rectifier blocked by barium, a transient A-current, a sustained L-type calcium current enhanced by Bay K 8644, and a fast, tetrodotoxin-sensitive sodium current. Each of these four currents was found in both horizontal cell somata and axon terminals. 3. The current-voltage relations of axon terminals and somata were similar, but, in the normal operating range of the cell (-30 to -50 mV), the mean slope resistance of the axon terminal was higher (1.38 G omega) than that of the soma (0.26 G omega). 4. Exposure to either glutamate, kainate, or quisqualate induced a sustained inward current in horizontal cell axon terminals. The reversal potential for this current was -3 mV when tested with voltage steps and +9.1 mV when measured by a voltage ramp. The same horizontal cells were insensitive to N-methyl-D-aspartate. 5. A continuum model was developed to compute the degree of signal transfer between a horizontal cell body and its axon terminal. The model consisted of a network of electrically coupled somata that communicates with a network of electrically coupled axon terminals through the connecting axons. The specific membrane resistances used for the model derived from the patch-clamp measures. 6. We computed the voltage change elicited in either the layer of somata or of axon terminals by a static light stimulus of arbitrary dimensions. The amplitude of a spot response as a function of its radius was given by the weighted sum of two Bessel functions with different space constants. 7. The computed responses of the cell body were dominated by the Bessel function with the smaller space constant, whereas those of the axon terminal depended primarily on the Bessel function with the larger space constant. 8. The model predicts that, in contrast to the findings in teleost retina, there is little signal transfer between the somata and axon terminals of horizontal cell in the turtle retina.


Nicotinic acetylcholine receptors are directly affected by agents used to study protein phosphorylation.

1. Messenger RNAs for the subunits of the muscle nicotinic acetylcholine receptor (nAChR) were expressed in Xenopus oocytes. A two-electrode voltage clamp was used to measure the acetylcholine (ACh)-induced macroscopic currents. In addition, patch-clamp techniques were used to study nAChR channels in whole cells and in outside-out patches excised from BC3H-1 cells and in patches from oocytes. The single-channel and macroscopic currents were modified by compounds that are usually used to study protein phosphorylation. 2. IBMX (3-isobutyl-1-methylxanthine) is a phosphodiesterase inhibitor. Because it elevates the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP), IBMX is often used to indirectly activate cAMP-dependent protein kinase. H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] is mainly used as a rather nonspecific inhibitor of protein kinase activity. Both IBMX and H-7 directly inhibit ACh-induced currents independent of their action on phosphorylation. This direct effect of these compounds is similar to the previously reported inhibition of nAChRs and K+ channels by forskolin, which is commonly used to elevate intracellular cAMP. 3. Macroscopic currents induced in the oocytes by 50 microM ACh had an average peak current of 605 nA, and the currents decayed biexponentially with tau of 15 and 225 s. When 300 microM H-7 was added simultaneously with the ACh, the average peak current was 228 nA and the tau were 1 and 108 s. When 500 microM IBMX was added simultaneously with the ACh, the average peak current was 308 nA and the tau were 9 and 237 s. H-7 and IBMX decreased the peak current induced by ACh, and the compounds increased the decay rate of the current. Under these experimental conditions, the IC50 for reduction of peak amplitude at -30 mV was 160 microM for H-7 and 475 microM for IBMX. 4. H-7 preferentially inhibits the open conformation of the nAChR channel, but there is also some inhibition of the closed channel. The inhibition is voltage dependent: inhibition decreases e-fold per 34 mV depolarization. H-7 does not become trapped within the closed channel and does not significantly alter desensitization under our experimental conditions. 5. H-7 and IBMX interrupt or terminate single-channel openings in membrane patches excised from oocytes or BC3H-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)


The functional anatomy of middle-latency auditory evoked potentials: thalamocortical connections.

1. An 8 x 8-channel microelectrode array was used to map epicortical field potentials from a 4.375 x 4.375-mm2 area in the right parietotemporal neocortex of four rats. Potentials were evoked with bilaterally presented click stimuli and with electrical stimulation of the ventral and dorsal divisions of the medial geniculate body. 2. Epicortical responses to click stimuli replicated earlier findings. The responses consisted of a positive-negative biphasic waveform (P1a and N1) in the region of primary auditory cortex (area 41) and a positive monophasic waveform (P1b) in the region of secondary auditory cortex (area 36). Two potential patterns, one at the latency of the N1 and the other at the latency of the P1b, were used to represent activation of cells within areas 41 and 36. A linear combination of these patterns was sufficient to explain from 90 to 94% of the variance of the evoked potential complex at all latencies. 3. In the same animals, epicortical responses to electrical stimulation of the ventral and dorsal divisions of the medial geniculate body were also localized to areas 41 and 36, respectively. A linear combination of potential patterns from these separate stimulation conditions was sufficient to explain from 80 to 93% of the variance of the original click-evoked potential complex at all latencies. 4. These data provide functional evidence for anatomically defined topographical thalamocortical projections to primary and secondary auditory cortex. They suggest that short-latency cortical evoked potentials (10-60 ms poststimulus) are dominated by parallel thalamocortical activation of areas 41 and 36.


5-HT modulation of hyperpolarization-activated inward current and calcium-dependent outward current in a crustacean motor neuron.

1. Serotonergic modulation of a hyperpolarization-activated inward current, Ih, and a calcium-dependent outward current, Io(Ca), was examined in the dorsal gastric (DG) motor neuron, with the use of intracellular recording techniques in an isolated preparation of the crab stomatogastric ganglion (STG). 2. Hyperpolarization of the membrane from rest with maintained current pulses resulted in a slow time-dependent relaxation back toward rest and a depolarizing overshoot after termination of the current pulse. In voltage clamp, hyperpolarizing commands negative to approximately -70 mV caused a slowly developing inward current, Ih, which showed no inactivation. Repolarization back to the holding potential of -50 mV revealed a slow inward tail current. 3. The reversal potential for Ih was approximately -35 mV. Raising extracellular K+ concentration ([K+]o) from 11 to 22 mM enhanced, whereas decreasing extracellular Na+ concentration ([Na+]o) reduced the amplitude of Ih. These results indicate that Ih in DG is carried by both K+ and Na+ ions. 4. Bath application of serotonin (5-HT; 10 microM) caused a marked increase in the amplitude of Ih through its active voltage ranges. 5. The time course of activation of Ih was well fitted by a single exponential function and strongly voltage dependent. 5-HT increased the rate of activation of Ih. 5-HT also slowed the rate of deactivation of the Ih tail on repolarization to -50 mV. 6. The activation curve for the conductance (Gh) underlying Ih was obtained by analyzing tail currents. 5-HT shifted the half activation for Gh from approximately -105 mV in control to -95 mV, resulting in an increase in the amplitude of Gh active at rest. 7. Two to 4 mM Cs+ abolished Ih, whereas barium (200 microM to 2 mM) had only weak suppressing effects on Ih. Concomitantly, Cs+ also blocked the 5-HT-induced inward current and conductance increase seen at voltages negative to rest. In current clamp, Cs+ caused DG to hyperpolarize 3-4 mV from rest, suggesting that Ih is partially active at rest and contributes to the resting membrane potential. 8. Depolarizing voltage commands from a holding potential of -50 mV resulted in a total outward current (Io) with an initial transient component and a sustained steady-state component. Application of 5-HT reduced both the transient and sustained components of Io. 9. Io was reduced by 10-20 mM tetraethylammonium (TEA), suggesting that it is primarily a K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)


Pharmacological properties of N-methyl-D-aspartate receptors on ganglion cells of an amphibian retina.

1. The pharmacological characteristics of the N-methyl-D-aspartate (NMDA) receptors on amphibian retinal ganglion cells were studied to determine their similarities or differences from NMDA receptors found in mammalian central nervous system (CNS) cells. Cells were tested with a variety of NMDA antagonists acting at sites on the NMDA receptor/ion channel complex. 2. Whole-cell voltage-clamp recordings were obtained from ganglion cells of the larval tiger salamander with a retinal slice preparation. All cells responded with inward currents (Vhold = -70 mV) when exposed to bath applications of NMDA, kainate (KA), and glutamate (GLU). NMDA currents reversed near 0 mV and showed a negative slope conductance region in the presence of external Mg2+. 3. NMDA-evoked inward currents could be blocked by application of 300 microM D-2-amino-7-phosphonoheptanoate (DAP7), 100 microM Zn2+, 25 microM 7-chloro-kynurenate (7-cl-KYN), 1 microM MK-801, and 5 mM Mg2+. These results indicate that like mammalian NMDA receptors the amphibian NMDA receptor possesses binding sites for NMDA, glycine, zinc, dissociative anesthetics, and Mg2+. 4. NMDA responses were evoked in the presence of 1 mM extracellular Mg2+ in 100% of cells tested when held at -70 mV. Furthermore, there was a resting conductance at -70 mV and membrane current noise that could be attenuated by the application of NMDA-specific antagonists suggesting a tonic activation of NMDA receptors for cells at the resting potential.


Evidence for nitric oxide synthase inhibitor-sensitive and insensitive hippocampal synaptic potentiation.

1. Nitric oxide (NO) has been proposed as a retrograde messenger, mediating the postsynaptic to presynaptic transfer of the effects of conditioning stimulation, responsible for the initiation of hippocampal long-term potentiation (LTP). To further test this hypothesis, we inhibited nitric oxide synthase (NOS) to determine whether synaptic potentiation produced by different conditioning stimulus patterns and intensities was differentially affected by reduction of stimulation-dependent NO production. 2. Synaptic potentiation was produced in hippocampal slices from young F-344 rats by two different conditioning stimulation protocols. Conditioning stimuli were delivered to the Schaffer-collateral commissural system, and moderate levels of potentiation of the population excitatory postsynaptic potential (EPSP) in area CA1 were produced by a single 100 Hz, 1-s conditioning train delivered at half-maximal stimulus intensity. Higher levels of potentiation of the population EPSP were obtained by delivering two 100 Hz, 1-s conditioning stimulus trains, with a 60-s intertrain interval, at high stimulus currents. 3. Application of the nitric oxide synthase inhibitors NG-nitro-L-arginine (NOARG; 0.1-200 microM) and NG-monomethyl-L-arginine (NMMA; 100 microM) produced no significant direct effects on synaptic responses. 4. In slices that received a single conditioning stimulus train, both NOARG and NMMA were ineffective in blocking or reducing potentiation at concentrations between 0.1 and 200 microM. In slices receiving the more intense pair of conditioning stimulus trains, levels of potentiation in control slices were higher, and there was a very significant reduction by both NOARG (50 and 100 microM) and NMMA (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells' influence on circadian behavioral rhythms.

The product of the period (per) gene of Drosophila melanogaster is continuously required for the functioning of the circadian pacemaker of locomotor activity. We have used internally marked mosaics to determine the anatomical locations at which per expression is required for adult rhythmicity, and thus where the fly's circadian pacemaker is likely located in this holometabolous insect. We first provide a detailed description of the distribution and nature of per-expressing cells in the fly's CNS. Using an antibody to the per gene product, or to that of a reporter of per expression, in conjunction with an antibody to the embryonic lethal-abnormal visual system (elav) gene product--which is used as a marker of neuronal identity--we have experimentally confirmed previously proposed assignments of per-expressing cells to the neuronal and glial classes. Thus, we found that per expression and elav immunoreactivity colocalized in large cells located in the lateral cortex of the central brain, as well as in more dorsally located cells in the posterior central brain. In contrast, we found that cells located at the margins of the cortex and the neuropil, and within the neuropil, as well as smaller cortical cells found throughout the brain's cortex, were elav negative, supporting the notion that they are glial in nature. Using internally marked mosaics, we find that the pacemaker is located in brain but is not exclusive to the eyes, the ocelli, or the optic lobes, which is consistent with previous reports obtained in this and other insects of this class. Although the pacemaker may be a paired structure, we show that the functioning of one of them is sufficient for rhythmicity. Finally, we report that glial expression is sufficient for some behavioral rhythmicity to be manifest. However, the rhythmicities of animals for which per expression was confined to glia were weak, suggesting that neuronal per expression as well may be required for normal pacemaker function.


Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor.

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium-induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady-state mRNA levels (expressed on a "per nerve" basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring.


Childhood medical and behavioral consequences of maternal cocaine use.

Reviewed available studies of the impact of fetal cocaine exposure on child medical and developmental outcome, as well as the current status of clinical psychological interventions and research strategies. Current studies are inconclusive but suggest that prenatal exposure to crack-cocaine can have significant effects on the growth and neurological development of the infant, with the potential of later learning and behavioral disabilities. Social-environmental correlates of maternal cocaine use are confounding factors with known negative effects on child outcome. Large, population-based studies using multivariate analyses are needed to determine the independent effects of cocaine on child outcome relative to other confounding variables.


Transport and hydrolysis of maltose by Schwanniomyces castellii.

Hydrolysis and transport of maltose into Schwanniomyces castellii was studied under aerobic and anaerobic conditions. Amylase and glucosidase were not synthetized in presence of maltose in anaerobic conditions. Maltose permease was synthesized in anaerobiosis and its functioning is not inhibited. Glucose strongly repressed induction. The half-saturation constant for uptake of maltose was 0.06 mM. The rate of uptake of maltose was decreased by 2,4-dinitrophenol, antimycin or sodium azide. The significance of these results in relation to the Kluyver effect is discussed.


Discrimination of multiple binding sites for antagonists of the calcium release channel complex of skeletal and cardiac sarcoplasmic reticulum.

The mechanisms by which ruthenium red (RR), neomycin and FLA 365 ([2,6-dichloro-4-aminophenyl]isopropylamine) inhibit calcium channels of skeletal and cardiac sarcoplasmic reticulum (SR) are characterized. Neomycin and FLA 365 inhibit ryanodine-enhanced calcium release from skeletal SR vesicles in a dose-dependent manner. The apparent affinity of [3H]ryanodine is reduced in a dose-dependent manner by each inhibitor indicative of competitive mechanisms. Displacement studies with skeletal and cardiac SR demonstrate that the order of inhibitory potency is RR greater than neomycin greater than FLA 365 and RR greater than FLA 365 greater than neomycin, respectively. Neomycin is 100-fold less potent in cardiac SR and inhibition of [3H]ryanodine binding is biphasic in both tissues. Neomycin induces a greater proportion of [3H]ryanodine binding states recalcitrant to inhibition in cardiac SR. The ability of neomycin to increase the apparent affinity of [3H]ryanodine for its binding sites is potentiated by RR and attenuated by FLA 365. Kinetic binding studies reveal that increasing neomycin concentrations decreases the association of [3H]ryanodine as predicted for competitive inhibition. However, high (much greater than Kn) neomycin increases [3H]ryanodine binding affinity by slowing dissociation of the radioligand demonstrating that, like micromolar ryanodine, neomycin induces allosterism. Studies with combinations of antagonists demonstrate the existence of two non-overlapping inhibitor recognition sites within the ryanoid site, one polycationic inhibitor site and one FLA 365 inhibitor site. These results suggest that aminoglycoside-induced muscle paralysis may be mediated by direct block of pre- and postsynaptic calcium release channels of endoplasmic reticulum.


Differential effects of glyburide on premature beats and ventricular tachycardia in an isolated tissue model of ischemia and reperfusion.

Possible anti- and proarrhythmic effects of glyburide, an ATP-sensitive K+ channel blocker, were assessed in an isolated tissue model of reperfusion. Transmembrane electrical activity was recorded from endo- and epicardium of isolated segments of guinea pig right ventricular free walls, or two sites on papillary muscles with microelectrodes. An electrocardiogram was recorded by two electrodes placed at opposite ends of the tissue bath. Regular stimulation was delivered to endocardium. Tissues were exposed to simulated ischemia for 15 min and then were reperfused with normal Tyrode's solution. Rapid sustained or nonsustained ventricular tachycardia, bigeminy or trigeminy with characteristics of transmural re-entry occurred early in reperfusion in 50% of free walls. Triggered arrhythmias with characteristics of oscillatory afterpotentials (delayed afterdepolarizations) occurred in 20%. Arrhythmias were accompanied by prolongation of transmural conduction times and abbreviation of endocardial effective refractory periods and action potential durations. Glyburide (3 or 30 microM) significantly attenuated abbreviation of action potential durations and effective refractory periods during ischemic conditions and early reperfusion. Neither endocardial nor transmural conduction times were modified by glyburide; however, glyburide significantly decreased the incidence of transmural conduction block during ischemic conditions. Glyburide abolished reperfusion arrhythmias with characteristics of re-entry, but potentiated oscillatory afterpotentials in papillary muscles and triggered arrhythmias with characteristics of oscillatory afterpotentials in free walls. Identical effects were seen with glyburide present in ischemic solution, or in both ischemic and reperfusion solutions, but no effect was observed with glyburide present only in reperfusion. Our study demonstrates possible cellular mechanisms underlying simultaneous pro- and antiarrhythmic drug effects exerted on late premature beats and rapid arrhythmias and closely coupled premature beats.


Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts.

Phospholipase A2 (PLA2) activity results in the formation of lysophospholipids and free fatty acids which may contribute to ischemic myocardial dysfunction. We evaluated the cardioprotective activity of two putative PLA2 inhibitors, quinacrine and 7,7-dimethyleicosadienoic acid (DEDA), in isolated globally ischemic rat hearts. Pretreatment with 1, 5 and 50 microM quinacrine before ischemia did not alter coronary flow but did cause significant cardiodepression. Twenty five minutes of global ischemia and 30 min of reperfusion caused severe myocardial dysfunction and lactate dehydrogenase release. Quinacrine significantly improved reperfusion contractile function and reduced lactate dehydrogenase release, indicative of cardioprotection. In contrast, 30 to 100 microM DEDA produced neither preischemic cardiodepression nor cardioprotective activity. PLA2 inhibition was inferred from measurements of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha in the coronary effluent and myocardial palmitoyl-lysophosphatidylcholine. Quinacrine and DEDA reduced both 6-keto-prostaglandin F1 alpha and palmitoyl-lysophosphatidylcholine by similar degrees. These results suggest that the cardioprotective activity of quinacrine is independent of PLA2 inhibition. A possible role of calcium inhibition was investigated in rat aortic smooth muscle strips. Norepinephrine-, KCl- and BAY K8644-induced contractions were antagonized in the presence of 5 and 50 microM quinacrine, but were unaffected by 30 to 60 microM DEDA. The ability of quinacrine to inhibit calcium was investigated further in cardiac ventricular myocytes. Measurement of mean whole cell calcium currents showed that quinacrine (5 microM) could inhibit this current up to 70%. Thus, these results suggest that quinacrine-induced cardioprotection may not be due to PLA2 inhibition, but may be related to calcium entry blocking activity.


Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells.

Human megakaryoblastic cells (Meg-01) were found to possess constitutive and express inducible nitric oxide (NO) synthase activities. The constitutive NO synthase was Ca+(+)- and NADPH-dependent, as is the NO synthase found previously in human platelets. Stimulation of Meg-01 cells by the cytokines interleukin-1 beta (0.15-32.5 ng/ml) and tumor necrosis factor-alpha (0.15-10 ng/ml) resulted in expression of the inducible, Ca+(+)-independent, NO synthase. This activity was increased by the addition of NADPH, tetrahydrobiopterin and sepiapterin as cofactors. Induction of this enzyme was accompanied by a decrease in the constitutive NO synthase activity, a phenomenon which was prevented or reversed by dexamethasone (1 microM). Thus, human early differentiated megakaryocytic cells can synthesize NO from L-arginine by both the constitutive and the inducible NO synthases. These findings indicate that these enzymes may play an important biological role in megakaryocyte and platelet functions.


Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone.

The N-deacetylation of thiacetazone, an antitubercular drug possessing hepatotoxic side effects, by an exclusively cytosolic arylacylamidase has been identified in the liver and kidney of rat by monitoring the appearance of its metabolite p-aminobenzaldehydethiosemicarbazone spectrophotometrically. Studies toward its characterization in liver cytosol revealed that the hydrolase possesses a broad pH optimum ranging from 6.0 to 9.0. The Km and Vmax values for the N-deacetylation of thiacetazone are 5.7 x 10(-4) M and 0.123 nmol of p-aminobenzaldehydethiosemicarbazone formed/min/mg cytosolic protein, respectively. The ability to metabolize thiacetazone was the same in the livers of cat, mouse and human, but lagged significantly in that of rat. Among the biodegradable esters examined as potential rivals of thiacetazone, only aspirin competitively inhibited thiacetazone hydrolysis (Ki = 2.1 x 10(-4) M). Discrimination of cytosolic thiacetazone N-deacetylase from nonspecific p-nitrophenylacetate esterase on the basis of their differential reactivity toward various inhibitors and activators disclosed that low concentrations of p-chloromercuribenzoate, AgNO3 and CuSO4 selectively undermine the activity of thiacetazone N-deacetylase, whereas SKF 525-A, ZnSO4 and FeCl3 are effective inhibitors of p-nitrophenylacetate esterase. However, divalent ions (Ca++ and Mg++) and EDTA failed to alter the activity of the enzyme. Besides, thiacetazone metabolism was significantly retarded upon exposure to malathion. Notably, Nal/Kl stimulated the N-deacetylase activity as a function of iodide concentration. The hydrolysis of thiacetazone in the liver and kidney remained uninduced by phenobarbital, 3-methylcholanthrene or benzo(a)pyrene (80 mg/kg, p.o., 8 days).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of H1 receptor blockade on the early and late response to cutaneous allergen challenge.

We investigated whether cutaneous antigen-induced inflammatory cell infiltration and mediator release were modified by H1 receptor antagonists. Three chemically unrelated antihistamines (cetirizine, promethazine and chlorpheniramine) were tested in three groups of allergic subjects in a double-blind, crossover design. Chamber fluids were collected for 12 hr and histamine release, prostaglandin D2 production and cellular infiltration were quantified. Cetirizine significantly decreased late leukocyte migration into antigen-challenged chambers: eosinophils by 68% (P less than .04), basophils by 64% (P less than .04) and neutrophils by 72% (P less than .04), whereas mononuclear cells were not significantly affected. No alteration in the numbers of peripheral blood leukocytes or eosinophils occurred while on cetirizine treatment, suggesting that the decrease in inflammatory cells during the late phase reaction in the skin is not secondary to alterations in the peripheral leukocyte pool. In contrast, neither promethazine nor chlorpheniramine induced any significant alteration in inflammatory cell infiltration. All three antihistamines caused significant inhibition of the immediate reaction to antigen without any significant alteration in late phase reaction cutaneous reactivity. None of the three antihistamines caused any significant alteration in histamine or prostaglandin D2 levels. Thus, cetirizine may be an antihistamine uniquely capable of downregulating the late phase reaction inflammatory cell milieu without altering either early or late mediator production. The mechanisms involved and the clinical relevance of these findings remain to be explored.


Synthesis and excitatory amino acid pharmacology of a series of heterocyclic-fused quinoxalinones and quinazolinones.

As part of our program aimed at the development of potent excitatory amino acid antagonists, we synthesized and evaluated a series of substituted 1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-ones, 4, tetrazolo[1,5-a]quinoxalin-4(5H)-ones, 5, and pyrazolo[1,5-c]quinazolin-5(6H)-ones, 6, and an imidazo[1,2-a]quinoxalin-4(5H)-one, 7. In general, the same heterocycles which demonstrated the best affinity for the AMPA receptor also demonstrated the best affinity for the glycine site on the NMDA receptor complex. 1-Propyl-7,8-dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4d, was found to bind with the greatest affinity to the AMPA receptor with an IC50 of 0.83 microM and antagonized 40 microM AMPA-induced depolarization in the cortical slice preparation with an IC50 of 44 microM. 7,8-Dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4a, and 7,8-dichloroimidazo[1,2-a]quinoxalin-4(5H)-one, 7, possessed the best affinity for the glycine site with IC50 values of 0.63 and 1.26 microM, respectively. It is noteworthy that the SAR for the heterocyclic compounds did not directly parallel that of known quinoxalinediones (e.g. DNQX, 2, and DCQX, 15) at the AMPA receptor nor that of the kynurenic acids at the glycine site on the NMDA receptor complex.


Atomic force microscopy of conventional and unconventional nucleic acid structures.

Images of conventional (Watson-Crick base paired) and unconventional (G4 RNA) nucleic acid structures have been obtained by atomic force microscopy. The images are reproducibly generated from samples deposited on freshly cleaved mica. Periodic substructural features are evident in fibres observed in both cases. In the case of G4 RNA, tip-induced formation of large fibres is observed.


Insulin-like growth factor-I counteracts bFGF-induced survival of nitric oxide synthase (NOS)-positive spinal cord neurons after target-lesion in vivo.

We have used nitric oxide synthase (NOS) histochemistry as a perikaryal viability marker to trace the retrograde reaction of spinal cord intermediolateral (IML) sympathoadrenal projection (SAP)-neurons to target-removal, i.e., selective adrenomedullectomy and local administration of either insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF) or a combination of both. Counting of NOS-positive preganglionic spinal cord neurons 4 weeks post surgery indicated that more than 80% of stained neurons were lost from the IML-cell column. This percentage loss corresponds to the numerical loss of NOS-stained SAP-neurons labeled retrogradely with Fast-blue prior to adrenomedullectomy. Basic FGF-supplementation at the site of lesion resulted in maintenance of the majority of NOS-positive IML-neurons, a finding confirmed by the survival rate of Fast-blue prelabeled SAP-neurons. Thus, besides maintenance of the structural integrity of SAP-neurons, bFGF prevents loss of intracellular NOS-activity which may reflect unaltered cell metabolism (and function) of these neurons following target-removal in vivo. By contrast, IGF-I failed to alter the rate of disappearance of NOS-staining and labeling index of neurons within the IML-cell column postlesion, suggesting that IGF-I is not neurotrophic for SAP-neurons by itself. Combined treatment with both factors resulted in a more widespread loss of NOS-stained and Fast-blue-prelabeled SAP-neurons than registered after bFGF-only treatment. No co-trophic effect of bFGF and IGF-I was evident; rather, the pronounced bFGF-induced rescuing effect was significantly suppressed by exogenous IGF-I in vivo, supporting the idea that this or another molecule induced by the treatment enhances rather than prevents retrograde degeneration and neuronal death within the adult lesioned IML-adrenal pathway.


Alterations in slow transport kinetics induced by estramustine phosphate, an agent binding to microtubule-associated proteins.

Estramustine phosphate (EP) disassembles microtubules by binding to microtubule-associated proteins (MAPs) rather than tubulin. In this study, EP-induced alterations of MAP integrity caused a unique form of axonal atrophy in rats. Initially, EP-induced axonal atrophy occurred in both proximal and distal axons of the sciatic nerve, characterized by an increase in neurofilament packing density, associated with a decrease in axonal area. In chronic exposure, distal axonal atrophy was associated with decreased numbers of microtubules, while the neurofilament number remained unaltered for the myelin spiral length. Continued exposure caused enlargement of proximal axons associated with an increase in neurofilament content. Correlative slow transport studies done at two different times, 7 and 14 days after [35S] methionine injection showed that EP retards the transport of cytoskeletal proteins migrating with both components of slow transport (SCa and SCb). However, there was a differential effect on SCb which showed progressive slowing along the nerve while the rate of SCa stayed relatively constant. In this model, the early occurring distal axonal atrophy can best be explained by reduced cytoskeletal components, particularly those traveling in SCb. Later in the course of intoxication, a relatively constant rate of SCa permitted continuous transport of neurofilament triplets, accounting for unaltered numbers of neurofilaments in distal axons with increased packing density. This model of axonal atrophy is unique because spacing of neurofilaments, not numbers determined axon size. Furthermore, EP-induced dissociation of the SCa and SCb kinetics suggests that MAPs play a role in the orderly, cohesive migration of slow transport components, essential for the normal organization of cytoskeleton.


Sub-chronic exposure to opiates in the rat: effects on brain levels of substance P and calcitonin gene-related peptide during dependence and withdrawal.

Substance P (SP) and calcitonin gene-related peptide (CGRP) are putative transmitters in the central and peripheral (sensory) nervous systems. In this study, we examined the effects of dependence on and withdrawal from morphine and methadone on brain SP and CGRP content. Female Long Evans rats (70-100 g) were provided with plain drinking water or solutions containing opiate. No choice of drinking fluid was allowed. The maintenance level of each opiate (0.8 and 0.4 mg/ml for morphine and methadone, respectively) was continued for 4 days. Following an injection with naloxone (10 mg/kg i.p.) or saline, animals were decapitated 0, 20, or 60 min later and regional brain peptide content was measured by specific radioimmunoassays. SP and CGRP content in opiate-maintained and naive animals were similar following saline injection. However, following naloxone injection in morphine-maintained animals, SP content was elevated in the hypothalamus and midbrain at 20 min, but by 60 min was no longer distinguishable from basal (0 min) level. CGRP content was increased in the medulla oblongata and followed a comparable time course but, unlike SP, was not altered in the hypothalamus or midbrain. No alterations were observed in methadone-maintained animals. These results correlated with the peak of the behavioral morphine withdrawal syndrome and were consistent with the comparatively milder abstinence encountered in methadone medication.


Isolation and characterization of periaxolemmal and axolemmal enriched membrane fractions from the rat central nervous system.

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP-1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl- channels, which based on channel sizes and open times, are largely distinct from one another.


Sickle cell/beta-thalassemia in North Jordan.

Clinical and haematological features of 50 patients with sickle cell/beta-thalassemia (SB0 or SB+) are investigated. Total haemoglobin value was not significantly different (P greater than 0.05) in both types. Haemoglobin F and S were significantly higher (P less than 0.05) in SB0 than SB+ while haemoglobin A2 level was lower in SB0 than SB+. One SB0 case with exceptionally high HbF (32 per cent) with severe clinical course was found. In this case, the high HbF did not ameliorate the clinical severity. Heterogeneity in each type of sickle cell/beta-thalassemia is discussed. The differentiation of SB0 and sickle cell disease is best made on the basis of family study.


Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.


Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.


Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.


Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages, not in peripheral blood monocytes.

In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells collected from horses on various days during the first 2 weeks postinfection with the Wyoming strain of EIAV failed to detect any viral RNA in these cells. For the two horses described here, serum reverse transcriptase activity correlated directly with the degree of replication detected in tissue macrophages on the day of sacrifice. These results suggest that unlike other lentivirus infections in which mature tissue macrophages accumulate cytoplasmic viral RNA to a high level but fail to produce infectious virions, mature tissue macrophages are the likely primary source of the high titer of viremia present during acute infection with EIAV. No significant posttranscriptional block of viral replication in tissue macrophages appears to occur with EIAV.


Poliovirus-specific major histocompatibility complex class I-restricted cytolytic T-cell epitopes in mice localize to neutralizing antigenic regions.

A major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) response is induced in BALB/c mice upon immunization with poliovirus serotype 1 (Mahoney strain). A similar class I-restricted response is also induced upon immunization with purified VP1 capsid proteins. Thus, poliovirus-specific MHC class I CTL responses can be induced independently of viral infection in murine hosts. In experiments using recombinant vaccinia virus vectors expressing different segments of the poliovirus capsid proteins and synthetic peptides, two regions of the VP1 capsid protein appear to contain epitopes recognized by this bulk CTL population. These epitope regions contain a Kd-restricted peptide-binding motif. Interestingly, each of these CTL epitopes is located near previously defined neutralizing antigenic sites.


Worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects.

The natural occurrence of lentiviruses closely related to feline immunodeficiency virus (FIV) in nondomestic felid species is shown here to be worldwide. Cross-reactive antibodies to FIV were common in several free-ranging populations of large cats, including East African lions and cheetahs of the Serengeti ecosystem and in puma (also called cougar or mountain lion) populations throughout North America. Infectious puma lentivirus (PLV) was isolated from several Florida panthers, a severely endangered relict puma subspecies inhabiting the Big Cypress Swamp and Everglades ecosystems in southern Florida. Phylogenetic analysis of PLV genomic sequences from disparate geographic isolates revealed appreciable divergence from domestic cat FIV sequences as well as between PLV sequences found in different North American locales. The level of sequence divergence between PLV and FIV was greater than the level of divergence between human and certain simian immunodeficiency viruses, suggesting that the transmission of FIV between feline species is infrequent and parallels in time the emergence of HIV from simian ancestors.


Identification of a feline immunodeficiency virus gene which is essential for cell-free virus infectivity.

Feline immunodeficiency virus (FIV) contains at least three small open reading frames (ORFs) in the genome, in addition to the three structural genes. Two of these ORFs (putative vif and ORF-A) have unknown functions. Northern (RNA) blot analysis of mRNAs from an FIV-infected cell line showed that the putative-vif-specific mRNA was expressed as a 5.2-kb species. To examine the function of the putative vif gene, we constructed mutants carrying a deletion in either the vif-like gene or the rev gene from an infectious molecular clone of FIV. Although the vif mutant produced virion-associated reverse transcriptase at a normal level upon transfection, cell-free virus prepared from the transfected cells could not infect feline CD4+ cells. The infectivity of the vif mutant, however, was demonstrated in a coculture of the transfected cells and feline CD4+ cells. We conclude that FIV contains the vif gene, which is structurally and functionally similar to that of the primate lentiviruses.


3-Methylglutaconic aciduria: a marker for as yet unspecified disorders and the relevance of prenatal diagnosis in a 'new' type ('type 4').

The Mendelian disorder known as 3-methylgutaconic aciduria (McKusick 250950) gives evidence of allelic and locus heterogeneity. Type 1 has a mild clinical phenotype and confirmed 3-methylgutaconyl-CoA hydratase deficiency; inheritance is autosomal recessive. Other forms have major clinical manifestations and subdivide into X-linked (type 2), a form in Iraqi Jews with optic atrophy (so-called type 3); and untyped (putative autosomal recessive) forms without identified enzyme defects. In the latter, 3-methylglutaconic aciduria may simply be a marker for another metabolic disorder. We describe a male proband with 3-methylglutaconic aciduria designated here as 'type 4' (autosomal recessive, with severe psychomotor phenotype and cerebellar dysgenesis). He is the offspring of Italian consanguineous parents. Born with congenital malformations, he has been followed for 18 years, showing profound developmental delay and cerebellar dysgenesis. Measures of hydratase activity in cultured fibroblasts from the proband and 11 additional patients (two with type 1 disease, 9 with either type 2 or an unspecified form) revealed deficient enzyme activity in type 1 cases and normal activity in the proband and the other 11 cases. Two of the untyped cases probably have 3-methylglutaconic aciduria of the type described here. Prenatal diagnosis in the form described here may be feasible by analysis of amniotic fluid metabolites in pregnancies at risk if the mother does not entirely remove elevated concentrations. A female sibling of the proband had normal metabolite values in amniotic fluid. Postnatal follow-up confirmed absence of the disease. We give the normal values for amniotic fluid and results on these additional fetuses at risk (none affected).


Direct assessment of the role of NK cells in autoimmune diabetes.

Considerable indirect evidence implicates participation of natural killer cells (NK) in the pathogenesis of diabetes in BB rats. The most convincing evidence derives from studies showing that anti-CD8 antibody effectively prevents both primary disease onset and autoimmune damage to transplanted islets. However, anti-CD8 treatment depletes both NK and cytotoxic T cells (CTL) since both cell types express the CD8 marker. To study directly the role of NK in diabetic BB rats we used MCA 3.2.3, a monoclonal antibody which selectively depletes normal Lewis rats of NK cells but not CTL. A regimen of ip injected antibody achieved rapid reduction of NK cells in diabetic and nondiabetic BB rats by FACS analysis. NK cell activity remained low in rats treated weekly as evidenced by YAC tumor cell killing. We next studied the effect of NK depletion on disease incidence in diabetes-prone BB rats of which about one half are expected to develop diabetes. Onset and incidence of diabetes in 3.2.3-treated and control antibody-treated aged matched litter mates were equal. These studies suggest that NK cells are not necessary for autoimmune islet destruction in spontaneously diabetic BB rats and support a role for CTL in pathogenesis of the disease.


Mucosal glutamine utilization after small-bowel transplantation: an electrophysiologic study.

A short course of FK 506 after small bowel transplantation averts rejection in the rat and achieves indefinite survival of the recipient whose nutritional status is dependent on the function of the intestinal graft. Ex vivo electrophysiologic studies using the Ussing Cell were conducted to delineate functional competence of the graft by evaluating mucosal ion transport and glutamine utilization. Orthotopic small-bowel transplantation was performed in Lewis (LEW) rats as recipients of either Brown-Norway (BN) allografts or LEW syngeneic grafts. Allograft recipients received FK 506 either as a short course (2 mg/kg on Day 0-4 after transplantation) or continuously (2 mg/kg Day 0-4, then 0.5 mg/kg weekly). Ileal mucosa was harvested from small bowel grafts 9 and 60 days after transplantation and mounted in the Ussing Cell containing Hanks' balanced salt solution with/without L-glutamine (20 mM). Transmembrane potential difference (PD), which represents mucosal active ion transport, and mucosal resistance, an index of membrane integrity, were recorded. Nine days after transplantation, mucosal PD was the same in the ileum from syngeneic grafts, allografts treated with FK 506 and normal LEW and BN rats, and the addition of glutamine increased PD equally in all groups. In comparison, PD was markedly decreased in allografts undergoing rejection, and the glutamine response was blunted. Sixty days after transplantation, mucosal PD was reduced in allografts treated with a short course of FK 506, but normal in allografts receiving continuous immunosuppression with FK 506 and in syngeneic grafts. A decrease of mucosal resistance was not a feature of rejection nor a sequel of limited FK 506 therapy. Our data indicate that allograft rejection results in a significant decrease in mucosal PD and a poor response to glutamine. Control of rejection by FK 506 preserves normal electrophysiologic responses of the allograft mucosa.


Intraoperative liver radiation after partial hepatectomy in a rat model.

Hepatic resection of metastatic tumor is a treatment option in selected patients. Resection margin is a prognostic factor of hepatic recurrence and survival. Although intraoperative radiation therapy (IORT) has been clinically useful in some gastrointestinal cancers, there is little information regarding its use following hepatic metastasectomy. In this study, a rat model was employed to evaluate histological changes and DNA synthesis as an indication of hepatic regenerative capacity following hepatectomy and liver IORT. All rats (N = 40) had a partial hepatectomy and were divided into four random groups: a nonradiated group and three groups of 1000, 2000, and 3000 cGy given by IORT. The only deaths occurred in the 3000 cGy group. Routine H and E staining of liver sections after 3, 6, and 10 days suggested progressive hepatocyte damage notably in the 3000 cGy group. Comparison of the final average liver weights at 10 days confirmed a diminished liver mass in the 2000 and 3000 cGy animals. DNA synthesis in hepatocytes measured by [3H]-thymidine label incorporation 3, 6, and 10 days after hepatectomy and IORT indicated a comparative and overall decrease in Day 6 peak activity between the three IORT groups. This study demonstrated delayed but substantial hepatic regeneration in the post-resected liver within the clinically useful IORT dose range (1000-2000 cGy) needed to control minimal residual tumor. This model has importance concerning the feasibility of IORT to the hepatic resection bed for patients where resection margins are inadequate.


The interrelationships among filtration surface area, blood pressure, and chronic renal disease.

A primary role for the kidney in hypertension has long been recognized, but the pathogenetic interactions among renal hemodynamics, hormonal and hereditary factors, and dietary sodium intake remain ill defined. Reduction in the filtration surface area, whether acquired in the course of intrinsic renal disease or after surgical renal ablation, leads to systemic hypertension as well as to progressive renal insufficiency, sequellae made even more severe by dietary sodium excess. Moreover, hypertension and progressive renal disease occur in some individuals born with a solitary kidney, and occur almost invariably with more severe degrees of dysgenesis. Hypertension is also commonly observed in certain inbred rat strains in which the filtration surface area is congenitally deficient. Based on these and other lines of evidence reviewed herein, we postulate that a renal abnormality that contributes to essential hypertension in the general population is a reduced number of glomeruli and tubules, the consequences of which are limitations in the ability to excrete sodium and thus salt-sensitive hypertension. Furthermore, congenitally decreased filtration surface area may explain why only some, but not all, patients exposed to potentially injurious renal stimuli eventually manifest chronic nephropathy, and may also account for the susceptibility of subsets of type I and type II diabetics to develop overt glomerulopathy. Clinically, tests of renal reserve capacity may serve as a useful guide to identification of those patients at risk for the development of hypertension and progressive renal disease.


Cognitive function and angiotensin-converting enzyme inhibitors in comparison with other antihypertensive drugs.

This review mainly considers randomized studies where angiotensin-converting enzyme (ACE) inhibitors were compared with other antihypertensive drugs in terms of objectively assessed cognitive function, subjectively evaluated cognitive function, work performance, and general vitality. An overall assessment of the data suggests that ACE inhibitors may improve performance on tests of alertness but it is unlikely that they produce either deleterious or beneficial effects on memory. On the other hand, methyldopa interferes with cognitive function and work performance. Propranolol was also associated with impairment of activity and satisfaction at work. In subjective evaluation of cognitive function, two trials suggest an increased reporting of impairment with nifedipine but this has not yet been confirmed. Vitality was reduced on both methyldopa and propranolol. It is concluded that the ACE inhibitors, in common with atenolol and verapamil, do not appear to have any major effects on cognitive function in hypertensive patients.


Alpha-adrenergic and angiotensin II pressor sensitivity in hypertensive patients treated with an angiotensin-converting enzyme inhibitor.

We investigated pressor sensitivity to infused phenylephrine (PE), 0.05 to 0.4 micrograms/kg/min, and angiotensin II (Ang II), 2.5 to 10 ng/kg/min, in 35 patients with mild-to-moderate hypertension, before and at the end of a 4-week treatment period with the angiotensin-converting enzyme (ACE) inhibitor, cilazapril, 2.5 or 5.0 mg/day. Cilazapril lowered the mean systolic and diastolic blood pressure by 10.6/3.5 mm Hg, but had no effect on the dose-response curves of dose of PE or Ang II vs. the increase in systolic, diastolic, or mean blood pressure, or heart rate. There were also no significant effects of cilazapril on PD20 values, i.e., the dose of PE or Ang II required to increase mean arterial blood pressure (MAP) by 20 mm Hg, or on delta R-R/delta MAP (ratio of the increase of the ECG R-R interval to the increase in mean arterial blood pressure) as a measure of baroreflex sensitivity. Plasma renin activity was significantly increased by cilazapril therapy, but there were no changes in plasma concentrations of Ang II or atrial natriuretic factor. We conclude that cilazapril, an ACE inhibitor, does not alter alpha 1-adrenoceptor and Ang II receptor sensitivity to selective agonists, nor does it affect baroreflex sensitivity.


Effects of chronic cilazapril treatment on cardiovascular control: a spectral analytical approach.

In 11 subjects with mild hypertension (sitting arterial pressure of 146 +/- 5/97 +/- 2 mm Hg), the effects of chronic angiotensin-converting enzyme (ACE) inhibition (cilazapril, 5 mg p.o. once daily for 4 weeks) were studied at rest and during active standing by means of spectral analysis. Sympathetic vascular control was inferred from the power of the low-frequency (LF) component of the systolic arterial pressure (SAP) variability, assessed noninvasively with a plethysmographic technique. Simultaneously, quantitative indices of neural control of the sinoatrial (SA) node were obtained with the power of the LF and of the high-frequency (respiration linked) component of R-R variability. Before treatment, active standing produced a clear increase in the LF components of R-R and SAP variabilities. At the end of the 4 weeks of cilazapril treatment, the LF component of SAP variability during standing was significantly lower than prior to treatment, while the increase in the LF component of R-R variability was left unchanged. These findings suggest an inhibitory effect of chronic ACE inhibition upon vasomotor sympathetic control, as assessed non-invasively by this technique.


Twenty-four-hour ambulatory noninvasive continuous finger blood pressure measurement with PORTAPRES: a new tool in cardiovascular research.

PORTAPRES model 1 is a portable 24 h continuous noninvasive blood pressure recorder based on the same principles as FINAPRES, the volume-clamp method of Peñaz and the physiocal criteria of Wesseling. In addition, PORTAPRES measures two adjacent fingers in alternation every 30 min and automatically corrects hydrostatic effects due to height changes of the measured fingers. The device measures 255 x 210 x 60 mm and weights about 3,000 g, including a lithium battery pack and a TEAC cassette FM instrumentation tape recorder to record the finger pressure wave form, the height signal, and beat-to-beat derived systolic, mean and diastolic pressure as well as heart rate. It appears to be an excellent new tool for cardiovascular research in humans. In a randomized, placebo-controlled, double-blind, crossover study the effect of oral administration of 2.5 mg cilazapril, a new potent long-acting, nonsulfhydryl-group angiotensin converting enzyme (ACE) inhibitor, given once daily for 7 days, was investigated in 16 healthy young men (mean age 25.3 +/- 1.6 years). Finger blood pressure and heart rate were measured with PORTAPRES for 24 h during everyday life and during standardized laboratory tasks, once about 1 to 2 h A.M. and once about 10 to 11 h P.M. once about 1 to 2 h A.M. and once about 10 to 11 h P.M. after drug administration. Physical activity was controlled by integrated thigh-EMG. Using stepwise multiple linear regression analysis it was shown that based on 64 s mean values, this measure of physical activity explains 34-77% of the heart rate variance within 24 h (median 53%), 10-52% (31%) of systolic, and 4-38% (25%) of diastolic blood pressure variance when up to 20 time lags of the EMG signal were introduced as possible predictors. This indicates that varying degrees of physical activity have a great impact on everyday blood pressure and heart rate. After one week cilazapril did not alter 24-h means of systolic and diastolic blood pressure or heart rate significantly, but reduced 30 min averages of both systolic and diastolic blood pressure by 6-10 and 4-6 mm Hg between 1 1/2 and 4 or 6 h after drug administration respectively during everyday life (p = 0.0067-0.066) without changing heart rate. On adjusting cardiovascular variables for the effects of physical activity, this blood pressure reduction was confirmed.(ABSTRACT TRUNCATED AT 400 WORDS)


Renoprotective action of angiotensin-converting enzyme inhibition in diabetes mellitus.

Roughly 40% of all diabetics, whether insulin dependent or not, develop persistent albuminuria, a decline in their glomerular filtration rate, and elevated blood pressure, i.e., diabetic nephropathy. Diabetic nephropathy is the single most important cause of end-stage renal disease in the Western world, accounting for over one-quarter of all end-stage renal disease. Systemic/glomerular hypertension plays a role in the initiation and progression of diabetic glomerulopathy. Angiotensin-converting enzyme (ACE) inhibitors are superior to conventional antihypertensive drugs in preventing the development of glomerular lesions in insulin-treated streptozotocin diabetic rats. Lowering of glomerular hypertension may be the crucial factor involved. Human studies suggest that ACE inhibitors postpone the progression to clinical overt diabetic nephropathy in normotensive diabetic patients with persistent microalbuminuria. ACE inhibitors combined with a diuretic reduce albuminuria and postpone renal insufficiency in hypertensive diabetics with overt nephropathy. No treatment modality other than antihypertensive treatment has yet been proven to be effective in protecting renal function in diabetic nephropathy. All previous reports dealing with the natural history of diabetic nephropathy have demonstrated a cumulative death rate between 50 and 77% 10 years after the onset of proteinuria. Effective antihypertensive treatment has reduced the cumulative death rate to 15-20% 10 years after the onset of nephropathy.


Renal effects of angiotensin II in normotensive subjects on short-term cilazapril treatment.

In 13 normotensive subjects on a normal sodium diet, we studied renal vascular, blood pressure, and hormonal changes induced by infusion of exogenous angiotensin II (Ang II) (5 ng/kg/min) during baseline conditions and after 5 days of cilazapril administration. In both conditions, Ang II elicited a slight pressor response accompanied by renal hemodynamic and hormonal changes that were similar. This suggests that hourly changes in Ang II concentrations and/or in Ang II receptor density after short-term administration of the angiotensin-converting enzyme (ACE) inhibitor cilazapril does not significantly affect renal vascular reactivity. In addition, these results also indirectly suggest that most of the renal effects of ACE inhibition are mediated through suppression of Ang II formation.


The antiproteinuric effects of blood pressure-lowering agents: differences between nondiabetics and diabetics.

The mechanism of the antiproteinuric effect of angiotensin-converting enzyme (ACE) inhibitors in diabetic and nondiabetic renal disease is as yet unknown. A meta-analysis of studies on the effects of ACE inhibitors and other antihypertensive drugs on proteinuria, blood pressure, and renal hemodynamics in nondiabetic renal disease revealed that ACE inhibitors lower proteinuria more than other antihypertensives. Moreover, a close correlation (p less than 0.01) between changes in urinary protein loss and in filtration fraction was found, whereas such a correlation could not be detected between changes in proteinuria and in blood pressure. This suggests that, at least in nondiabetic renal disease, the fall in proteinuria during ACE inhibition is the consequence of the intrarenal effect of the drug more than the systemic effect. Data on the mechanism of action of ACE inhibitors in diabetic microalbuminuria and in diabetic overt proteinuria are less consistent. A fall in proteinuria on antihypertensive drugs in these patients can be observed also without a significant fall in blood pressure, and without any change in filtration fraction. We therefore conclude that one should be cautious in extrapolating the data from studies in diabetic renal disease to patients with nondiabetic nephropathies. Moreover, we argue also that nonhemodynamic effects of ACE inhibitors also could be involved in the antiproteinuric effect of these drugs.


Angiotensin-converting enzyme inhibition and diabetic nephropathy.

Hypertension and diabetes mellitus are strongly associated conditions from epidemiologic, genetic, and pathophysiologic points of view. The prevalence of hypertension is high in patients with diabetes, and, conversely, many patients with essential hypertension are glucose intolerant. Proteinuria appears in 40-50% of patients with insulin-dependent diabetes mellitus and 20-30% of patients with non-insulin-dependent diabetes mellitus. Progressive renal failure occurs in 30-40 and 3-8% of patients, respectively, hypertension being a leading factor in its rate of progression. In various animal experiments, ACE inhibitors are able to prevent proteinuria and glomerular sclerosis, presumably by lowering transglomerular capillary pressure. In the diabetic human, ACE inhibitors are powerful antihypertensive drugs, devoid of metabolic side effects. Clinical studies indicate that ACE inhibitors reduce proteinuria and possibly slow the rate of decline in renal function. Such an effect is not observed with beta-blockers. Large-scale studies are needed to confirm this very important hypothesis.


Renal prostaglandin synthesis and angiotensin-converting enzyme inhibition.

Renal prostaglandins (PGs) help maintain renal blood flow and glomerular filtration rate when the kidney is exposed to a vasoconstrictor stress. In addition, they aid pressure natriuresis and blunt the antidiuretic effect of vasopressin. Angiotensin-converting enzyme (ACE) inhibitors could decrease renal PG synthesis by reducing angiotensin II (Ang II) formation or increase it by preventing kinin inactivation. Additionally, they could affect PG synthesis or catabolism directly. The effects of ACE inhibitors on blood pressure and renal hemodynamics appear to be largely independent of changes in renal PG synthesis. Similarly, there is no evidence that pressure natriuresis is modified by ACE inhibitors. A kinin induced increase in collecting duct PG synthesis may account for the water diuresis seen clinically with ACE inhibitors. A possible beneficial interaction between thromboxane synthesis inhibitors and ACE inhibitors may exist. Thromboxane synthetase inhibitors can reduce renal vascular resistance by redirecting PG endoperoxide synthesis toward prostacyclin. This effect may be offset by a prostaglandin-induced increase in renin release and Ang II formation. ACE inhibitors, by preventing Ang II synthesis, may increase the vasodilation due to thromboxane synthesis inhibition.


Renal effects of antihypertensive agents in parenchymal renal disease and renovascular hypertension.

Treatment of hypertension by conventional antihypertensive medications usually has no significant effect on renal function in patients with essential hypertension and normal glomerular filtration rate. In this condition, new agents such as angiotensin-converting enzyme (ACE) inhibitors and calcium-channel blockers (CCBs) also do not appear to modify renal function. Reduction of arterial pressure is crucial for renal protection in patients with chronic renal disease and the effect of the new agents on the progression of renal failure remains unknown despite some promising reports. In patients with renovascular hypertension, mainly those with bilateral stenosis or stenosis of a solitary kidney, the use of ACE inhibitors may be associated with reversible renal deterioration, particularly in subjects with already impaired renal function and receiving diuretics. In this situation, reduction in arterial pressure by CCBs does not appear to lead to renal deterioration.


The brain renin-angiotensin system: localization and general significance.

This report summarizes the present data about the existence of components of the renin-angiotensin system in the rat brain. Angiotensinogen mRNA, mas proto-oncogene mRNA, angiotensin II (Ang II), and Ang II receptors have been mapped in the brain by using in situ hybridization, immunocytochemistry, and receptor autoradiography. These markers turned out to be widely distributed throughout the brain and to be not only restricted to areas related to cardiovascular control, but also to be present in functionally different areas, suggesting also other functions of angiotensin peptides. The distribution patterns of these components were correlated with data on the distribution of angiotensinogen, renin, angiotensin converting enzyme, and angiotensin fragments that revealed substantial topological mismatches. Using the model of "volume transmission," possible explanations for these mismatches are proposed. In this regard, a possible involvement of angiotensin fragments and the mas proto-oncogene in the functioning of the brain renin-angiotensin system is also discussed, demonstrating the increasing complexity of this central regulatory system.


Angiotensin-converting enzyme inhibition, angiotensin, and cognition.

In these studies, we have investigated possible cognition-enhancing effects of angiotensin-converting enzyme (ACE) inhibition, and putative neurochemical correlates for these actions. In a mouse habituation model, ACE inhibitors improved basal performance and antagonized scopolamine-induced deficits. The performance of aged mice and those with lesions of the nucleus basalis was also improved. ACE inhibition also improved scopolamine-impaired performance of rats in a swim-maze model. Neurochemical studies showed that a low dose (10 micrograms/kg i.p.) of ceranapril caused significant alterations in ex vivo rat brain catecholamine levels in the nucleus accumbens, amygdala, and septum. In further studies, angiotensin II (Ang II) was shown to decrease potassium-stimulated [3H] acetylcholine release from slices of rat entorhinal and human temporal cortex, an effect that could be antagonized by the angiotensin receptor antagonist [1-sar,8-thr]Ang II. It is concluded that ACE inhibition can improve both basal and impaired performance in animal models of learning, and that this improvement may be in part a consequence of the removal by ACE inhibition of an inhibitory tone on central acetylcholine release, and/or an effect on central catecholaminergic function.


Role of brain angiotensin in cardiovascular regulation.

The brain is one of the organs where an intrinsic renin-angiotensin system (RAS) has been described. Stimulation of circumventricular or brainstem angiotensin II (Ang II) receptors engenders a distinct pattern of cardiovascular, endocrine, and behavioral responses featuring blood pressure increase, attenuation of the baroreceptor reflex, drinking, release of pituitary hormones such as vasopressin, oxytocin, and ACTH, and natriuresis. In contrast to most of the other central actions of Ang II, the natriuretic effect cannot be elicited by Ang II as a circulating hormone. Recently, we have shown that stimulation of Ang II AT-1 receptors in the circumventricular organs causes a selective release of norepinephrine (NE) in the paraventricular nucleus (PVN) and in the supraoptic nucleus (SON). As vasopressin is also released from the PVN and SON, it is possible that the Ang II-NE interaction is involved in the release of vasopressin, thereby contributing to central blood pressure regulation and volume control. Finally, a substantial body of results suggests that an overactivity of the brain renin-angiotensin system is one of the contributors to genetic hypertension. However, this idea needs further confirmation.


Diabetes and hypertension.

Arterial hypertension is more common in diabetes mellitus than in nondiabetic subjects, and many metabolic and hemodynamic features of diabetes mellitus contribute to the etiology of hypertension. Control of hypertension in diabetes mellitus is extremely important as high blood pressure accelerates both macrovascular and microvascular complications of this disease. Most classes of antihypertensive agents are effective in blood pressure control in diabetes mellitus, so the choice of antihypertensive therapy is based on the differences in adverse effects of these agents on metabolic control and their effect on other cardiovascular risks.


Interaction between the renin-angiotensin-aldosterone and sympathetic nervous systems.

The renin-angiotensin-aldosterone system is mainly involved in the regulation of arterial blood pressure and fluid balance. One of the main stimuli for the secretion of renin present in the renal juxtamedullary cells, but also in some other tissues, is provided by the sympathetic nervous system via the action of norepinephrine on beta 1-adrenoceptors. There is good evidence in animal experiments that angiotensin II (Ang II) facilitates sympathetic neurotransmission by several mechanisms, all of which seem to involve distinct, but perhaps heterogeneous, Ang II receptors. Acting within the central nervous system, angiotensin augments sympathetic nerve outflow directly, but probably also by inhibiting the reflex decrease in sympathetic nerve activity following an increase in arterial pressure. Ang II also stimulates adrenomedullary and ganglionic transmission as well as enhances the release of sympathetic transmitter by a presynaptic action. In addition, there is some evidence that angiotensin can inhibit norepinephrine reuptake and augment its biosynthesis and responses mediated via both extrasynaptic alpha 2- and intrasynaptic alpha 1-adrenoceptors. Angiotensin-converting enzyme inhibitors, particularly when the endogenous renin-angiotensin activity is high, attenuate sympathetic neurotransmission. Despite a clear demonstration that the renin-angiotensin system augments the activity of the sympathetic nervous system in animals, evidence for such a role in humans is tenuous. This is probably mainly due to the difficulty in quantitatively monitoring and assessing the autonomic function in humans. It is possible that in congestive heart failure, where the renin-angiotensin system can be highly activated, sympathetic facilitation by angiotensin as well as its attenuation by converting enzyme inhibitors may be important.


Regulation of cerebral blood flow in health and disease.

A review is given of the normal regulation of cerebral blood flow (CBF) and its pathophysiology in hypertension and stroke. In otherwise healthy hypertensive patients, the absolute level of CBF is the same as in normal subjects. CBF autoregulation, however, is shifted towards higher pressure, thus impairing the tolerance to hypotension. In most patients, this does not interfere with the beneficial effect of treatment, i.e., stroke prevention. Cerebral ischemia, however, may be provoked by overzealous pressure lowering in selected clinical settings: initial or intensified treatment of very severe hypertension, treatment of hypertension in the elderly, and treatment of hypertension in acute stroke. In the latter, a complicated sequence of brain ischemia and hyperemia makes antihypertensive intervention difficult in the early phase, when blood pressure is probably best allowed to decrease spontaneously.


Protective effect of cilazapril on the cerebral circulation.

The goal of an antihypertensive treatment is to prevent "end-organ" damage. Cerebral vascular complications are among the most important because they are life threatening and can occur even at an early stage of the disease. Recently, it has been shown that cilazapril can decrease the mortality of stroke-prone rats, suggesting a decrease in the incidence of strokes, which occur spontaneously in these animals. The present article reviews the different functional and morphological changes that may explain the cerebral protective effects of cilazapril, such as the normalization of cerebral vascular reserve, decrease in the media, increase in the external diameter, and normalization of the mechanics and endothelial function of cerebral arterioles. In addition, the inhibition by cilazapril of injury-induced proliferation of smooth muscle cells and the infiltration of the endothelium by macrophages could prevent the development of atherosclerosis.


Immunocytochemical detection of factor XIII A--subunit in acute leukemia.

Factor XIII (FXIII) is a plasma pro-transglutaminase consisting of A and B subunits in a tetrameric structure. A cellular form of FXIII consisting exclusively of A subunits exists in platelets and monocytes: monocyte FXIII may be involved in connective tissue organization. To evaluate the expression and diagnostic significance of FXIII A subunit (FXIIIA) in acute leukemia, we performed an immunocytochemical study (PAP technique) with rabbit antiserum against FXIIIA on leukemic blasts of 48 cases. FXIIIA was detected only in myelomonocytic (M4), monocytic (M5) and megakaryocytic (M7) cases: in M4 and M5 samples the amount of blast cytoplasmic FXIIIA was closely correlated with the expression of monocyte-specific antigenic and cytochemical markers. Our data show immunocytochemical detection of FXIIIA to be useful for acute leukemia characterization.


Single-channel and whole-cell recordings from non-dissociated sympathetic neurones in rabbit coeliac ganglia.

A procedure is described for performing patch-clamp recordings on mammalian sympathetic neurones within intact ganglia. The plasma membrane of superficial neurones was cleaned by blowing (1.5-3 h) a gentle stream of Ringer saline onto ganglia, the connective sheath of which was previously softened by a short protease treatment. This procedure preserved the intraganglionic connectivity so that the neurones could be activated either synaptically or antidromically by stimulating the appropriate nerves. Depending on the duration of the mechanical cleaning step, recordings were performed on either the neurones or the satellite glial cells covering the neuronal cell bodies. The applicability of the various configurations of the patch-clamp technique to studying sympathetic neurones is illustrated by recordings of whole-cell voltage, whole-cell currents and single-channel currents in cell-attached and excised patches. With these techniques, the resolution of the membrane current recordings is higher than with conventional microelectrodes. The results obtained show that mammalian sympathetic neurones have a very high input resistance (0.5 G omega), are electronically compact and may display pacemaker activity. These techniques provide a useful tool for studying the synaptic transmission and neuromodulation mechanisms operating within the sympathetic ganglia.


A computerized 2-dimensional vibrating probe for mapping extracellular current patterns.

We describe a computer-assisted 2-dimensional vibrating probe system for mapping endogenous electric current patterns in biological preparations. This system overcomes some of the main limitations of the original 1-dimensional vibrating probe design and adds several new capabilities. Two piezo-electric bender elements mounted perpendicularly are used to vibrate the probe in a circle by applying 2 sine waves (1 to each element) that are 90 degrees out-of-phase with each other. The circular rotation of the probe allows it to detect simultaneously the 2 orthogonal components of a current in the horizontal plane. The voltages measured by the probe are digitized and analyzed by a computer and are used to calculate a current vector. A graphical representation of the current vector is then superimposed on a video image of the experimental preparation. This probe system responds to known currents in the expected manner and exhibits a low inherent noise level. Also included in this paper are some preliminary measurements made with this instrument on neurulating Xenopus embryos and on transected larval sea lamprey (Petromyzon marinus) spinal cords.


Effect of different convulsants on calmodulin levels and proto-oncogene c-fos expression in the central nervous system.

In the present study, a relationship between convulsant activity and two cellular events, changes in calmodulin (CaM) concentration and proto-oncogene c-fos expression has been considered. c-fos has been found activated after the administration of the organochlorine insecticide lindane, the Ca2+ channel agonist Bay K, and N-methyl-D-aspartate (NMDA). The administration of the voltage-dependent Ca2+ channel antagonist nifedipine was able to block the expression elicited by lindane. The effect of lindane on c-fos expression could not be blocked by prior administration of MK-801, a non-competitive antagonist of the NMDA receptor. These results suggest a possible role for the voltage-dependent Ca2+ channels in the mechanism of action of lindane. By means of in situ hybridization, the different patterns of c-fos expression after the administration of the mentioned compounds have been described. A possible modification of the levels of CaM has also been investigated. Among all the subcellular fractions considered, only levels of nuclear CaM appeared to be affected after the different treatments. The changes observed seemed to follow a similar pattern to that described for c-fos induction. Calcium entry through these voltage-dependent calcium channels would be the link between membrane depolarizing events and expression of c-fos and/or increase in nuclear CaM.


Altered amounts of G-protein mRNA and cAMP accumulation after long-term opioid receptor stimulation of neurons in primary culture from the rat cerebral cortex.

Primary neuronal enriched cultures were incubated with mu (morphine, 10(-5) M), delta (DPDPE, 10(-6) M) and kappa (U-50,488H, 10(-5) M) receptor agonists for 5 days, respectively. Thereafter the acute inhibitory actions of mu, delta or kappa receptor agonists on forskolin stimulated cAMP accumulation was assayed. The effect of long term opioid treatment on the steady-state level of G-protein mRNA (G alpha s, G alpha i-1 and G alpha i-2) was analyzed using an RNAase protection hybridization assay. Incubation for 5 days with kappa receptor agonist resulted in an attenuated ability to decrease the accumulation of cAMP by kappa receptors, as well as mu and delta receptors, which was also observed after 5 days of incubation with the delta receptor agonist. Furthermore, the adenylate cyclase responsiveness to forskolin stimulation was markedly reduced in cultures treated with either delta or kappa receptor agonists. Five days of incubation with kappa receptor agonist resulted in an increase in the levels of G alpha s and G alpha i-2 mRNAs. No effects on the amounts of G alpha s mRNA, G alpha i-1 mRNA or G alpha i-2 mRNA were detected after 5 days of delta receptor stimulation. On the other hand, 5 days of mu receptor stimulation decreased the amounts of G alpha s, G alpha i-1 and G alpha i-2 mRNA. Incubation with kappa receptor agonist for 24 h resulted in a significant decrease in the forskolin-stimulated accumulation of cAMP. The stimulatory effect of forskolin was further decreased after 3 days incubation with kappa receptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic administration of NMDA antagonists induces D2 receptor synthesis in rat striatum.

Dopamine D2 receptor gene expression was examined in rat striatum after chronic treatment with N-methyl-D-aspartate (NMDA) receptor antagonists (ketamine at 15 mg/kg/day or MK-801 at 0.1, 0.2 and 0.4 mg/kg/day per os, for 50 days). The long-isoform mRNA, as well as the total D2 mRNA expression were induced. No change was noticed in striatal dopamine release or turnover. D2 binding studies carried out in MK-801 chronically treated (0.3 mg/kg/day per os, for 50 days) and control rats revealed an increased receptor density in treated animals without a significant change in receptor affinity. These results suggest that the synthesis of both striatal D2 receptor isoforms is postsynaptically regulated at the transcriptional level, by events triggered by glutamate through the NMDA-type receptor.


Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms.

Sequences from homologous regions of the nuclear and mitochondrial small-subunit rRNA genes from 10 members of the mushroom order Boletales were used to construct evolutionary trees and to compare the rates and modes of evolution. Trees constructed independently for each gene by parsimony and tested by bootstrap analysis have identical topologies in all statistically significant branches. Examination of base substitutions revealed that the nuclear gene is biased toward C-T transitions and that the distribution of transversions in the mitochondrial gene is strongly effected by an A-T bias. When only homologous regions of the two genes were compared, base substitutions per nucleotide were roughly 16-fold greater in the mitochondrial gene. The difference in the frequency of length mutations was at least as great but was impossible to estimate accurately because of their absence in the nuclear gene. Maximum likelihood was used to show that base-substitution rates vary dramatically among the branches. A significant part of the rate inconstancy was caused by an accelerated nuclear rate in one branch and a retarded mitochondrial rate in a different branch. A second part of the rate variability involved a consistent inconstancy: short branches exhibit ratios of mitochondrial to nuclear divergences of less than 1, while longer branches had ratios of approximately 4:1-8:1. This pattern suggests a systematic error in the branch length calculation. The error may be related to the simplicity of the divergence estimates, which assumes that all base positions have an equal probability of change.


Use of stage II-III Xenopus oocytes to study voltage-dependent ion channels.

Stage II-III Xenopus oocytes represent a useful extension of the standard Xenopus oocyte expression system. The oocytes are smaller; the reduction in membrane area, and therefore capacitance, leads to a faster settling time during a voltage clamp step. In addition, there appears to be less Ca(2+)-activated chloride current; this may render the stage II-III oocyte a useful system for studying K+ channels, where chloride currents can be a significant problem. Conversely, though, these early stage oocytes may not be useful for expression of neurotransmitter receptors coupled to phospholipase C, for such receptors are often monitored by activation of the Cl-current. The injections of RNA are technically more difficult in stage II-III oocytes. This can, however, be overcome with some simple modifications of the injection apparatus, mainly inclusion of a pump or similar device for actual injections.


Ion channels. Preventing artifacts and reducing errors in single-channel analysis.

The power of single-channel analysis techniques has rapidly expanded during the past few years, giving investigators increased ability to identify models and estimate parameters while reducing error and artifacts. At present, however, there is no single best method, as even the most advanced techniques have various limitations which depend on the experimental data and models being examined. Consequently, for the examined models and experimental data, the most critical part of single-channel analysis is to estimate errors and evaluate the ability of the methods used to discriminate among possible gating mechanisms. The magnitudes of the errors and the ability to identify models and estimate parameters depend on the models being examined as well as the experimental conditions and data. Consequently, the evaluation of the errors associated with each method needs to be repeated when the experimental data and examined models change.