[The temporal organization of the functional connection of the motor cortex neurons in the cat]. Conditioned food-procuring response to time (2 minutes interval) was elaborated in cats, multiunit activity of the motor cortex being recorded. On the basis of single spike trains discriminated from the multiunit activity the cross-correlation histograms were built and the spikes composing their peaks were analysed in real time. This secondary analysis of the histograms allowed to ascertain the dynamics of functional connections between the neurons during the phase of active waiting according to the distribution of coincident impulses. A concentration of coincident impulses of simultaneously recorded cells was observed in different moment of time. In some neuronal pairs the concentration of coincident impulses was revealed to the end of the conditioned interval. The data obtained are considered as a manifestation of the conditioned reaction at the level of neuronal interaction.


[The effect of chronic activation and block of the dopamin- and enkephalinergic systems of the neostriatum on conditioned-reflex behavior abd dopamine metabolism in the nigrostriatal system of rats]. Were studied the effects of bilateral daily intrastriatal microinjections in the course of two weeks of amphetamine (45 mcg), haloperidol (5 mcg), naloxone (5 mcg), and enkephalin synthetic tetrapeptide analogue (15 mcg) on behaviour and the level of dopamine and its metabolites in the striatum and substantia nigra of rats. Amphetamine improved but haloperidol impaired conditioned avoidance response realization in a shuttle-box and produced parkinsonian-like akinetic status. Naloxone was behaviourally uneffective but the tetrapeptide produced the obvious cataleptic status with plastic rigidity of the skeletal muscles. Both amphetamine and haloperidol lowered significantly striatal dopamine level and increased the level of its metabolites. The tetrapeptide produced the opposite neurochemical effect. Possible origin of discoordination between behavioural and neurotransmitter changes are discussed.


[The spontaneous neuronal activity of the hippocampus during the modulation of the theta rhythm by cholinergic substances]. Background activity of the hippocampal neurons, extracellularly recorded in waking chronic rabbits, was analysed in control state and after systemic injection of physostigmine and scopolamine. Similar analysis was done in the hippocampus chronically deprived of ascending brain stem afferents. Cholinergic drugs controlled the number of hippocampal neurons with theta-modulation and the degree of its stability but not the frequency. Activation of cholinergic theta-rhythm resulted also in regularization of activity with suppression of delta-modulation and complex spike discharges; its blockade was accompanied by the opposite changes. Both drugs shifted the level of background activity in the majority of neurons, but the overall mean frequency did not vary between the states. Regression analysis demonstrated significant negative correlations with dominating decrease in the level of activity in high-frequency neurons ( > 25 sp/s) and its increase in low-frequency ones ( < 25 sp/s) after injection of both drugs. Stability of the overall mean frequency and uniformity of its shifts presumably indicate that the frequency, unlike the pattern of the background activity, is not directly controlled by the cholinergic septal input.


[The neuronal activity of suspension grafts of the neocortex]. Were compared electrophysiological characteristics of suspension (SUS) and solid (SOL) tissue grafts of embryonic neocortex transplanted into the barrel field of adult rats. The level of survival of SUS and SOL grafts did not differ significantly (89% and 95% correspondingly). All SUS grafts were integrated with the host brain judging by histological and electrophysiological criteria. Reactivity of neurons to electrical stimulation of the recipient's brain and to sensory (tactile) stimulation, as well as the latencies of on-responses were similar in both types of the grafts. However, proportion of neurons with on-responses was nearly twice lower in SUS grafts, while the number of neurons responding to tactile stimulation by primary suppression of activity was significantly higher than in SOL ones. It is suggested that the derangement of primary architectonics unavoidable during preparation of suspensions is responsible for the described differences between SUS and SOL grafts.


[A comparative analysis of the change in the EEG spectral characteristics of rats during the activation and suppression of the catecholaminergic systems]. To find EEG-markers of catecholaminergic activation shifts EEG power spectra of white rats were computed before and after intraperitoneal injections of propranolol, metaproterenol, haloperidol, amantadine, or isotonic sodium chloride solution. Differential spectral characteristics were undergone to factor analysis and discriminant analysis. Factors with similar structure for both catecholaminergic systems were revealed in EEG-reactions to mutually antagonistic injections and relatively specific factors as well. The leading factor of adrenotropic injections described the augmentation of the spectral power in the range of 9-16 Hz induced by propranolol and its reduction by metaproternol. Similar factor was also revealed in reactions to dopaminotropic injections with the smaller value of discriminant function coefficient. One more common feature of EEG-reactions to catecholaminergic disturbances was found to consist of the reciprocal narrow-band shifts in the theta- and delta-diapasons. The leading factor for the recognition of dopaminotropic disturbances described the increase of EEG power in the band of 19-30 Hz at activation and its reduction at suppression of the transmitter system.


Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast. Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, suggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amino acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.


[Pregnancy after successful therapy of HBV-associated hepatocellular cancer--a case report]. Malignant tumors are a difficult problem in obstetrics, both in pregnancy but also after an successful therapy, especially, if the patient wishes to be pregnant. However, there are borderline cases of medical and ethical regions. In the present paper the case of a young women (21 years old) with HCC is described. In the age of 16 years a solid hepatocellular carcinoma was found and treated by partial resection of the right lobus hepaticus. Anamnestically a blood exchange transfusion caused by a neonatal Rh-incompatibility and a hepatitis B in the age of 11 months are known. 3 and 6 years after the treatment, she was successfully pregnant and is today mother of 2 children.


[The frequency composition of the brain electrical activity in rats after the use of the delta-sleep peptide and its analogs]. Frequency spectra of brain electrograms in the course of 1 h after peripheral and central administration of the delta-sleep peptide (DSIP) or two its analogues were studied in freely moving rats. In autumn series of experiments carried out on 18 animals was revealed the phase action of DSIP being manifested in initial (up to 20 min after the injection) suppression of fast (20-26 Hz) oscillations in electrocorticograms and their augmentation in subsequent intervals. Under the identical conditions analogues of DSIP induced the effects characteristic for different phases of DSIP action. In spring-summer series of experiments carried out in 6 animals was revealed a significant increase of the delta-waves in electrical activity of the Putamen after intraperitoneal injection of DSIP and its first analogue. Under the conditions of intraventricular injection DSIP induced stable augmentation of oscillations in a diapason of 14-16 Hz in the neocortex, and its analogues induced similar changes in a nearby frequency diapason of 9.6-11 Hz.


Progressive myoclonic ataxia without ragged red fibres: Unverricht-Lundborg disease vs Ramsay Hunt syndrome. We describe eight patients from three families presenting with myoclonus, ataxia, infrequent seizures and minimal intellectual impairment. All were Arabs from different parts of the Arabian peninsula. The new consensus on terminology, genetic and clinical definition of Baltic myoclonus, Ramsay Hunt syndrome and Unverricht-Lundborg disease suggests that our group are best categorised under the term of progressive myoclonic ataxia of the Unverricht-Lundborg type. Moreover, this report reinforces the existence of this syndrome outside Scandinavia.


[Vision in the edible snail. The morphology and total electrical activity of the retina]. Recording of the integrative electrical activity of the retina of the snail and electron microscopic study of its eye have shown the following. The electrical activity of the photoreceptors is a source of the retinogram (ERG) of the snail. ERG reaction form is characterized by two phases of a response: the initial spike and the following slow fading. For each given value of the photometric brightness of a light signal there exists a low limit of its length, beginning from which the ERG reaction of the snail assumes the form described. The value of the ERG response is a logarithmic function of brightness of the light stimulus.


[The influence of epiphysectomy and melatonin on the antidepressive effect in disordered function of the beta-adrenergic mechanisms in rats]. After chronic administration of beta-adrenoblocker propranolol (1 mg/kg, 14 days) reorganization of time course of forced swimming with decrease of rhythmical index of depression was observed. Pinealectomy slightly attenuated, and melatonin (1 mg/kg) potentiated this effect. As proposed, antidepressive properties of beta-adrenoblockade primarily are not connected with pharmacological denervation of the pineal gland.


Deblocking effect of TRH-T in three cases of chronic progressive multifocal neuropathy. Three cases of acquired demyelinating multifocal neuropathy with persistent conduction blocks are reported. In one of them protyreline tartrate (TRH-T) had an evident deblocking effect; similar but milder effects were seen in the other two cases. The course and consistency of this effect is analyzed on the grounds of clinical and electromyographic findings. The nature of the THR-T responsive conduction block is analysed as a mild variant of CIDP with regard to its occurrence in a late and chronic phase without signs of active immune processes.


[The effect of self-stimulation of the lateral hypothalamus on the sleep-waking cycle in rats during alcoholization]. Directed activation of a system of positive emotional reinforcement induces regulatory effect on limbic-neocortical mechanisms of the sleep-waking cycle organization in rats after chronic alcoholization carried out in periods of decreased and increased circadian rhythms of emotional activity. In animals with high level of positive emotional drive after the alcoholization self-stimulation of the lateral hypothalamus suppresses hypersynchronous paroxysmal activity in waking EEG, decreases the content of waking in the sleep-waking cycle, restores the paradoxical phase of sleep. In animals with inhibition of positive emotional drive in consequence of alcoholization self-stimulation of the lateral hypothalamus has no essential effect on the mechanisms of regulation of the sleep-waking cycle.


Antihypoxic treatment at an early stage of diabetic neuropathy: an electrophysiological study with sabeluzole. Thirty-seven non-IDDM patients at an early stage of polyneuropathy, defined as the presence of symptoms for less than two years, as well as an abnormal perception threshold and/or abnormal thermal discrimination threshold, were treated with sabeluzole, a new antihypoxic drug, or placebo for 1 year in a double-blind, placebo-controlled study. They were examined neurophysiologically every 3 months, when motor (tibial, ulnar) nerve and sensory (sural, ulnar) nerve conduction velocities, H-reflex of the soleus muscle, SF-EMG of the anterior tibial muscle, static and dynamic pupillometry were measured. Statistical analysis did not show significant differences in nerve function between the sabeluzole group and the placebo group. There were also no significant changes within each group over the 1-year period. The results of the present study show no beneficial effect of sabeluzole on peripheral nerve function in patients at an early stage of diabetic polyneuropathy.


Leukotrienes in the cerebrospinal fluid of multiple sclerosis patients. The concentration of the leukotrienes B4 (LTB4) and C4 (LTC4) was measured in the cerebrospinal fluid (CSF) of 38 multiple sclerosis (MS) patients and 51 with other neurological diseases. The LTB4 and LTC4 levels were significantly elevated in MS compared with the controls. The findings suggest that lipoxygenase products might play a pathogenetic role in the early, encephalitogenic phase of MS. The administration of lipoxygenase inhibitors or leukotriene antagonists might well open new perspectives for the treatment of MS.


IgG paraproteins in neurological diseases: lack of association with neurotropic viral/bacterial antigens. In this study we tested the hypothesis that IgG paraproteins in neurological diseases might be regarded as an anti-idiotypic response raised by a nervous system antigen associated with certain neurotropic agents. After initial ELISA screen, positive samples from 34 neurological patients who had paraproteins in their CSF and serum on routine IEF investigation, were tested by immunoblot technique for the presence of specific monoclonal immunoglobulin G (IgG) against eight different neurotropic antigens. Only one patient had specific monoclonal IgG against herpes simplex virus. The results of this study did not confirm our hypothesis. Further study of IgM/IgA paraproteins is indicated.


Type 3 GM1 gangliosidosis: characteristic MRI findings correlated with dystonia. We describe three brothers with type 3 GM1 gangliosidosis presenting as dystonia. The ages of the patients when examined were 28, 31, and 33. They had developed dysarthria with facial grimacing since early childhood. The common neurological sign was generalized dystonia. Both dystonic postures and dystonic movements resulting from varying degrees of fixed rigidity of each muscle involved did not disappear when the patients were lying or sitting relaxed. There was no correlation between the severity of dystonia and the residual activities of acid beta-galactosidase. Magnetic resonance imaging (MRI) showed bilaterally symmetric high intensity lesions only in the putamen on T2-weighted and proton density images. Selective putaminal changes on MRI may be the lesions most responsible for symptomatic dystonia in this disorder.


Detection of latent herpes simplex virus DNA and RNA in human geniculate ganglia by the polymerase chain reaction. By using the polymerase chain reaction (PCR) we detected latent herpes simplex virus type 1 (HSV-1) in human geniculate and trigeminal ganglia obtained from autopsy cases. A pair of primers which were specific for a part of the HSV-1 thymidine kinase domain were used for detection of HSV DNA. We also examined the latency-associated transcript (LAT), known as latency-specific RNA, by means of reverse transcription-PCR with a pair of LAT-specific primers. HSV-1 DNA was detected in 16 of 17 (94%) trigeminal ganglia and in 15 to 17 (88%) geniculate ganglia of adults. We also demonstrated HSV-1 RNA derived from the LAT in both types of ganglia. These findings suggest that HSV-1 latently infects the majority of geniculate and trigeminal ganglia of adults, and that PCR and reverse transcription-PCR are useful tools for analysis of HSV latency.


Effects of dauricine and anisodamine on LTs and PAF induced DNA synthesis in bovine anterior cerebral arterial smooth muscle cells. The effects of leukotrienes (LTs) and platelet activating factor (PAF) on DNA synthesis in bovine anterior cerebral arterial smooth muscle cells were studied. The results showed that LTB4, LTD4 and PAF were all stimulatory to the cells. The maximal rates of stimulation were 32%, 29% and 77%, respectively, for LTB4, LTD4 and PAF at 10(-7) mol/L. Both dauricine and anisodamine at concentrations of 10(-7) to 10(-4) mol/L exhibited strong inhibitory effects on LTs and PAF induced DNA synthesis.


Effects of acute and long-term atropine treatment on levels, release and response to VIP and PHI in the submandibular gland of cat and rat. We have studied the effects of acute and long-term treatment of cats and rats with atropine on the levels, release and effects of two peptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), that probably co-exist with acetylcholine in the parasympathetic nerves supplying the submandibular gland. Atropine treatment (progressively increasing doses from 2 to 15 mg kg-1 injected s.c.) for 14 days did not alter the contents of VIP- or PHI-like immunoreactivity (-IR) in the cat submandibular gland or in three other tissues (nasal mucosa, trachea and tongue). Acute as well as long-term atropine treatment decreased the vasodilation following low-, but not high-, frequency parasympathetic nerve stimulation. During prolonged stimulation (60 min) there was a decreased vasodilatation response following both acute and long-term atropine treatment. The overflow of VIP-IR and PHI-IR following parasympathetic nerve stimulation was markedly increased by acute, but not by long-term atropine treatment. The VIP- or PHI-induced stimulation of cyclic AMP (cAMP) accumulation in the cat submandibular gland was not altered after long-term atropine treatment. Similarly, treatment of male Sprague-Dawley rats with atropine (20 mg kg-1) or imipramine (20 mg kg-1) for 14 days did not alter the sensitivity to VIP or to PHI of cAMP accumulation in the submandibular gland, nor was there any change in VIP-IR or PHI-IR content. In conclusion, although atropine treatment causes an acute increase in the overflow of VIP and PHI evoked by parasympathetic nerve stimulation, there is no depletion of peptide stores upon long-term treatment, nor is there any change in the effect of exogenous VIP and PHI on cAMP-accumulation.


Effects of a six-week, low-fat diet on serum cholesterol, body weight, and body measurements. This study was done to determine if significant weight loss, decrease in body measurements, and lowering of serum lipids and blood pressure could be shown in people on a low-calorie, low-fat, high-fiber diet over a period of six weeks. Forty-one people (33 females and 8 males) between the ages of of 37 and 55 years old participated in a six-week calorie-controlled, low-fat, low-sodium, high-fiber diet using foods available in supermarkets. Participants also exercised moderately. The average weight loss was 10.3 pounds, or 5.8% of the original average weight. The body mass index decreased 1.7-a 6.0% decrease for women and a 5.3% decrease for men. The average of five body measurements decreased 4.9%. Total cholesterol decreased 16%, LDL-C decreased 12%, and HDL-C decreased 18%. All of these decreases were statistically significant at P < .001. No significant difference was noted in blood pressures at the start and completion of the study, as both values were in the normal range. This relatively short period of dieting using 1200 calories for women and 1500 calories for men, along with moderate exercise, shows that reasonable weight loss and a tendency toward lowering lipid values can be obtained. Weekly monitoring by physicians and their office staffs can enhance this process. Continued accountability and support for lifelong behavior changes are probably necessary to maintain reasonable body weight in these patients.


Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR-106 osteosarcoma cells. In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca(2+)-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the alpha-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the alpha-subunit of Gi. The alpha-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a +/- 40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1 alpha/Gi2 alpha and Gi3 alpha could be immunoprecipitated, respectively, but immunoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)


Two hereditary defects related to vitamin D metabolism map to the same region of human chromosome 12q13-14. We have localized the locus for the vitamin D receptor (VDR) responsible for hypocalcemic vitamin D-resistant rickets (HVDRR), close to the pseudovitamin D-deficient rickets (PDDR) locus, another disorder related to impaired vitamin D metabolism. PDDR (formerly vitamin D dependency type I, VDD1) was recently mapped to human chromosome 12q14 by linkage analysis. Here we report on the assignment of VDR to 12q13-14 by in situ hybridization and by linkage analysis. Linkage analysis between VDR, PDDR, and several RFLP markers show close linkage, with no recombination (theta = 0) between VDR and PDDR (Z = 1.94), a COL2A1 haplotype (Z = 4.03), ELA1 (Z = 0.98), and D12S15 (Z = 4.17). The analysis of extended haplotypes in one of the PDDR families provides evidence for recombination between VDR and PDDR and localizes VDR together with COL2A1 proximal to PDDR. Complete allelic association detected between VDR and COL2A1 loci on PDDR chromosomes and lower association between VDR and PDDR suggests a VDR location very close to COL2A1 and one more distant to PDDR. We propose the following order of loci: (VDR, COL2A1), (PDDR, ELA1, D12S15), D12S4, (D12S14, D12S17), D12S6. Thus, two clearly distinct loci involved in the control of vitamin D activity map close to each other in the region 12q13-14.


The influence of bacterial collagenase on regeneration of severed rat sciatic nerves. Regeneration of peripheral nerve fibers is impeded by the formation of scar tissue at the site of injury. The possible beneficial effect of collagenase on nerve regeneration was studied using clinical, neurophysiological (evoked potentials) and histological (nerve fiber counts) methods. The sciatic nerves of rats were transected and the severed ends abutted and sewn together. In one series, the area about the lesion was covered with fibrin adhesive and infused with either isotonic saline (controls) or collagenase (treatment group). In the other series, the severed ends of the nerve were inserted into a silicone tube and separated by a collagen plug, which was infused with either saline or collagenase. Compared to the controls, the treated animals showed a significant improvement of clinical and neurophysiological parameters. After 3 months of observation, the collagen content of the transection site was reduced, and in the silicone series, the total number of myelinated axons 5 mm distal to the site of transection was increased, while the fiber diameter distribution was unchanged.


Increase in omega 3 (peripheral-type benzodiazepine) binding site densities in different types of human brain tumours. A quantitative autoradiography study. The density of omega 3 (peripheral type benzodiazepine) binding sites, a marker of reactive and tumoural cells, has been measured in different types of human brain tumours; omega 3 sites were quantified autoradiographically in sections from biopsy or autopsy specimens labelled with the specific radioligand 3H-PK 11195. Compared to normal brain parenchyma, up to 12-fold increase in omega 3 site densities were found in apparently viable areas of high grade astrocytoma and glioblastoma specimens, whereas more limited increases (2 to 3-fold) in this marker were observed in areas of necrosis. Low grade gliomas (astrocytomas) and meningiomas exhibited only moderate increases (2 to 3-fold) in this autoradiographic marker. Metastases of lung or kidney origin were characterized by greatly elevated (up to 20-fold) omega 3 site densities as compared to normal brain parenchyma. In every case, there was a good spatial correspondence between the histopathological limits of the tumour and the anatomical location of the increase in omega 3 site densities. These results suggest that omega 3 site densities in human brain tumours reflect their proliferative activity and point to a possible future usefulness of positron or gamma-ray emitting omega 3 site ligands for the clinical investigation and detection of human brain proliferative diseases.


[Retroperitoneal giant malignant fibrous histiocytoma: report of a case]. A case of histiocytoma in a 63-year-old woman with general fatigue is described. Ultrasonography and radiological examination revealed a large mass at the retroperitoneum displacing the right kidney to medial anterior abdomen. Exploration of the tumor with right kidney was done via a transperitoneal approach because the tumor had invaded the hilar fat tissue of the right kidney. The total resected weight was 3,200 g. Histological study showed malignant fibrous histiocytoma, storiform-pleomorphic type. Post-operative chemotherapy consisting of cyclophosphamide, vincristine, adriamycin, dimethyl triazeno-imidazole carboxamide (CYVADIC) was performed and the patient is doing well without any evidence of recurrence or metastasis.


[Metastatic bladder tumor from gastric carcinoma: a case report]. A 63-year-old man with a two-month history of nocturia and dysuria consulted his family doctor. As renal dysfunction and bilateral hydronephrosis were indicated, he was admitted to our hospital on November 28, 1988. Cystoscopy revealed a non-papillary and flat tumor from the ureteral orifice to the back wall of the bladder. A biopsy of the bladder wall revealed signet-ring cell carcinoma. A metastatic bladder tumor was suspected and laboratory tests of tumor markers showed a carcinoembryonic antigen value (CEA) of 1,000 ng/ml and CA19-9 of 12,210 U/ml. Upper gastrointestinal examination revealed carcinomatosis involving the stomach. A biopsy specimen of the stomach revealed the same pathological finding as the bladder wall. A metastatic bladder tumor was confirmed. The patient died of pulmonary emboli on December 11, 1988.


Electrophysiological properties of cultured outer medullary collecting duct cells. Whole cell patch-clamp techniques were used to characterize the electrophysiological properties of cells from the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture. With pipette and bathing solutions mimicking intracellular and extracellular fluid, the resting membrane voltage was -30 to -40 mV. The whole cell conductance exhibited slight outward rectification, and at the resting membrane voltage the cell conductance averaged 2.58 +/- 0.49 nS (n = 17). The major conductive ion species was Cl-. The Cl- conductance was also found to have a significant permeability to HCO3- and was inhibited by the Cl(-)-channel blockers diphenylamine carboxylic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A small K+ conductance was also present, but no Na+ conductance was detected. Current generated by the H(+)-adenosinetriphosphatase (H(+)-ATPase) was quantitated. This current was dependent on the presence of ATP in the pipette. Dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin A1, inhibitors of the vacuolar H(+)-ATPase, also reduced this outward current in an ATP-dependent manner. The inhibitor-sensitive component of the outward current, a measure of the current generated by the H(+)-ATPase, was in the range of 35-100 pA/cell.


Activation of endothelin ETB receptors increases glomerular cGMP via an L-arginine-dependent pathway. The effects of endothelins (ET) on guanosine 3',5'-cyclic monophosphate (cGMP) levels in intact rat glomeruli were examined. ET-3 produced a rapid approximately fivefold increase in cGMP levels with the maximum effect occurring at 1 min. The ET-3-induced increase in cGMP accumulation occurred in the absence and presence of 3-isobutyl-1-methylxanthine. ET-1, ET-2, ET-3, and the structurally related toxin, sarafotoxin S6c, all increased glomerular cGMP levels in a concentration-dependent manner and with similar potencies (EC50 approximately 15-30 nM). The L-arginine analogue, N omega-nitro-L-arginine (L-NNA), reduced basal levels of cGMP and also totally inhibited ET-induced increases in cGMP as did methylene blue, an inhibitor of soluble guanylate cyclase. The effect of L-NNA was attenuated by L-arginine but not by D-arginine. The stimulation of cGMP accumulation by ET-3 was dependent on extracellular Ca2+ and was additive to atriopeptin III but not to acetylcholine. The ETA-selective antagonist, BQ 123, had no effect on ET-3-induced formation of cGMP. Glomerular membranes displayed high-affinity (Kd = 130-150 pM) and high-density (approximately 2.0 pmol/mg) binding sites for 125I-ET-1 and 125I-ET-3. ET-1, ET-3, and sarafotoxin S6c displaced 125I-ET-1 binding to glomerular membranes with similar affinities. BQ 123 had no effect on 125I-ET-1 binding. We conclude that ET increases cGMP levels in glomeruli by stimulating the formation of a nitric oxide-like factor that activates soluble guanylate cyclase. This effect of ET appears to be mediated by activation of ETB receptors and may serve to modulate the contractile effects of ET.


Control of renal hemodynamics after protein feeding: role of calcium channels. This study was designed to test the hypothesis that the changes in renal hemodynamics that occur after a high-protein meal involve voltage-dependent calcium channels. In chronically instrumented female dogs, a 10-g/kg meal of raw beef caused plasma alpha-amino nitrogen levels to increase from 3.9 +/- 0.2 to 6.7 +/- 0.5 mg/dl after 90 min, and glomerular filtration rate (GFR) rose from 59 +/- 6 to 85 +/- 7 ml/min, effective renal plasma flow (ERPF) rose from 130 +/- 22 to 213 +/- 36 ml/min, and sodium excretion rose from 21 +/- 4 to 84 +/- 20 mu eq/min. On another day the dogs were pretreated with verapamil (0.075 mg/kg + 0.005 mg.kg-1.min-1 iv), which did not change arterial pressure significantly but increased GFR by 25 +/- 7% and ERPF by 23 +/- 5%. After a subsequent meat meal, plasma alpha-amino nitrogen increased from 4.0 +/- 0.3 to 6.6 +/- 0.1 mg/dl but GFR, ERPF, and sodium excretion did not change significantly. On the other hand, pretreatment with dopamine, which caused a similar degree of vasodilation to that caused by verapamil, did not prevent the response to a subsequent meat meal. Thus verapamil specifically prevented the normal increases in renal hemodynamics after protein feeding, suggesting that protein-stimulated renal vasodilation requires intact voltage-dependent calcium channels.


DOCA-enhanced sites of vasopressin-stimulated cAMP formation in rat cortical collecting tubule. In deoxycorticosterone acetate (DOCA)-NaCl hypertension, the effects of vasopressin (VP) in the cortical collecting tubule (CCT) are exaggerated. These include both the biochemical effect of VP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in the CCT and physiological effects of VP-mediated sodium and water retention. In this study, we examined the mechanism of enhanced VP-stimulated cAMP formation in the CCT. We compared cAMP formation in response to activators (following in parentheses) of the VP receptor (VP), of the stimulatory guanine nucleotide binding (Gs) protein [guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S); F-], and of the catalytic subunit of adenylyl cyclase (forskolin, Mn2+) between control and DOCA-NaCl-treated rats. The effects of VP and forskolin were enhanced in CCT of DOCA-NaCl-treated animals by 201 and 139%, respectively, compared with control animals. Other activators, Mn2+ (150%), F- (142%), and GTP gamma S (156%), also caused augmented cAMP formation in the CCT of DOCA-NaCl-treated rats. The DOCA-NaCl-induced increment in cAMP response to VP remained after pretreatment of the rats with pertussis toxin (171 and 169% increase in response in DOCA-NaCl and control rats, respectively), suggesting that altered inhibitory guanine nucleotide binding (Gi) protein function is not the mechanism for the altered response to VP in the CCT. Further evidence that Gi function is intact in DOCA-NaCl animals is that epinephrine (via alpha 2-adrenoceptor stimulation) inhibited VP-stimulated cAMP accumulation to a similar degree in DOCA-NaCl and control rats (86 and 76%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanisms of rubidium permeation by rabbit cortical collecting duct during potassium restriction. To examine the pathways of K permeation in the cortical collecting duct (CCD) from K-restricted rabbits, we studied the effects of the following three maneuvers: 1) luminal amiloride addition; 2) luminal Ba addition; and 3) luminal Na removal. Luminal addition of amiloride (1 mM) significantly increased the 86Rb lumen-to-bath efflux coefficient (KRb), and this effect was fully blocked by the presence of 2 mM luminal Ba. Addition of 2 mM luminal Ba reduced KRb both in the absence of luminal Na and in the presence of luminal Na. In contrast to the effect of amiloride addition, removal of luminal Na significantly increased KRb, but neither 2 mM luminal Ba nor 4 mM luminal Ba totally abolished this effect. However, simultaneous addition of luminal Ba and Sch 28080 (10 microM) fully inhibited the increase in KRb upon luminal Na removal, indicating that luminal Na removal enhances Rb efflux in part via H-K-adenosinetriphosphatase (H-K-ATPase). To test whether Na acts as a partial agonist for cation efflux via the H-K-ATPase we examined the effect of Sch 28080 on the 22Na lumen-to-bath efflux coefficient (KNa). These studies were conducted in the presence of 0.1 mM luminal amiloride to block fully apical conductive Na efflux, and the effect of Sch 28080 on KNa was examined at two different ambient K concentrations. In the presence of 0.5 mM K, Sch 28080 (10 microM) significantly inhibited KNa from 47.6 +/- 4.8 to 35.0 +/- 6.8 nm/s (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of acid-base transporters in cultured outer medullary collecting duct cells. Cells from the inner stripe of the rabbit outer medullary collecting duct (OMCDi) were grown in primary culture, and their acid-base transport properties were characterized using intracellular pH (pHi) measurements with the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal pHi in HCO(3-)-buffered solutions was 7.28 +/- 0.04 (n = 20). The presence of a Cl-/HCO(3-)-antiporter was demonstrated by reversible alkalinization on bath Cl- removal. The mean alkalinization seen on Cl- removal was 0.16 +/- 0.02 pH units (n = 20) and was inhibited 92% by 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Studies were also performed to determine the presence of an Na+/H+ antiporter and an H(+)-adenosinetriphosphatase (H(+)-ATPase). After an NH4Cl acid load the cells exhibited both Na(+)-dependent and Na(+)-independent pHi recovery mechanisms. The Na(+)-dependent mechanism was inhibited by amiloride. The Na(+)-independent mechanism was completely inhibited by 10(-3) M N-ethylmaleimide or 2.5 x 10(-9) M bafilomycin A1, but was not significantly altered by removal of bathing solution K+. Thus, the Na(+)-dependent recovery mechanism exhibited characteristics of an Na+/H+ antiporter, whereas the Na(+)-independent recovery mechanism was consistent with the presence of an H(+)-ATPase.


Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. We tested the hypothesis that intracoronary administration of L-arginine (L-Arg), the physiological nitric oxide (NO) precursor, during reperfusion would attenuate postischemic damage by L-Arg NO-pathway mechanisms. Open-chest, anesthetized dogs underwent 60 min of left anterior descending coronary arterial (LAD) occlusion followed by 270 min of reperfusion. Dogs received intracoronary 10 mM L-Arg (n = 9 dogs), intracoronary 10 mM D-arginine (D-Arg, n = 7), or saline vehicle (Veh, n = 10) in the LAD during the first 120 min of reperfusion using an extracorporeal system. After 270 min of reperfusion, segmental systolic and diastolic function were comparably impaired in all three groups. Infarct size (triphenyltetrazolium chloride) expressed as a percentage of the area at risk (An/Ar) was significantly (P < 0.05) reduced in the L-Arg group (17.7 +/- 3.2%) compared with the Veh group (34.8 +/- 2.4%); D-Arg reversed this cardioprotection (48.8 +/- 5.2%, P < 0.05 vs. L-Arg, Veh). Cardiac myeloperoxidase activity, an index of neutrophil accumulation (U/100 mg tissue), was significantly (P < 0.05) lower in the necrotic tissue of the L-Arg group (0.88 +/- 0.26) than in the Veh group (2.46 +/- 0.38). Furthermore, responses to endothelium-dependent vasodilators acetylcholine and A23187 in isolated ischemic-reperfused LAD rings were significantly (P < 0.05) greater in the L-Arg group than in the other two groups. We conclude that intracoronary infusion of L-Arg during the early phase of reperfusion reduced neutrophil accumulation and infarct size and the infusion preserved endothelial function, possibly by increasing NO release or production by the endothelium.


Phospholipid peroxidation deacylation and remodeling in postischemic skeletal muscle. Reperfusion of ischemic skeletal muscle is associated with white blood cell (WBC) sequestration and hydroperoxy-conjugated diene (HCF) formation, a marker of free radical-mediated phospholipid peroxidation. The purpose of this study was to define the kinetics of phospholipid fatty acyl peroxidation, deacylation, and remodeling in postischemic skeletal muscle during prolonged reperfusion in vivo, and to determine whether reperfusion with WBC and plasma-depleted blood would attenuate postischemic phospholipid peroxidation and myocyte necrosis. The isolated, paired, canine gracilis muscle model was used. After 5 h of ischemia, muscles underwent unaltered reperfusion or initial reperfusion with WBC-deficient blood cells resuspended in hydroxyethyl starch, followed by return to normal circulation (modified reperfusion). The concentration of native fatty acids and HCDs of linoleic acid extracted from muscle phospholipids was quantified by gas chromatography and positively identified by mass spectrometry. Ischemia and reperfusion resulted in phospholipid deacylation and a selective increase in phospholipid stearic acid content, but had no effect on total phospholipid phosphorus. Modified reperfusion decreased 1) early HCD formation (54%) and 2) postischemic skeletal muscle necrosis (49%). These data suggest that reperfusion results in phospholipid deacylation and remodeling, and that the initial oxidant stress during reperfusion may be a significant determinant of ultimate muscle necrosis.


Divergent regulation of atrial natriuretic factor receptors in high-output heart failure. We have investigated whether binding parameters and subtypes of glomerular, papillary, and vascular atrial natriuretic factor (ANF) receptors differ in rats with moderate high-output heart failure [aortocaval (AC) shunt] from their sham-operated controls. Body weight was lower and relative heart weight was higher in the AC shunt group than in the control group. Plasma renin activity (PRA) was also greater in AC shunt rats. Plasma COOH- and NH2-terminal ANF levels were higher in AC shunt animals than in their control counterparts. Total atrial ANF content was elevated in both the right and left atria of the AC shunt group. Glomerular and papillary ANF receptor density (Bmax) and ANF receptor affinity (Kd) were similar in both AC shunt and control rats. Vascular ANF receptor density and affinity were lower in AC shunt (Bmax = 65 +/- 13 fmol.mg protein; Kd = 467 +/- 52 pM) than in control rats (Bmax = 188 +/- 34 fmol.mg protein; Kd = 278 +/- 11 pM). Irreversible cross-linking of 125I-labeled ANF followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and radioautography demonstrated that both high- and low-molecular weight receptors were unchanged in glomerular membranes and downregulated in vascular membranes from AC shunt animals. However, guanosine 3',5'-cyclic monophosphate (cGMP) production by the isolated glomeruli of AC shunt rats was lower than that of controls. We conclude that in the presence of elevated plasma ANF levels, glomerular, papillary, and vascular ANF receptors may be regulated differently.


cAMP and extrarenal vasopressin V2 receptors in dogs. The effect of vasopressin analogues on plasma adenosine 3',5'-cyclic monophosphate (cAMP) concentration was examined in a group of five conscious dogs instrumented for the measurement of arterial pressure and cardiac output (electromagnetic flowmeter). These dogs were infused for 20 min with a selective antidiuretic (V2) agonist, desamino-8-D-arginine vasopressin (DDAVP, 10 ng.kg-1 x min-1). This infusion was repeated on another day in the presence of the combined V1-V2 antagonist d(CH2)5-D-Tyr(Et)-4-valine,8-arginine vasopressin. The dogs also received an infusion of the selective V1 agonist 2-phenylalanine,8-ornithine oxytocin (Phe-OrnOT) at a rate of 10 ng.kg-1 x min-1. The effect of these infusions was compared with those of an isotonic saline infusion. Plasma cAMP measured in the aorta remained unchanged during all infusions but that of the selective V2 agonist DDAVP alone, during which it increased significantly from 22.4 +/- 0.8 to 32.6 +/- 4.6 and 37.0 +/- 4.1 pmol/ml after 10 and 20 min, respectively. In the plasma sampled from the inferior vena cava caudal to the renal veins, cAMP increased during DDAVP infusion from 22.2 +/- 2.5 to 39.2 +/- 3.8 and 36.0 +/- 4.0 pmol/ml after 10 and 20 min, respectively. The infusion of DDAVP was later given to the same dogs under anesthesia after bilateral nephrectomy, which did not modify the effect of DDAVP on arterial plasma cAMP. In another group of four conscious dogs, infusion of DDAVP at the same rate did not induce significant changes in plasma catecholamines.(ABSTRACT TRUNCATED AT 250 WORDS)


Antiadrenergic effect of M-cholinoceptor stimulation on human ventricular contractility in vivo. The relevance of an indirect negative inotropic effect of pharmacological or vagally mediated M-cholinoceptor stimulation in the human ventricle in vivo is unknown. The inotropic response induced by isoproterenol (isoprenaline, 20 ng.kg-1 x min-1), measured by m-mode echocardiography as the increase of fractional shortening in seven healthy subjects, was reduced from 17.1 +/- 4.3 to 9.1 +/- 3.9% (P < 0.01) by M-cholinoceptor stimulation using intravenous injection of 3.6 micrograms/kg carbachol. However, the inotropic response of increasing doses of isoprenaline (5-20 ng.kg-1 x min-1) did not differ in 13 healthy subjects without and after M-cholinoceptor blockade (atropine, 0.015 mg/kg i.v.); the increase of fractional shortening amounted to 17.4 +/- 4.0 vs. 19.5 +/- 4.8% (NS) in response to 20 ng.kg-1 x min-1 isoprenaline. It is concluded that pharmacological M-cholinoceptor stimulation of the human ventricle significantly reduces the inotropic response of beta-adrenoceptor stimulation. This may offer a new therapeutic approach to attenuate an increased cardiac sympathetic tone. However, M-cholinoceptor blockade, i.e., interruption of parasympathetic influences, does not augment the inotropic response to beta-adrenoceptor stimulation. Therefore, in healthy resting humans a physiological role of the parasympathetic nervous system in mediating an indirect negative inotropic effect on ventricular contractility seems to be absent.


Sepsis- and endotoxin-induced increase in organ glucose uptake in leukocyte-depleted rats. Both gram-negative infection and bacterial endotoxin (lipopolysaccharide, LPS) produce a marked neutropenia and increase glucose disposal by peripheral tissues. The purpose of the present study was to determine whether leukocyte depletion before these insults would diminish the commonly observed increases in tissue glucose uptake. Rats were depleted of circulating and marginated leukocytes with cyclophosphamide (CPA). Under basal postabsorptive conditions the subcutaneous injection of live Escherichia coli into control animals enhanced whole body glucose disposal that resulted in part from a stimulation of glucose uptake by the liver, spleen, intestine, and lung. These increases in tissue glucose uptake were not associated with an increase in neutrophil number, as assessed by myeloperoxidase (MPO) activity. CPA-induced leukopenia did not alter the sepsis-induced increase in glucose uptake by these tissues and whole body glucose use remained elevated. In contrast, skin and muscle proximal to the site of infection showed an increase in both glucose uptake and MPO activity. Furthermore, leukocyte depletion attenuated the elevated glucose uptake by skin and muscle near the inflammatory focus. The intravenous injection of LPS also increased whole body glucose disposal and enhanced glucose uptake by the lung, liver, spleen, intestine, and skin in saline-treated rats. Of these tissues the lung, liver, and spleen had a corresponding increase in neutrophil number. The LPS-induced increases in tissue glucose uptake in leukopenic rats were comparable, with the exception of liver and lung. In these tissues the incremental increase in glucose uptake after LPS was reduced 40-50% in leukopenic animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Central oxytocin inhibition of angiotensin-induced salt appetite in rats. In several models of salt appetite in the rat, stimulated NaCl intake can be severely blunted by treatments associated with pituitary release of oxytocin (OT). Central administration of the potent dipsogen angiotensin II (ANG II) is known to elicit a limited salt appetite as well as thirst, but it has also been reported to stimulate pituitary OT secretion. These results suggest the possibility that the expression of ANG II-induced salt appetite in rats may be inhibited by a simultaneous central release of OT in response to this stimulus. To investigate this possibility, rats were given intracerebroventricular injections of OT-receptor antagonists before administration of 5 ng ANG II intracerebroventricularly in a 1-h two-bottle (water and 0.3 M NaCl) drinking test. This pretreatment resulted in a three- to fourfold potentiation of ANG II-induced saline ingestion, which was most prominent during the first 15 min of the test. OT-receptor antagonism did not, however, interfere with the dipsogenic properties of ANG II, nor did it stimulate saline ingestion alone in the absence of ANG II. Immunocytochemical studies demonstrated that central administration of ANG II at this dose caused pronounced c-fos expression in hypothalamic magnocellular OT and vasopressin neurons and also in OT neurons in parvocellular subdivisions of the paraventricular nucleus. These results therefore demonstrate that central administration of small doses of ANG II activates both magnocellular and parvocellular OT neurons in rats and indicate that some of the activated central OT pathway(s) may mediate an inhibitory effect that limits the salt ingestion induced by this treatment.


[Diagnostic and prognostic value of specific immune response to Cytomegaloviruses in pregnant women with a history of habitual abortion]. The authors analyze the usefulness of specific serologic tests for anti-CMV antibodies of the IgG and IgM classes for the prediction of the course of pregnancy and choice of immunocorrective therapy of women suffering from habitual abortions. Detection of specific IgM antibodies to cytomegalovirus with the use of Abbott diagnostic system was found highly informative, for it permitted a differential diagnosis of the cause of habitual abortions and monitoring of the treatment efficacy. Enzyme immunoassay of TBH in blood serum samples of pregnant women helped monitor the course of pregnancy.


[Congenital cytomegalic inclusion disease--clinico-anatomical characteristics]. Analysis of labor histories, case histories, and autopsy records over a decade, 1980-1990, has revealed 32 cases of cytomegalic inclusion disease in children. Eleven placentas of seropositive mothers were histologically examined. The findings evidence that cytomegaloviral infection was mostly combined with other bacterial and viral diseases. The central nervous system, kidneys, and lungs were the most sensitive to the virus effects. The DIC syndrome was present in all the cases.


Determination of dietary fibre as non-starch polysaccharides by gas-liquid chromatography. An improved method is described for the measurement of total, soluble and insoluble dietary fibre as non-starch polysaccharides (NSP). An established procedure is modified to allow more rapid removal of starch and hydrolysis of NSP. In its present form the procedure is simpler and more robust than those previously published. In the modified method starch is removed enzymically within 50 min and NSP is precipitated with ethanol and then hydrolysed by treatment with sulfuric acid for 2 h. The constituent sugars can in turn be measured by gas-liquid chromatography, high-performance liquid chromatography or more rapidly by colorimetry. The improved procedure described here for the removal of starch and hydrolysis of NSP applies to all three techniques, but only the method for measurement of sugars by gas-liquid chromatography is described here in full.


In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression. We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.


Prooxidant-induced Ca2+ release from liver mitochondria. Specific versus nonspecific pathways. Ca2+ release from mitochondria can be induced by a variety of chemically different prooxidants. Release induced by these compounds is possibly regulated by protein mono(ADP)ribosylation, and leaves mitochondria initially intact. Excessive "cycling" (continuous release and uptake) of Ca2+ by mitochondria leads to their damage, as shown by a decreased membrane potential, fast Ca2+ release, and impairment of ATP synthesis. When cycling is prevented by Ca2+ chelators or by inhibition of the uptake route with ruthenium red, prooxidants still induce Ca2+ release but mitochondria remain intact. It has recently been suggested that formation of a "pore" in the inner mitochondrial membrane participates in the Ca2+ release mechanism. We find that the prooxidant-induced Ca2+ release is not paralleled by sucrose entry into, or K+ release from, or swelling of mitochondria, provided Ca2+ cycling is prevented. Thus, the prooxidant-induced Ca2+ release does not require formation of a "pore." We conclude that the release occurs via a specific pathway.


In vitro demonstration of transport and delivery of antibiotics by polymorphonuclear leukocytes. Several antibiotics are concentrated inside polymorphonuclear leukocytes (PMN). To investigate whether PMN could act as vehicles for delivery of antibiotics, we combined an assay measuring PMN chemotaxis under agarose with a bioassay measuring levels of antibiotic in agar. Double-layer plates were made by pouring a layer of chemotaxis agarose into tissue culture plates and then adding a thin layer of Trypticase soy agar. Neutrophils were incubated with antibiotic for 1 h and then were washed and placed in wells made in the plates. After allowing PMN to migrate under the agar toward a chemoattractant well containing formyl-methionine-leucine-phenylalanine for 3 h, Streptococcus pyogenes was streaked on top of the agar and grown overnight. PMN migration and zones of inhibition of bacterial growth were measured. Neutrophils migrated 2.51 +/- 0.16 mm toward the chemoattractant well and 1.48 +/- 0.12 mm toward the medium well; migration was not significantly affected by any of the antibiotics used. Plates with PMN incubated without antibiotic showed insignificant inhibition of bacterial growth toward chemoattractant and medium wells (0.38 +/- 0.18 and 0.14 +/- 0.12 mm, respectively; for both, P > 0.05 for difference from 0). PMN incubated with oxacillin (3 micrograms/ml), a drug not concentrated in PMN, caused a similar lack of inhibition (0.28 +/- 0.09 mm toward chemoattractant; 0.14 +/- 0.03 mm toward medium). Incubation with 30 microns of ciprofloxacin per ml resulted in inhibition that was similar in both directions (1.40 +/- 0.16 versus 1.18 +/- 0.13 mm). However, for PMN incubated with azithromycin (3 micrograms/ml), an agent highly concentrated inside phagocytes, there was a large degree of inhibition which was significantly greater in the direction of chemoattractant than in the direction of medium (3.47 +/- 0.30 versus 1.89 +/- 0.25 mm; P < 0.001), indicating that release of bioactive azithromycin by neutrophils occurred after migration. Likewise, after incubation with rifampin (10 micrograms/ml), which is also concentrated by PMN, inhibition was significantly greater in the direction of chemoattractant than in the direction of medium (1.54 +/- 0.24 versus 0.81 +/- 0.28 mm; P = 0.001). We conclude that for certain antibiotics, PMN may act as vehicles for transport and delivery of active drug to sites of infection.


Induction of topoisomerase II-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin. Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a reaction mixture containing these naphthoquinones and topoisomerase II at an elevated temperature (65 degrees C) resulted in a great reduction in DNA cleavage, suggesting that the mechanism of the topoisomerase II-mediated DNA cleavage induced by these naphthoquinones is through formation of a cleavable complex, as seen with antitumor agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide and demethylepipodophyllotoxin ethylidene-beta-glucoside. Lawson and lapacol, which are structurally related plant metabolites with naphthoquinone moieties, could not induce topoisomerase II-mediated DNA cleavage. Plumbagin and shikonin induced a similar DNA cleavage pattern with topoisomerase II which was different from the cleavage patterns induced with other known topoisomerase II-active drugs. A DNA-unwinding assay with T4 DNA ligase showed that shikonin, lawson, and lapacol did not intercalate into DNA, while plumbagin and 2-methyl-1,4-naphthoquinone intercalate into DNA, but to a lower degree than 4'-(9-acridinylamino)methanesulfon-m-anisidide does.


Daptomycin or teicoplanin in combination with gentamicin for treatment of experimental endocarditis due to a highly glycopeptide-resistant isolate of Enterococcus faecium. Using an experimental endocarditis model, we studied the activity of daptomycin used alone or in combination with gentamicin against an Enterococcus faecium strain that was highly resistant to glycopeptides and susceptible to gentamicin. In vitro, the MIC of daptomycin was 1 micrograms/ml. In vivo, daptomycin appeared to be effective only when it was used in a high-dose regimen, i.e., 12 mg/kg of body weight every 8 h (-2.5 log10 CFU/g versus controls; P < 0.05), particularly when it was combined with gentamicin (-5.0 log10 CFU/g versus controls; P < 0.01). Since the distribution of daptomycin into cardiac vegetations, as evaluated by autoradiography, appeared to be homogeneous, the poor in vivo activity of daptomycin was considered to be related to its high degree of protein binding, as suggested by killing curves studies. Since the MIC of teicoplanin for the vancomycin-resistant E. faecium strain used in the study was only 64 micrograms/ml and since an in vitro synergy between teicoplanin at high dose and gentamicin was observed, a high-dose regimen of teicoplanin, i.e., 40 mg/kg every 12 h, was also assessed in vivo. This treatment provided marginal activity only when it was combined with gentamicin (-2.3 log10 CFU/g versus controls; P < 0.05). These results suggest that the levels of daptomycin or teicoplanin in serum required to cure experimental endocarditis caused by a highly glycopeptide-resistant strain of E. faecium would not be achievable in humans.


Relationships among antibacterial activity, inhibition of DNA gyrase, and intracellular accumulation of 11 fluoroquinolones. A series of 11 fluoroquinolone antibacterial agents, including 8 newly synthesized molecules and 3 reference compounds (pefloxacin, ciprofloxacin, and sparfloxacin), were tested for their MICs against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The intracellular accumulation of fluoroquinolones by these microorganisms was measured by centrifugation through silicone oil and a fluorescence assay. The minimal effective dose (MED) was determined for all agents in a supercoiling assay with E. coli DNA gyrase. The hydrophobicities of the quinolones were determined and expressed as the logarithm of the coefficient of distribution (log D) between 1-octanol and phosphate buffer (pH 7.2). No correlation was found between MICs and cell accumulation for the quinolones studied. A correlation was found between log D and accumulation by S. aureus (r = 0.71, n = 11), and an inverse correlation was found between log D and accumulation by E. coli (r = 0.73, n = 11) and P. aeruginosa (r = 0.64, n = 10). The correlation coefficients between MICs and MED for E. coli, which were 0.60, 0.64, and 0.74 (n = 11) for E. coli, P. aeruginosa, and S. aureus, respectively, rose to 0.85, 0.74, and 0.74 (n = 11) for the same microorganisms, respectively, when the accumulation of the drug by the cell was taken into account. It was concluded that the inhibitory activity against DNA gyrase remains the most important parameter for quinolone potency, but that intracellular accumulation must be taken into account, since, for a given organism, both parameters are under the control of the physicochemical properties of the quinolones.


Relationship between antibiotic concentration in bone and efficacy of treatment of staphylococcal osteomyelitis in rats: azithromycin compared with clindamycin and rifampin. We examined the effect of azithromycin (CP-62,993), a new oral macrolide-like antibiotic, alone and in combination with rifampin, as treatment for experimental staphylococcal osteomyelitis. Clindamycin was used as a comparison drug. Rats (n = 10 to 15 per group) were infected by direct instillation of Staphylococcus aureus into the tibial medullary cavity. After 10 days, 21-day treatments with azithromycin (50 mg/kg of body weight, once daily, by the oral route), rifampin (20 mg/kg, once daily, subcutaneously), or clindamycin (90 mg/kg, three times daily, by the oral route) were started. The drugs were used singly or in combination (azithromycin plus rifampin or clindamycin plus rifampin). Peak azithromycin concentrations in bone were > 30 times higher than levels in serum, but the drug had little effect on final bacterial titers (5.13 +/- 0.46 log10 CFU/g of bone; for controls, 6.54 +/- 0.28 log10 CFU/g). Clindamycin was more active than azithromycin (3.26 +/- 2.14 log10 CFU/g of bone; 20% of sterilized bones), but rifampin was the most active single drug (1.5 +/- 1.92 log10 CFU/g; 53% of sterilized bones). Therapy with rifampin or clindamycin alone was associated with the emergence of resistance. Rifampin plus azithromycin (0.51 +/- 1.08 log10 CFU/g of bone; 80% of sterilized bones) and rifampin plus clindamycin (0.87 +/- 1.34 log10 CFU/g of bone; 66% of sterilized bones) were the most active regimens. Thus, azithromycin is ineffective as a single drug for the treatment of experimental staphylococcal osteomyelitis, despite high levels in bone that markedly exceeded the MIC, but it may be an attractive partner drug for rifampin.


The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine. The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.


In vivo efficacy of a new fluoroquinolone, sparfloxacin, against penicillin-susceptible and -resistant and multiresistant strains of Streptococcus pneumoniae in a mouse model of pneumonia. The increasing emergence of penicillin-resistant and multiresistant strains of Streptococcus pneumoniae may pose a problem in coming years. We therefore compared sparfloxacin, a new fluoroquinolone with improved potency against streptococci, with amoxicillin, the "gold standard" in this setting, and another fluoroquinolone, ciprofloxacin, in a mouse pneumonia model. Their efficacies against penicillin-susceptible (serotype 3), macrolide-resistant (serotype 1), penicillin-resistant (serotype 23), and multiresistant (serotype 6) S. pneumoniae strains were evaluated. Immunocompetent Swiss mice (serotypes 1 and 3) and leukopenic mice (serotypes 6 and 23) were infected by peroral tracheal delivery of 10(4) to 10(6) CFU. Subcutaneous injections of antibiotics were initiated at 6, 18, 48, or 72 h after infection (six injections at 12-h intervals). In the immunocompetent mice, 100% survival was obtained with sparfloxacin (50 mg/kg) and amoxicillin (5 mg/kg) against both penicillin-susceptible and macrolide-resistant strains; ciprofloxacin gave significantly lower survival rates. Two to four injections of sparfloxacin completely cleared bacteria from lungs and blood; the most rapid eradication was achieved with amoxicillin. Sparfloxacin also fully protected leukopenic mice against penicillin-resistant strains. The dose of amoxicillin (50 mg/kg) required to protect mice and eradicate penicillin-resistant and multiresistant strains was 10 times higher than that effective against penicillin-susceptible strains. The microbiological and pharmacokinetic properties of sparfloxacin (e.g., the time during which concentrations exceed the MIC of the test pathogen) accounted for its efficacy against susceptible and resistant strains of S. pneumoniae in this model.


In vitro pharmacodynamic effects of concentration, pH, and growth phase on serum bactericidal activities of daptomycin and vancomycin. Clinical trials with daptomycin were halted in December 1990 because of treatment failures including two resistant Staphylococcus aureus strains. High protein binding of daptomycin (> 90%) and the lower-than-expected concentrations in serum with the dosage regimen of 3 mg/kg of body weight every 12 h may have contributed to these failures. To evaluate the effect that higher concentrations would have on bactericidal activity measured by time-kill curves, peak and trough concentrations were estimated for dosage regimens of 3, 5, and 10 mg/kg every 12 h. MICs, MBCs, and killing curves for daptomycin and vancomycin were performed by using the estimated concentrations with four S. aureus strains obtained from patients who failed daptomycin therapy for endocarditis. MICs and MBCs of daptomycin demonstrated a greater inoculum effect than those of vancomycin; MICs and MBCs of daptomycin increased three- to fourfold, but those of vancomycin increased only one- to twofold when the inoculum was increased from 5 x 10(5) to 5 x 10(7) CFU/ml. No pH-dependent effect on MICs or MBCs was seen. Strenuous experimental conditions were chosen: high inoculums (5 x 10(7) CFU/ml), extremes of pH (6.4, 7.4, and 8), and stationary and exponentially growing organisms; and all experiments completed in the presence of pooled human serum. Daptomycin exhibited concentration-dependent killing and statistically faster kill rates than vancomycin against stationary- or exponential-growth-phase organisms. A pH-dependent decrease in activity with daptomycin was also demonstrated. Daptomycin and vancomycin produced higher kill rates against exponentially growing organisms. A pH-dependent decrease in activity with daptomycin was also demonstrated. Daptomycin and vancomycin produced higher kill rates against exponentially growing organisms. The results indicate that the use of higher dosage regimens with compounds similar to daptomycin may be capable of overcoming the effects of pH, high inoculum, and protein binding.


Pre- and postexposure chemoprophylaxis: evidence that 3'-azido-3'-dideoxythymidine inhibits feline leukemia virus disease by a drug-induced vaccine response. The benefits of postexposure 3'-azido-3'-dideoxythymidine (AZT) prophylaxis following human immunodeficiency virus exposure are unknown. We describe a comprehensive assessment of pre- and postexposure AZT therapy in the feline leukemia virus (FeLV)-cat model for AIDS which included in vitro testing, an in vivo dose-response titration, a postexposure treatment study, plasma drug concentration determinations, and evaluation of the immune response to FeLV. In in vitro studies, AZT prevented FeLV infection of a feline T-lymphoid cell line, giving 50 and 90% inhibition concentrations of 4.6 and 11.1 mM, respectively. In all of the in vivo efficacy studies, AZT was administered by continuous subcutaneous infusion for 28 days. AZT toxicity was excessive at a dosage of 120 mg/kg of body weight per day, causing acute anemia, but AZT was tolerable at 60 mg/kg/day. In preexposure studies, AZT was efficacious in preventing chronic antigenemia at a dosage of > or = 15 mg/kg/day, at which plasma AZT concentrations averaged between 0.51 and 0.81 micrograms/ml (2.13 and 3.03 microM). As a postexposure treatment, at 60 mg/kg/day, AZT prevented chronic FeLV antigenemia when treatment was started up to 96 h post-virus inoculation (p.i.), but not when treatment was started at 192 h p.i. The 4-day period between 96 and 192 h p.i. appears to be critical for establishing chronic viremia. It is presumed that the increase in virus load between 4 and 8 days p.i. was able to overwhelm the immunologic functions responsible for containment of FeLV infection, even though AZT therapy effectively controlled viremia during the treatment period. The antibody response to FeLV varied depending on the time of AZT treatment initiation relative to virus challenge. When AZT treatment was started 48 h before or 8 h after FeLV challenge, antibodies to FeLV were not detected until after AZT treatment was discontinued at 28 days p.i. Following AZT treatment, however, antibody titers rapidly increased at a rate suggestive of a secondary immune response. When AZT treatment was initiate at later time points relative to virus challenge (24, 48, and 96 h p.i.), antibodies to FeLV became detectable during the treatment period. These results indicate that AZT treatment does not completely prevent FeLV infection, even when treatment begins before virus challenge, and that immune sensitization to FeLV proceeds during the prophylactic drug treatment period.


Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate inhibited HSV-1 and HSV-2 DNA polymerase competitively with dGTP, the Ki values being 8.5 and 5.8 microM, respectively, whereas for acyclovir-triphosphate, the Ki value was 0.07 microM for the two enzymes. Both compounds had relatively low levels of activity against the cellular DNA polymerase alpha, with Ki values of 175 and 3.8 microM, respectively. (S)-Penciclovir-triphosphate did inhibit DNA synthesis by HSV-2 DNA polymerase with a defined template-primer, although it was not an obligate chain terminator like acyclovir-triphosphate. These results provide a biochemical rationale for the highly selective and effective inhibition of HSV-2 and VZV DNA synthesis by penciclovir and for the greater activity of penciclovir than that of acyclovir when HSV-2-infected cells were treated for a short time.


Piperacillin-tazobactam versus imipenem-cilastatin for treatment of intra-abdominal infections. In order to compare the clinical and microbiological efficacies and safety of piperacillin plus tazobactam with those of imipenem plus cilastatin, 134 patients with intra-abdominal infections (73 patients with appendicitis) participated in an open randomized comparative multicenter trial. A total of 40 men and 29 women (mean age, 53 years; age range, 18 to 92 years) were enrolled in the piperacillin-tazobactam group and 40 men and 25 women (mean age, 54 years; age range, 16 to 91 years) were enrolled in the imipenem-cilastatin group. The patients received either piperacillin (4 g) and tazobactam (500 mg) every 8 h or imipenem and cilastatin (500 mg each) every 8 h. Both regimens were given by intravenous infusion. A total of 113 patients were clinically evaluable. Of 55 patients who received piperacillin-tazobactam, 50 were clinically cured, while 40 of 58 patients in the imipenem-cilastatin group were clinically cured. The differences were significant (Wilcoxon test; P = 0.005). There were 4 failures or relapses in the piperacillin-tazobactam group and 18 failures or relapses in the imipenem-cilastatin group. The microorganisms isolated were eradicated in similar proportions in the two patient groups. Adverse reactions, mainly gastrointestinal disturbances and nausea, were noted in 13 patients who received piperacillin-tazobactam and in 14 patients who received imipenem-cilastatin. Results of the present study show that piperacillin-tazobactam is effective and safe for the treatment of intra-abdominal infections.


Comparative study of penetration of lomefloxacin and ceftriaxone into transudative and exudative pleural effusion. We investigated the transpleural penetration of lomefloxacin (LFLX) and ceftriaxone (CTRX). LFLX (200 mg) was administered orally to three patients with transudative fluid and four with exudative fluid, and 2 g of CTRX was administered by drip infusion to four patients with transudative fluid and three with exudative fluid. For both groups that received LFLX and CTRX, blood samples were drawn at time zero and 1, 2, and 6 h after drug administration. Thoracocentesis of each group was performed at 6 h after drug administration. The mean ratios of concentrations in pleural fluid/maximum concentrations in serum (P/S max) of LFLX were 66% in patients with transudative fluid and 69% in patients with exudative fluid. The mean ratios of P/S max of CTRX were 9.1% in patients with transudative fluid and 13.5% in patients with exudative fluid. The P/S max ratios for the penetration of LFLX were five to six times higher than those for CTRX. In addition, there was less differentiation in concentrations of LFLX in pleural fluid between the transudative and exudative effusions than there was in the concentrations of CTRX.


DNA topoisomerases from pathogenic fungi: targets for the discovery of antifungal drugs. DNA topoisomerases, a class of enzymes that change the topological structure of DNA, have been shown to be the target of many therapeutic agents, including antibacterial agents (quinolones) and anticancer agents. These drugs inhibit the enzyme in a unique way so that the enzyme is converted into a cellular poison. Candida albicans and Aspergillus niger are two major opportunistic fungal pathogens. Our results show that these fungi have high levels of both type I and type II topoisomerases (with a minimum of 5 x 10(5) ATP-independent relaxation units and 2 x 10(5) P-4 unknotting units per liter of wild-type C. albicans). The ATP-dependent type II topoisomerase (termed C. albicans topoisomerase II) was purified by approximately 2,000-fold from C. albicans cells by using a simple isolation scheme that consists of three column procedures: hydroxylapatite, phosphocellulose, and heparin-agarose chromatographies. The responses of the Candida and the calf thymus topoisomerase II to some known topoisomerase II inhibitors were measured. Etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide, compounds known to inhibit catalysis and to enhance DNA breakage by mammalian topoisomerase II, and A-80198, an etoposide derivative, enhanced cleavage by both enzymes at similar concentrations of these compounds, with the response of the calf thymus topoisomerase II from slightly to fourfold higher in magnitude than the response of the Candida enzyme in the same concentration range. In contrast, A-75272 (a cytotoxic tricyclic quinolone) shows a slightly stronger DNA cleavage enhancement effect with the Candida enzyme than with the mammalian counterpart. The abundance of the enzyme in cells and the different drug responses of the host enzyme and the fungal enzyme suggest that the fungal topoisomerase may serve as a target for the discovery of effective and safe antifungal agents.


Activity of glutaraldehyde at low concentrations against capsid proteins of poliovirus type 1 and echovirus type 25. The activity of glutaraldehyde (GTA) against capsid proteins of poliovirus type 1 and echovirus type 25 was studied to understand the mode of action of this reagent against enteroviruses. The viruses were treated with GTA concentrations ranging from 0.005 to 0.10%. In the poliovirus particles, high-molecular-weight products were formed by 0.05% GTA, whereas in the echovirus particles, they were formed at 0.005% GTA. These products consist of complexes composed essentially of VP1 and VP3. There seemed to be differences in the composition of the complexes in the two viruses. Cross-linkings between the two polypeptides of the poliovirus capsid may be due to the accessibility to GTA of lysine residues on the loops of VP1 and VP3, which twist out from the surface of the shell.


[Beta adrenergic receptors and experimental left ventricular hypertrophy]. The effects of sinoaortic denervation (SAD) on the development of left ventricular hypertrophy (R: heart weight/total body weight; LVT : left ventricular thickness), myocardial beta-adrenergic receptivity ([125I]CYP binding; adenylate cyclase activity) and plasma catecholamine levels (HPLC) were investigated in both normotensive (group 1) and hypertensive dogs evaluated (group 2) 1 and 18 months (group 3) after SAD. Noradrenaline (NA) and adrenaline (A) plasma levels were 461 +/- 54 and 85 +/- 45 pg/ml in controls, 861 +/- 185 and 191 +/- 23 pg/ml in group 2 (P < 0.05) and were normal in group 3 (426 +/- 132 and 110 +/- 16 pg/ml). R and LVT values were higher (p < 0.05) in SAD dogs (R = 7.7 +/- 0.1 and 7.8 +/- 0.2; LVT = 13.6 +/- 1.3 and 14.2 +/- 0.9 mm in groups 2 and 3 respectively) than in group 1 (R = 6.7 +/- 0.1, LVT = 9.3 +/- 0.8 mm). In group 1, the total number of beta-adrenoceptors (beta-AR) was 37 +/- 11 and 29 +/- 6 fmol/mg prot in left ventricule (LV) and right auricle (RA) respectively. Bmax was significantly lower in group 2 (LV: 10 +/- 3; RA: 13 +/- 2 fmol/mg prot, p < 0.05) than in group 1 but was normal in group 3 (LV: 37 +/- 3 and RA: 31 +/- 3 fmol/mg prot).(ABSTRACT TRUNCATED AT 250 WORDS)


[Myocardial beta-adrenergic receptors and left ventricular hypertrophy induced by pressure overload in man]. In experimental models, the characteristics of beta-adrenoceptors in left ventricular hypertrophy (LVH) due to pressure overload remain controversial and no data are still available in man. We investigated right auricular (RA) and left ventricular (LV) beta-adrenoceptors characteristics (125 I cyanopindolol binding) in two groups of patients undergoing valve replacement without heart failure (LV ejection fraction > 55%). Height patients with mitral stenosis (mean age: 64 +/- 4 years) and without LVH (LV mass index < 120 g/m2) constituted the control group and 13 patients with aortic stenosis (mean age: 66 +/- 4 years) and LVH (LV mass index > 150 g/m2) the study group. The results are: [table: see text] These results show that, in man, LVH due to pressure overload does not induce variation of total number beta-adrenoceptors, but is associated with a selective decrease in left ventricular beta 1-adrenoceptors.


[Activity of platelet sodium-proton exchanger, microalbuminuria and insulin-dependent diabetes]. The plasma membrane Na+/H+ exchanger is a ubiquitous system which plays a role in the regulation of intracellular pH and the control of cell growth. In order to assess the potential role of this system in the pathogenesis of diabetic nephropathy, we investigate 42 normotensive insulin-dependent diabetic patients with or without microalbuminuria. We tested the platelet Na+/H+ exchange as the rate of amiloride sensitive and sodium dependent volume gain of cells suspended in sodium propionate. Urinary albumin excretion (UAE) was assayed by radioimmunoassay on a 24 h sample; the glomerular filtration rate (GFR) and the renal plasma flow were determined by 99 m Tc-DTPA and 1231 l-hippuran respectively. Thirty patients (group 1) had EUA > 30 mg/24 h (m +/- sd: 11 +/- 7 mg/24 h), 12 patients (group 2) had microalbuminuria (62 +/- 30 Mg/24 h, range from 35 to 136 mg/24 h). The platelet Na+/H+ exchange rate was significantly increased in patients of group 2: 0.34 +/- 0.01 versus 0.26 +/- 0.06 s-1 x 10(-2) (p < 0.005). There was no significant difference between these two groups regarding blood pressure (116 +/- 14/71 +/- 7 versus 119 +/- 12/73 +/- 5 mmHg), age, diabetes duration, glycated hemoglobin or fructosamine levels. On the whole population, we found a significant positive correlation between the platelet Na+/H+ exchange rate and the UAE (r = 0.57, p < 0.001) and with the glomerular filtration fraction (r = 0.43, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


[Increase of activity of angiotensin-converting enzyme in insulin-dependent diabetic patients with permanent microalbuminuria]. Angiotensin I Converting Enzyme (ACE), which is synthesized by vascular endothelial cells, can be elevated in some diabetic subjects. To study if serum ACE can be elevated in subjects with high risk for malignant microangiopathy, 34 normotensive type I, insulin-dependent diabetic subjects with persistent microalbuminuria (30-300 mg/24 h) were compared for serum ACE activity (Liebermann's method) with 30 normotensive, normoalbuminuric type I, insulin-dependent diabetic subjects of same age (33 +/- 15 (M +/- SD) vs 39 +/- 14 years), sex (13 F/21 M vs 15 F/15 M), stage of retinopathy (14 vs 16 nil/11 vs 7 background/6 vs 4 preproliferative/3 vs 3 proliferative), HbA1c (7.7 +/- .9 vs 8.2 +/- 1.0%). Serum ACE activity of diabetic subjects were also compared with 120 age and sex related healthy controls. Serum ACE activity was higher in type I, insulin-dependent diabetic subjects with microalbuminuria than in those with normoalbuminuria (406 +/- 114 vs 359 +/- 97 IU/l; p = 0.05), or in controls (307 +/- 95 IU/l; p = 0.0001). Normoalbuminuric subjects also had higher ACE activity than controls (p = 0.02). In diabetic subjects, serum ACE activity was not related to diabetes duration (r = 0.1; ns), stage of retinopathy (r = 0.06; ns), HbA1c (r = 0.02; ns), or to blood pressure (r = 0.03; ns), but was related to urinary albumin excretion (r = 0.28; p = 0.03) in diabetic subjects. However, stage of retinopathy was related to diabetes duration (r = 0.74; p = 0.0004) and to age (r = 0.42; p = 0.003) in these subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


[Effect of dietary sodium in hypertension not treated with drugs]. The usefulness of salt restriction in essential hypertension is still now disputed. This study was designed to test the influence of a diet with and without salt restriction in 19 untreated essential hypertensives (12 with and 7 without family history of hypertension) and free of cardiovascular and renal complications. Each patient was examined after a placebo period, after 1 month of salt restriction, and after 1 month of salt supplementation. Weight, blood pressure, 24 hours urinary sodium excretion and red blood cell ionic fluxes were measured. In patients with hypertensive heredity, the blood pressure did not change. The intracellular sodium concentration, the cotransport and the countertransport remained stable. The ouabain sensitive sodium pump slightly increased during salt restriction and remained stable after salt supplementation. In patients without such hypertensive heredity (who were older and heavier), sodium restriction period was characterized by significant decrease in blood pressure, weight, intracellular sodium concentration and increase in sodium pump activity. When salt was increased, all the parameters remained stable. A more balanced diet with sodium restriction decreases the blood pressure in relation to age, weight and the blood pressure level. Hypertensive heredity does not seem to be a parameter of salt sensitivity. The blood pressure decrease is also related to the quantitative importance of sodium restriction. The ouabain sensitive pump activity changes during diet especially in relation to weight loss and decreasing salt intake.


Acrosome reaction changes the pattern of cyclic nucleotide phosphodiesterases in human sperm. Three categories of cyclic nucleotide phosphodiesterases (cNPDE) are distinguished at present: type 1 with high affinity to cyclic GMP; type 2 with low affinity to cyclic AMP and to cyclic GMP; and type 3 with high affinity to cyclic AMP. For the evaluation of normal values in human spermatozoa 50 semen samples with normal classical semen parameters were investigated. The activities (means +/- SD) of the cNPDE types (10(-11) mol/10 min x 10(8) spermatozoa) of washed human spermatozoa amounted to 47 +/- 22 (type 1), 3350 +/- 1537 (type 2), and 70 +/- 38 (type 3). A significant inhibition of type 3 by cyclic GMP could not be detected. One milligram protein of the spermatozoa hydrolyzed about 20-fold the amount of cyclic nucleotides compared with 1 mg protein of the seminal plasma. Furthermore, the cNPDE of the spermatozoa and of the seminal plasma differed in the influence of type 3 by cyclic GMP and in the pattern of activities. The acrosome reaction (AR) induced by the cold shock method led to an activation of type 2 and 3 unlike the initiation of the AR by the digitonin method. The latter did not cause significant differences of the cNPDE activities before and after the AR.


[Increase of angiotensin converting enzyme activity during oral load of glucose]. Elevated serum angiotensin I-converting enzyme activity may occur in diabetic subjects. This may signal alteration of vascular endothelium. To study the effect of acute glucose change on serum Angiotensin Converting Enzyme (ACE), we performed an oral glucose tolerance test in 17 obese subjects (7M/10F), (Body Mass Index, (BMI): 31 +/- 1 kg/m2), aged 48 +/- 3 years. We measured serum ACE activity (Lieberman's method), active renin (RIA Pasteur kit), and aldosterone (RIA, Cis-International kit), before and 2 hours after oral glucose intake (75 g), and plasma glucose and insulin every 30 min. After oral glucose tolerance test, subjects were classified as 6 Non Insulin-Dependent Diabetic (NIDD), 8 Glucose intolerant (GI), and 3 NormoGlycaemic (NG) subjects. Active renin did not vary after glucose loading (14 +/- 2 vs 15 +/- 2 pg/ml) nor aldosterone (104 +/- 14 vs 133 +/- 18 pg/ml), while ACE activity rose significantly (229 +/- 25 vs 277 +/- 28 IU/l; p = 0.02). Serum ACE activity were different in the 3 groups before glucose loading (NIDD: 266 +/- 37, GI: 252 +/- 32, NG: 90 +/- 21 IU/l; Kruskal-Wallis H = 7.03; p = 0.03), but not after 2 hours (NIDD: 297 +/- 42, GI: 275 +/- 36, NG: 204 +/- 113 IU/l; ns).(ABSTRACT TRUNCATED AT 250 WORDS)


[Modulating effects of atrial natriuretic peptide on vascular permeability and blood pressure by inhibition of cyclic phosphodiesterase GMP in the rat]. Atrial natriuretic peptide lowers arterial pressure and increases hematocrit through reduction in plasma volume caused by a transcapillary shift of plasma fluid and protein toward the interstitium. Cyclic GMP, the second messenger of atrial natriuretic peptide is catabolized by cGMP-phosphodiesterase; therefore we examined the consequences of inhibition of the phosphodiesterase on these responses using the specific cGMP inhibitor M&B 22.948. In anesthetized, bilaterally nephrectomized rats, a 45-min infusion of atrial natriuretic peptide (1 microgram/kg/min) reduced arterial pressure by 7.6 +/- 1.5% and increased hematocrit by 9 +/- 0.6% (both p < 0.01), leading to a calculated decrease in plasma volume of 14.4 +/- 0.9%. Infusion of M&B 22.948 (0.68 mg/kg/min) did not affect hematocrit and lowered arterial pressure by 8.1 +/- 0.5% (p < 0.01), an effect similar to that observed following administration of sodium nitroprusside (10 micrograms/kg/min). Simultaneous infusion of atrial natriuretic peptide and M&B 22.948 had additive arterial pressure lowering effects (-15.9 +/- 1.1%; p < 0.01 vs atrial natriuretic peptide or M&B 22.948 alone), while the increase in hematocrit of 9.4 +/- 0.7% was identical to that seen with atrial natriuretic peptide alone. Thus, M&B 22.948 amplified atrial natriuretic peptide effects on arterial pressure, but not on vascular permeability. These findings indicate differential regulation of atrial natriuretic peptide effects by inhibition of the cGMP-phosphodiesterase.


A biochemical and NMR spectroscopic study of hydrazine in the isolated rat hepatocyte. Using isolated rat hepatocytes the biochemical effects of hydrazine have been investigated using both conventional assay techniques and high resolution proton NMR. High resolution proton NMR revealed that hydrazine caused a significant increase in alanine and lactate levels in the incubation buffer, whereas levels of beta-hydroxybutyrate were decreased. NMR also detected metabolites of hydrazine notably acetylhydrazine and a cyclised hydrazone formed with alpha-ketoglutarate. Changes were detected in NADH and NADPH, ATP, succinate dehydrogenase (SDH) and total non-protein sulphydryl groups (TNPSH). However, the changes in pyridine nucleotides occurred at higher concentrations than those affecting succinate dehydrogenase and ATP. Similarly, the depletion of TNPSH occurred at a higher concentration and with a different time course to that seen with ATP depletion and inhibition of succinate dehydrogenase.


Airway response by neurokinin A.

Airway response by neurokinin A. We studied the effect of neurokinin A (NKA) on tracheal smooth muscle (TSM) contraction induced by the administration of acetylcholine (ACh) intra-arterially (ia) into the tracheal circulation or bilateral vagal stimulation in 18 mongrel dogs. In five dogs, 10(-12) to 10(-7) mol of NKA was injected selectively into the tracheal circulation. The tracheal contractile response to 10(-7) mol NKA was 3.12 +/- 0.48 g/cm, 6.3 +/- 0.7% AChmax. Plasma histamine concentration in the right atrium was measured at the peak response to NKA. Plasma histamine concentration was 3.33 +/- 2.05 ng/ml after 10(-7) mol NKA (vs 0.32 +/- 0.08 ng/ml for control, p > 0.10). In the other 5 dogs, dose-response was obtained with 10(-12) to 10(-7) mol ACh 10 min before and 10 min after administration of 10(-8) mol NKA. Frequency-response relationship also was induced by electrical stimulation of the distal cut ends of the cervical vagi in the range of frequencies 1-20 Hz (constant 30 V) at 15-s intervals 10 min before and after administration of 10(-8) NKA. Considerable potentiation of the tracheal contractile response to 10(-18) to 10(-7) mol ia ACh occurred 10 min after administration of 10(-8) mol NKA (p < 0.05). Significant potentiation of the tracheal contraction to vagal stimulation at 10-20 Hz was also observed 10 min after administration of 10(-8) mol NKA (p < 0.05). We demonstrate that the substantial contractile effects of NKA on canine tracheal smooth muscle are not related to histamine release from mast cells in the respiratory tract.(ABSTRACT TRUNCATED AT 250 WORDS)


[Inhibitory effect of IPD-1151T (suplatast tosilate) on mast cell induction from normal mouse spleen cells]. The effects of IPD-1151T (suplatast tosilate), a novel anti-allergic drug, on murine mast cell induction were examined by the in vitro cell culture technique. Spleen cells from BALB/c mice suspended in RPMI-1640 medium containing interleukin 3 were cultured in the presence or absence of IPD-1151T. Half the volume of the medium was changed every 4 days. Mast cell numbers increased as the culture time went on and reached a peak on the 16th day, when spleen cells were cultured without IPD-1151T. However, induction of mast cells from spleen cell cultures was inhibited by IPD-1151T in a dose dependent fashion. These results strongly suggest that IPD-1151T is a very useful agent for therapy in allergic diseases.


Early glucocorticoid induction of calmodulin and its suppression by corticotropin-releasing factor in pituitary corticotrope tumor (AtT20) cells. Glucocorticoids inhibit stimulus-evoked ACTH secretion by the rapid induction of new protein(s) that suppress intracellular free calcium signals. The present study examined whether the calcium receptor protein, calmodulin, is induced by glucocorticoids in the mouse pituitary corticotrope tumor (AtT20 D16:16) cell line. Treatment of AtT20 D16:16 cells with the synthetic glucocorticoid dexamethasone markedly (up to 10-fold) increased the level of a single (approximately 1.6kb) calmodulin mRNA 90 min after the application of steroid. Puromycin applied 15 min before and during dexamethasone treatment blocked the induction of this mRNA, suggesting that additional glucocorticoid induced transcription factor proteins may be required for enhanced calmodulin gene transcription. A two-fold increase in the intensity of an approximately 18K immunoreactive calmodulin protein band was detected by immunoblotting at 90 min after dexamethasone administration. Corticotropin releasing factor, added for 30 min at the start of steroid treatment, prevented the increase of calmodulin mRNA, as well as the suppression of corticotropin releasing factor-evoked ACTH release caused by dexamethasone. These data suggest that calmodulin may be involved in the early phase of glucocorticoid inhibition of pituitary ACTH release.


Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A. Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.


Interaction of the catalytic subunit of protein kinase A with the lung type V cyclic GMP phosphodiesterase: modulation of non-catalytic binding sites. We have previously demonstrated that the catalytic sub-unit of protein kinase A can catalyse a potent activation of partially purified Type V cyclic GMP-specific phosphodiesterase activity (Burns et al., 1992, Biochem. J. 283, 487-491). We now demonstrate that this phosphodiesterase most likely has a sub-unit mass of 90kDa, based upon 32P-cyclic GMP photo-affinity labelling, that activation of the phosphodiesterase does not require the prior binding of cyclic GMP to the phosphodiesterase, and that alkaline phosphatase can reverse the protein kinase A-dependent activation of phosphodiesterase activity. Zaprinast is a mixed inhibitor of non-activated cyclic GMP phosphodiesterase activity. However, inhibition of the protein kinase A-activated phosphodiesterase is competitive. These results suggest that protein kinase A can modulate the inhibitory effects of zaprinast via perturbations of a non-catalytic binding site.


The expression of milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system. We expressed milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system in which the cloned cDNA for the hVDR was expressed under the control of bacterial T7 polymerase. The hVDR protein comprised approximately 60% of total bacterial protein. It migrated on polyacrylamide-sodium dodecyl sulfate gels with an M(r) of 48,000. It had the predicted amino acid composition and amino acid sequence analysis. The expressed protein was bound by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a Kd in the nanomolar range. It sedimented on sucrose density gradients at 3.5S. Furthermore, the expressed protein bound to the osteocalcin vitamin D response element (VDRE) as assessed by a gel mobility shift assay. The expression of large amounts of hVDR protein should allow for the use of this protein in structure-function and x-ray crystallography studies.