Synergistic interaction between alpha 2-adrenergic agonists and benzodiazepines in rats.

Both alpha 2-adrenergic agonists and benzodiazepines exert anxiolytic and sedative effects when administered as preoperative medications. Clinical effects achieved with a combination of drugs, representative of these classes of compounds, is greater than that which could be expected from a simple additive response. Therefore, we investigated the nature of the interaction between dexmedetomidine, the highly-selective alpha 2-adrenergic agonist, and midazolam in a series of in vivo and in vitro studies in rats. Rats were administered midazolam, dexmedetomidine, or a combination of midazolam and dexmedetomidine intravenously to derive three dose-response curves for loss of righting reflex (LRR). LRR was determined in rats in a rotating cage (4 rotations/min) by observing whether the rat failed to maintain its upright posture for greater than or equal to 15 s exactly 2.5 min after drug administration. The effect of either flumazenil (benzodiazepine receptor antagonist) or atipamezole (the alpha 2-adrenergic antagonist) on the LRR was also determined. A probit analysis was performed and an isobologram for the ED50 was derived to assess the nature of the interaction. Rat brain membranes were prepared for receptor binding assays using [3H]-flumazenil and [3H]-rauwolscine to characterize the benzodiazepine and alpha 2-adrenergic receptors, respectively. The ability of either midazolam or dexmedetomidine to displace the radiolabeled ligand from the alternative receptor was assessed. To detect a possible kinetic interaction between the two drugs, separate cohorts of rats were administered the two drugs individually or in combination at the combination ED50 doses.(ABSTRACT TRUNCATED AT 250 WORDS)


A hypnotic response to dexmedetomidine, an alpha 2 agonist, is mediated in the locus coeruleus in rats.

Dexmedetomidine, the highly selective alpha 2-adrenergic agonist, produces a dose-dependent hypnotic response in rats through a central mechanism. Because the locus coeruleus (LC) contains pathways involved in the maintenance of vigilance and a high prevalence of alpha 2 adrenoceptors, we investigated the role of this brainstem nucleus in the hypnotic response to dexmedetomidine. The experimental model consisted of chronic, stereotactically cannulated rats (n = 157) in which the hypnotic response to dexmedetomidine was assessed by the duration of the loss of their righting reflex. Correct placement of the cannula was confirmed histologically at necropsy. The hypnotic response to dexmedetomidine 0.3-333.3 micrograms administered into the LC increased in a dose-dependent fashion. Dexmedetomidine 6.6 micrograms injected 2 mm lateral to the LC did not cause the animals to lose their righting response. Atipamezole 0.07 micrograms-12 micrograms, a selective alpha 2-adrenergic antagonist, blocked the hypnotic response to dexmedetomidine 6.6 micrograms when both were administered into the LC. Also, atipamezole 0.7-30 micrograms, administered into the LC, blocked in a dose-dependent manner the hypnotic response to intraperitoneal (ip) dexmedetomidine 50 micrograms.kg-1. Atipamezole injected into the LC did not block the hypnotic response to pentobarbital 40 mg.kg-1 ip. Prazosin, an alpha 1-adrenergic antagonist, 4.2 micrograms into the LC or 1.0 mg.kg-1 ip, did not alter the hypnotic response to dexmedetomidine 6.6 micrograms into the LC. The present data suggest that alpha 2-adrenergic receptors in the LC appear to be a major site for the hypnotic action of dexmedetomidine.(ABSTRACT TRUNCATED AT 250 WORDS)


Developmental toxicity of nondepolarizing muscle relaxants in cultured rat embryos.

Evidence of developmental toxicity of clinically used nondepolarizing muscle relaxants was sought in rat embryos grown in culture. Embryos were explanted at 8 AM on day 9 of gestation (presomite stage, plug day = day 0), and were cultured in rotating bottles with medium containing various concentrations of d-tubocurarine, pancuronium, atracurium, and vecuronium. At 10 AM on day 11 of gestation (forelimb bud stage), culture was terminated and embryos were examined for general morphology. Treatment with tested agents resulted in dose-dependent developmental toxicity; namely, growth retardation seen as decreased crown-rump length, decreased number of somite pairs, and morphologic abnormalities. However, the concentrations that caused toxicity were at least 30-fold greater than serum concentrations clinically achieved in the mother. We conclude that these muscle relaxants have a low potential for causing developmental toxicity during organogenesis.


Neurotransmitters in the vestibular commissural system of the cat.

The present study focused on the transmitters that control the vestibular neural activity and, in particular, that regulate commissural inhibition. Extracellular spikes of a single vestibular neuron were recorded in decerebrate cats. The seven barrels of the electrode, with the exception of the center barrel, were filled with transmitter candidates and their specific antagonists, while the center barrel was filled with 2 M NaCl for extracellular recording. After isolation of a type 1 neuron, chemicals were iontophoretically applied to examine their effects on its activity. The results were as follows: (1) GABA and glycine markedly decreased spontaneous firing of the neurons, while serotonin did not affect their activity. (2) Bicuculline abolished the inhibitory effects of GABA on the neurons. (3) Strychine abolished the effects of glycine. (4) Commissural inhibition induced by electrical stimulation of the contralateral labyrinth was not abolished by strychine but was abolished by bicuculline. We conclude that (1) vestibular type 1 neurons are controlled by GABAergic and glycinergic but not serotoninergic neurons, and (2) commissural inhibition is activated by the GABAA receptor, but not by the GABAB receptor.


Computer-assisted three-dimensional reconstruction and simulations of vestibular macular neural connectivities.

The macular neuroepithelium is morphologically organized as a weighted neural network for parallel distributed processing of information. The network is continuous across the striola, where some type II hair cells synapse with calyces containing type I cells with tufts of opposite directional polarities. Whether other hair cell to calyx appositions that lack synapses interact because of intercellular potassium accumulation remains an open question. A functionally important inference of macular organization is that just as arrays of hair cells communicate an entire piece of information to a nerve fiber, so do macular subarrays of nerve fibers (not single units) carry the whole coded message to the brain stem. Moreover, the size of the network subarray can expand or become more limited depending upon the strength and/or duration of the input. It is the functioning of the network and its subarrays that must be understood if we are to learn how maculas carry out their work and adapt to new environments. Simulations of functioning maculas, or subparts, based on precise morphology and known physiology are useful tools to gain insights into macular information processing. The current simulations of afferent collateral electrical activity are a prelude to development of a 3-D model. The simulations demonstrate a relationship between geometry and function, with the diameter of the stem apparently being a major determinant of electrical activity transmitted to the base in the case of collaterals with short stems. Thus, while changes in synaptic number and/or size may be an important adaptive mechanism in an altered g environment, changes in diameter of the stem is another means of altering outflow. Research on the effects of microgravity should be extremely useful in examining the validity of this and other concepts of neural adaptation, since maculas are biological linear accelerometers ideally suited to the task. Maculas are also extremely interesting to study in detail because of the richness of connectivities and submicroscopic organization they present. Many of their features are common with more complex parts of the brain. It seems possible that knowledge of the three-dimensional geometric relationships operative in a functioning macula will contribute much to the understanding of the dynamics underlying more complex behavior. Computerized approaches greatly facilitate this task and provide an objective method of analysis. It is likely that, in the end, simple rules will be found to govern optimal neural architectural organization, even at higher cognitive levels. The architecture only appears complex because we do not yet grasp its meaning.(ABSTRACT TRUNCATED AT 400 WORDS)


The natural course of multiple endocrine neoplasia type IIb. A study of 18 cases.

Multiple endocrine neoplasia (MEN) type IIb is an autosomal dominantly inherited disorder associated with medullary thyroid cancer, pheochromocytoma, and a characteristic phenotype. The present study was performed to investigate the natural course of the syndrome and to describe its expression. The medical records of 18 patients with MEN IIb, seven male and 11 female, were reviewed. The mean age at diagnosis of MEN IIb was 18 years (range, 8 to 41 years). All 18 patients had medullary thyroid cancer. In three patients, medullary thyroid cancer was diagnosed via screening. In two of these patients, the calcitonin value normalized after thyroidectomy. One patient died of metastases from medullary thyroid cancer at the age of 20 years (median duration of follow-up, 10 years). Eight of the 18 patients had pheochromocytomas. All of our patients had neuromas and bumpy lips, and all but one had a marfanoid habitus. A large proportion of the patients had intestinal abnormalities (75%), thickened corneal nerves (69%), skeletal abnormalities (87%), and delayed puberty (43%). The course of medullary thyroid cancer in MEN IIb is not always as aggressive as is generally thought. Periodic examination of relatives who are at risk may lead to early diagnosis and curative treatment. Intestinal abnormalities, skeletal abnormalities, and delayed puberty are commonly found in association with MEN IIb.


Acute syphilitic meningitis in a man with seropositivity for human immunodeficiency virus infection and normal numbers of CD4 T lymphocytes.

Coinfection with the human immunodeficiency virus (HIV) and Treponema pallidum may predispose to accelerated neurosyphilis. The mechanism underlying this interaction is undefined, but usually presumed to result from HIV-induced suppression of cell-mediated immunity as reflected in the CD4 T-lymphocyte count. We report a patient infected with HIV who developed aggressive neurosyphilis despite a CD4 count of 1000/mm3. The CD4 cells constituted 17% of his total lymphocytes. Our case suggests that while severe neurosyphilis can occur in HIV-infected persons with normal absolute CD4 counts, the percentage of CD4 cells may be a better indicator of the risk of neurosyphilis. These observations are relevant to designing treatment strategies for patients coinfected with HIV and T pallidum based on measures of their immunocompetence.


Transport of L-glutamine and L-glutamate across sinusoidal membranes of rat liver. Effects of starvation, diabetes and corticosteroid treatment.

There is increasing evidence that membrane transporters for glutamine and glutamate are involved in control of liver metabolism in health and disease. We therefore investigated the effects of three catabolic states [starvation (60 h), diabetes (4 days after streptozotocin treatment) and corticosteroid (8-day dexamethasone) treatment] associated with altered hepatic amino acid metabolism on the activity of glutamine and glutamate transporters in sinusoidal membrane vesicles from livers of treated rats. In control preparations, L-[14C]glutamine uptake was largely Na(+)-dependent, but L-[14C]glutamate uptake was largely Na(+)-independent. Vmax. values for Na(+)-dependent uptake of glutamine and/or glutamate exceeded control values (by about 2- and 12-fold respectively) in liver membrane vesicles from starved (glutamine), diabetic (glutamate) or steroid-treated (glutamine and glutamate) rats. The Km values for Na(+)-dependent transport of glutamine or glutamate and the rates of their Na(+)-independent uptake were not significantly altered by any treatment. Na(+)-independent glutamate uptake appeared to include a dicarboxylate-exchange component. The patterns of inhibition of glutamine and glutamate uptake by other amino acids indicated that the apparent induction of Na(+)-dependent amino acid transport in catabolic states included increased functional expression of systems A, N (both for glutamine) and X-ag (for glutamate). The results demonstrate that conditions resulting in increased secretion of catabolic hormones (e.g. corticosteroid, glucagon) are associated with increased capacity for Na(+)-dependent transport of amino acids into liver cells from the blood. The modulation of hepatic permeability to glutamine and glutamate in these situations may control the availability of amino acids for intrahepatic metabolic processes such as ureagenesis, ammonia detoxification and gluconeogenesis.


Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells.

P-glycoprotein plays a key role in multidrug resistance of tumor cells. In order to elucidate the possible quarternary structure/function relationship of P-glycoprotein, we treated multidrug-resistant human leukemia K562/ADM cells with the crosslinking reagent, disuccinimidyl suberate. In addition to 180K P-glycoprotein, a 340K protein was immunoprecipitated with an anti-P-glycoprotein monoclonal antibody, MRK-16. The 340K protein is most probably a dimeric P-glycoprotein, since only the 180K P-glycoprotein was immunoprecipitated with MRK-16 when K562/ADM cells were treated with the cleavable crosslinking reagent, dithiobis(succinimidylpropionate), and analysed under reduced conditions. The dimeric P-glycoprotein was photolabeled with [3H]azidopine like the 180K monomeric P-glycoprotein and the photolabeling was inhibited by excess amount of vincristine and verapamil. The dimeric P-glycoprotein could be a functionally active form of the protein involved in the transport of antitumor agents.


Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.


Effects of buthionine sulfoximine and diethyl maleate on glutathione turnover in the channel catfish.

Despite the growing use of fish in toxicological studies, little is known regarding glutathione (GSH) metabolism and turnover in these aquatic species. Therefore, we examined GSH metabolism in the liver and gills of channel catfish (Ictalurus punctatus), a commonly employed aquatic toxicological model. Treatment of channel catfish with L-buthionine-S,R-sulfoximine (BSO, 400 or 1000 mg/kg, i.p.), an inhibitor of GSH biosynthesis, did not deplete hepatic GSH in channel catfish. In addition, hepatic GSH concentrations did not fluctuate in catfish starved for 3 days, indicating relatively slow turnover of hepatic GSH. However, hepatic GSH concentrations were reduced significantly (P less than 0.05) after 7 days of starvation. Administration of the thiol alkylating agent diethyl maleate (DEM, 0.6 mL/kg, i.p.) resulted in depletion of 85% of hepatic GSH at 6 hr post-DEM, with complete GSH recovery observed at 24 hr post-DEM. Co-administration of BSO and DEM (1000 mg/kg, 0.6 mL/kg, respectively) substantially depleted gill GSH and eliminated detectable liver GSH. Following BSO/DEM, GSH recovery in hepatic mitochondria occurred more rapidly than did liver cytosolic GSH. gamma-Glutamylcysteine synthetase (GCS) activities were comparable in the 10,000 g supernatants of catfish liver and gills (204 +/- 21 and 268 +/- 20 nmol/min/mg protein, respectively) whereas gamma-glutamyltranspeptidase (GGT) activity was not detected in the 600 g post-nuclear fraction of either liver or gills. In conclusion, i.p. administration of DEM was an effective means for achieving short-term hepatic GSH depletion in channel catfish, whereas co-administration of BSO and DEM elicited prolonged and extensive hepatic GSH depletion in this species. Like rodents, channel catfish maintained physiologically distinct hepatic mitochondrial and cytosolic GSH pools, and also regulated hepatic GSH levels by in situ hepatic GSH biosynthesis. However, unlike rodents, there was no evidence for a labile hepatic cytosolic GSH pool in channel catfish. These similarities and differences need to be considered when designing toxicological studies involving the GSH pathway in channel catfish and possibly other fish species.


Major histocompatibility complex genes and susceptibility to systemic lupus erythematosus in southern Chinese.

To investigate the predisposing role of major histocompatibility complex (MHC) genes to systemic lupus erythematosus (SLE) in a Chinese population. Polymorphism in the HLA-DRB, DQB, complement component C4, and 21-hydroxylase genes was analyzed by restriction fragment length polymorphism analysis and oligonucleotide probing of in vitro-amplified DNA from 88 Chinese patients with SLE and 69 matched control subjects. HLA-DRw15 and DQw1 were significantly more frequent in patients (corrected P less than 0.006, relative risk 5.2), but none of the 9 sequence variants of DQw1 were increased. The C4A gene deletion usually associated with SLE in Caucasoid and black patients was absent from all Chinese subjects, but possession of other C4 deletions and of DRw15 conferred the greatest risk (relative risk = 8.3). Different MHC haplotypes predispose to lupus in Chinese than in other ethnic groups. Our data suggest that the susceptibility lies at, or telomeric to, the DR locus, and that DRw15 and C4 deletions may act synergistically in conferring disease susceptibility.


Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen.

To establish an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Ki antibody, using a bovine recombinant Ki antigen, and studying its specificity. Sera from 220 patients with various connective tissue diseases were screened, and a prospective study of fluctuations in anti-Ki antibody and clinical course of a woman with systemic lupus erythematosus (SLE) was analyzed, by ELISA: Anti-Ki antibodies were present in 18.9% of patients with SLE. The titer of anti-Ki antibody in the woman with SLE rose before the onset of pericarditis and pleuritis in this longitudinal study. ELISA using a recombinant Ki antigen is useful for the diagnosis of SLE, and it might be useful in estimating disease activity in patients with SLE.


Increased levels of circulating intercellular adhesion molecule 1 in Kawasaki disease.

We investigated whether levels of intercellular adhesion molecule 1 (ICAM-1) antigen shed into the circulation increase during acute Kawasaki disease (KD). We also compared ICAM-1 levels in acute KD with those in anaphylactoid purpura (AP) and in measles. Serum ICAM-1 levels were measured by a double-determinant immunoassay using 2 monoclonal antibodies in the FAST (Falcon assay screening test) system. Serum levels of tumor necrosis factor alpha (TNF alpha) were measured by a specific and sensitive sandwich enzyme immunoassay. Patients with KD, but not those with AP or measles, had increased levels of shed ICAM-1 antigen in serum samples obtained during acute stages. Moreover, during the acute stage, KD patients with coronary artery lesions (CAL) had still higher levels of shed ICAM-1 than did those without CAL. We found a positive correlation between serum levels of shed ICAM-1 and levels of TNF alpha during acute KD. Our findings suggest that the serum ICAM-1 level is an important immunologic parameter for determining the severity of vascular damage during acute KD.


[Association of a secreting oncocytic tumor of the kidney and a parathyroid adenoma].

An unusual association of a secreting renal oncocytoma and a parathyroid adenoma is described. The high level of hypercalcemia and of blood parathormone (PTH 44-68), partially reduced by nephrectomy and totally normalized by parathyroidectomy, as well as the renal tumor PTH evaluation and the ultrastructural features showing secretory granules in oncocytic cells of kidney, advocate for a double site of PTH secretion. Removal of both tumors permits a complete recovering.


Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.


[Location of ctDNA fragment related to CMS in Brassica napus L. var. xiangai].

The ctDNA of sterile line and its maintainer from Brassica napus L. var. xiangai were digested by restriction endonucleases EcoRI, BamHI, PstI and SmaI. Only one special fragment E3.2kb was observed in EcoRI restriction pattern of maintainer. After being eluted, this fragment was incubated with plasmid pUC9 and then transformed E. coli JM83. Through colour screening, clony hybridization and electrophoretic analyses, the special clone carrying E3.2 was obtained. The rRNA gene probe was used to hybridize with the EcoRI restriction pattern. The result showed that E3.2kb fragment is homologous to rRNA gene. And then, we use E3.2 fragment as probe to hybridize with rRNA gene digested by BamHI, EcoRI, SalII, BglII, HindIII and PstI. According to rRNA gene map, E3.2 fragment was located at the leader sequence of 16S rRNA gene, from +2.0kb to +5.4kb. Because this 3.4kb region was in the inverted repeat region of ctDNA and it is homologous to E3.2kb that related to CMS. So probably CMS is related to this 3.4kb region in the inverted repeat region of ctDNA.


Geographical subtypes demonstrated by RFLP following PCR in the LTR region of HTLV-I.

It has been demonstrated that specific mutations, localized in the long terminal repeat (LTR) region of HTLV-I, allowed to propose the existence of three HTLV-I subtypes. Because some of these mutations created or suppressed the restriction sites for ApaI, NdeI, DraI, SacI, MaeIII, and MaeII enzymes, these endonucleases were used for characterization of further 30 HTLV-I isolates. Seventeen proviral DNA from Japan, five from the Caribbean, one each from French Guyana, the United States, and China, two from Ivory Coast, and three from Zaire were tested. The DNA used were extracted from 26 in vivo peripheral blood mononuclear cell samples and from four cell lines obtained from two Japanese, one Chinese, and one North American patients. Digestions were performed on amplified DNA of the LTR region (nucleotides 31-768). The results confirm the existence of three subtypes of HTLV-I according to LTR sequences. Subtype I was observed only in patients from the Ivory Coast and Zaire. Subtype II was found in the patient from the French West Indies, and in 33% of the samples from Japan. Subtype III was most often observed in the Japanese but also in the Zairian patients.


Brucella abortus stimulates human T cells from uninfected and HIV-infected individuals to secrete IFN gamma: implications for use of Brucella abortus as a carrier in development of human vaccines.

Brucella abortus has been characterized as a T-independent type 1 antigen/carrier in human and murine antibody responses. In this report it is shown that BA can activate human CD3+ T cells to secrete interferon-gamma (IFN gamma). Unlike mitogens, such as phytohemagglutinin, this stimulation was associated with minimal T-cell proliferation or upregulation of interleukin-2 (IL-2) receptor. Monocytes inhibited BA-mediated IFN gamma secretion since their removal resulted in increased responses, whereas adding monocytes back to cultures caused inhibition. BA elicited IFN gamma from CD4+ and CD8+ T cells, although CD4+ T cells secrete significantly more (p less than 0.05) IFN gamma than CD8+ T cells. The ability of BA to elicit IFN gamma from human T cells was inhibited in the presence of anti-Tac, suggesting that BA also induces IL-2 secretion and that IL-2 is involved in BA-mediated IFN gamma secretion. Detectable IL-2 secretion was induced by BA in the presence of anti-Tac. Exogenous IL-2 acted synergistically with BA to enhance IFN gamma secretion, suggesting that the amount of IL-2 released by BA alone was insufficient for optimal IFN gamma release. Furthermore, addition of IL-2 to T cells from individuals with poor or absent responses to BA, including individuals infected with HIV-1, restored their ability to secrete IFN gamma in response to BA. These data indicate that BA is capable not only of activating human B cells but can also induce T cells, probably of the TH1 phenotype, to secrete IFN gamma.(ABSTRACT TRUNCATED AT 250 WORDS)


Pseudoaneurysm of the gastroduodenal artery arising within a pancreatic pseudocyst.

A case is presented of a 41-year-old man admitted for obstructive jaundice. Work-up revealed a pseudocyst of the pancreatic head which contained a large pseudoaneurysm of the gastroduodenal artery. After an unsuccessful attempt at embolization, the patient was treated with an uncomplicated pancreaticoduodenectomy.


Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.


Identification of an essential cysteine in the reaction catalyzed by aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli has been studied by oligonucleotide-directed mutagenesis. The focus of this investigation was to examine the role of a cysteine residue that had been previously identified by chemical modification with an active site directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104, 59-64). Substitution of this cysteine at position 135 with an alanine results in complete loss of enzyme activity. However, changing this cysteine to a serine yields a mutant enzyme with a maximum velocity that is 0.3% that of the native enzyme. This C135S mutant has retained essentially the same affinity for substrates as the native enzyme, and the same overall conformation as reflected in identical behavior on gel electrophoresis and in identical fluorescence spectra. The pH profile of the native enzyme shows a loss in catalytic activity upon protonation of a group with a pKa value of 7.7. The same activity loss is observed at this pH with the serine-135 mutant, despite the differences in the pKa values for a cysteine sulfhydryl and a serine hydroxyl group that have been measured in model compounds. This observed pKa value may reflect the protonation of an auxiliary catalyst that enhances the reactivity of the active site cysteine nucleophile in the native aspartate-beta-semialdehyde dehydrogenase.


Assay for N-acetylmuramyl-L-alanine amidase in serum by determination of muramic acid released from the peptidoglycan of Brevibacterium divaricatum.

A method is reported for the determination of N-acetylmuramyl-L-alanine amidase in serum. Muramic acid, released from the interpeptide bridges of Brevibacterium divaricatum peptidoglycan, is measured by a modified colorimetric method. Using this procedure, it was possible to determine N-acetylmuramyl-L-alanine amidase in aliquots of less than 10 microliters human serum with an incubation time of 10 min. Amidase activity was found in all the sera tested (n = 11). The relevance of this simple and fast assay is discussed.


Prophylaxis for Pneumocystis carinii pneumonia in patients infected with human immunodeficiency virus.

Following the initial observation by Dr. Margaret Fischl that trimethoprim-sulfamethoxazole can prevent Pneumocystis carinii infection in patients with Kaposi's sarcoma, initiating prophylaxis for pneumocystic infection in all patients with less than 200 CD4+ cells/mm3 has become accepted practice. This prophylactic intervention has been found not only to reduce the development of pneumonia due to P. carinii but also to prolong life. Drs. Henry Masur and Joseph A. Kovacs first reviewed prophylaxis for P. carinii pneumonia in patients infected with the human immunodeficiency virus for the AIDS Commentary 3 years ago. They have updated that initial review for this AIDS Commentary, placing currently available information into concise clinical perspective and detailing a rational plan for the clinician to follow based on results of recent studies.


A pharmacokinetic study of sulphasalazine and two new formulations of mesalazine.

We have examined the pharmacokinetics of enteric coated sulphasalazine compared with two new formulations of mesalazine. These consisted of microgranules of mesalazine coated with Eudragit S in a concentration of either 20 or 25% dry lacquer substance; these in turn were enclosed in capsules coated with Eudragit L. In-vitro dissolution studies of coated microgranules showed that drug release was pH dependent. Studies in 7 normal volunteers showed median peak concentrations of 5-amino-salicylic acid and N-acetyl-5-amino-salicylic acid occurred at about 6 hours with both microgranular preparations, compared with sulphasalazine at 15 h. The microgranule formulation coated with 20% Eudragit S gave serum levels and overall systemic absorption similar to values with sulphasalazine. This new formulation may be of value for delivering mesalazine and other therapeutic agents to the colon.


Effects of acetorphan, an antidiarrhoeal enkephalinase inhibitor, on oro-caecal and colonic transit times in healthy volunteers.

Acetorphan is a potent enkephalinase inhibitor displaying antidiarrhoeal activity attributable to its intestinal antisecretory action mediated by endogenous enkephalins. The effect of acetorphan on digestive motility was studied in 12 healthy volunteers. Oro-caecal transit time was evaluated using the sulphasalazine/sulphapyridine method and colonic transit times using radiopaque markers. These measurements were successively performed after one week treatment with an antidiarrhoeal dose of acetorphan (100 mg t.d.s.) or placebo. There was no significant modification in transit time linked to acetorphan treatment: total oro-caecal times were 303 +/- 32 min vs. 287 +/- 27 min and colonic transit times 25.8 +/- 5.8 h vs. 31.3 +/- 5.5 h after acetorphan and placebo, respectively (means +/- S.E.M.). There was no significant modification either in right colonic, left colonic or rectosigmoid segmental transit times, or in the mean number of stools. These results, consistent with those from animal studies, confirm that, unlike classical antidiarrhoeal mu opiate receptor agonists, which act by delaying intestinal transit, acetorphan does not affect the transit. Antidiarrhoeal activity not accompanied by a delayed intestinal transit could have beneficial therapeutic consequences in the management of infectious diarrhoea. In addition, we show that the sulphasalazine and radiopaque markers methods can be simultaneously applied in the same study.


Signal transduction in mesangial cells.

Phenotype, growth, and functional characteristics of glomerular mesangial "myofibroblasts" are under the control of multiple hormones, vasoactive agents, autacoids, and cytokines. Several parallel signal transduction pathways couple receptor occupancy with functional changes, including phospholipases C, A2, and D breakdown of membrane phospholipids, and adenylate/guanylate cyclase activation. Changes of cytosolic ion concentrations, cyclic nucleotide accumulation, and eicosanoid biosynthesis are currently interpreted as intracellular signals for protein kinase activation. Phosphorylation of multiple substrates by serine/threonine kinases C, A, and G or by tyrosine kinases directly coupled to receptors, is a final step in cell activation. Cross-talk between signal transduction pathways, along with the release of eicosanoids and cytokines acting as intercellular mediators, provides the potential for interactive regulation of glomerular cell functions.


Characterization of the gene encoding the plastid-located glutamine synthetase of Phaseolus vulgaris: regulation of beta-glucuronidase gene fusions in transgenic tobacco.

The gln-delta gene, which encodes the plastid-located glutamine synthetase of Phaseolus vulgaris, was cloned and its promoter region was sequenced. Primer extension analysis was used to map the two major transcription initiation sites which are about 90 nucleotides apart. A fusion of 2.3 kb of the upstream region of the gln-delta gene to the reporter gene uidA encoding beta-glucuronidase was shown to be expressed in the chlorophyllous cell types of leaves and stems and in the root meristem region of transgenic tobacco. Analysis of a series of three 5' promoter deletion fusions revealed the presence of a region essential for promoter activity between -786 and -327 and regions involved in tissue-specific regulation and light regulation between -786 and +43.


cDNA, amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1.

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317-327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3' end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15-20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.


Clinical meaning of GGT activity in follow-up of patients with alcohol-related liver injury and cholestasis.

The dynamics of GGT was investigated in three groups of patients after removing some primary causes of GGT increase. Group A included 34 patients with alcohol-related liver disease, group B included 16 patients with alcoholic liver injury and cholestasis, caused by concomitant alcoholic pancreatitis and group C included 17 patients with extrahepatic cholestasis, caused by choledocholithiasis. Follow-up assays of GGT were performed on the 7th, 14th and 30th days. Our results showed that the dynamics of GGT was more rapid after removing the cause for cholestasis than in stopping alcohol consumption in patients with chronic liver diseases. On the 14th day more than a 50% decrease in GGT activity was noted in 20% of the patients from groups A and B and in almost all cases from group C. On the 30th day, the reference range of GGT was not attained by any of the patients with liver disease nor in five patients from group C. No significant correlation was found between the severity of liver damage and the extent of GGT increase at the beginning and at the end of the follow-up period.


Haemodynamic effects of roxatidine, an H2-receptor antagonist.

The haemodynamic effects of roxatidine were investigated in 12 patients with congestive heart failure (New York Heart Association class II) in a placebo-controlled, double-blind, randomized, cross-over study. Impedance and mechanocardiography were determined following successive 7-day treatment periods with placebo and roxatidine 150 mg once daily. Comparison with placebo values showed roxatidine to significantly increase the preejection period (109.7 +/- 2.7 ms versus 117.3 +/- 2.7 ms, 1.5 h after administration). Heart rate and blood pressure remained the same. In contrast to other, newer H2-receptor antagonists, which decrease stroke volume and/or cardiac output, roxatidine did not reduce these parameters but increased the preejection period and the ratio of the preejection period to the left ventricular ejection time, indicating a slight negative influence on cardiac performance.


Disappearance of a pituitary tumor after 15 months of treatment with CV 205-502, a new dopamine agonist.

We report the case of a 27-year-old woman with a prolactin-secreting macroadenoma of the pituitary gland who was under treatment with a new dopamine agonist, Sandoz CV 205-502. Immediately after diagnosis of a 12 x 10 mm intrasellar prolactinoma, treatment with CV 205-502 was begun at a daily dose of 0.075 mg. Under this low dose, prolactin in the serum normalized after 4 weeks, the initial hormone value being 189.9 ng/ml. After 6 months of therapy there was still a 6 x 6 mm tumor, and after 15 months of treatment the pathological process could no longer be observed with magnetic resonance imaging. In the 20th month of therapy the patient became pregnant. An ovulatory menstrual cycle had been present for a few months.


Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants.

The haematological recovery time, infection rate and supportive care requirements of patients receiving recovery phase autologous peripheral blood stem cell transplants (APBSCT) (n = 38), autologous bone marrow transplants (autoBMT) (n = 13) and allogeneic bone marrow transplants (alloBMT) (n = 14) were compared with respect to the time post-transplant to reach 0.1, 0.5 and 2.0 x 10(9) neutrophils/l and 50 and 150 x 10(9) platelets/l, the length of hospitalization, fever and antibiotic use, the incidence of documented infection and the number of red cell and platelet transfusions. The APBSCT group had a significantly more rapid recovery of neutrophils and platelets and their supportive care requirements were significantly less than the autoBMT and the alloBMT groups. There was no difference between the latter two groups. The most significant variables contributing to the differences in haematological recovery times were the granulocyte-macrophage progenitor (CFU-GM) dose infused and, to a lesser extent, patient age. The APBSCT group received a higher CFU-GM dose of 87 +/- 12 x 10(4)/kg BW compared with 12 +/- 5 and 17 +/- 3 x 10(4)/kg BW in the autoBMT and the alloBMT groups, respectively (p = 0.0001). Patient age showed a negative correlation with the rate of recovery because the APBSCT group, which recovered faster was also older (48 +/- 2 years, compared with 33 +/- 3 and 31 +/- 2, respectively, p = 0.0001). On multivariate analysis, CFU-GM dose was the only variable to show a significant correlation with all the haematological recovery endpoints studied in these 65 patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Incidence of mixed chimerism using busulfan/cyclophosphamide containing regimens in allogeneic bone marrow transplantation.

The incidence of mixed chimerism (MC) following allogeneic bone marrow transplantation (allo-BMT) is in part a measure of the marrow ablative effect of preparative regimens. Although the incidence of MC has been reported for many patients treated with total body irradiation (TBI), limited data for busulfan/cyclophosphamide (BU/CY) recipients have been examined. We performed restriction fragment length polymorphism (RFLP) analysis on 68 peripheral blood samples from 26 patients treated with BU/CY prior to allo-BMT for chronic myelogenous leukemia or acute myeloid leukemia. MC was detected in four of 26 patients for an overall incidence of 15.4%. Three of four MC patients are alive with no evidence of disease at 263 to 795 days post-transplantation. A fourth patient is alive at day 501 but developed CNS relapse at day 274. The level of recipient origin cells was less than 10% in all samples and detectable MC was transitory with an RFLP pattern that reverted to full chimerism. These results are comparable to those reported for TBI-containing regimens in patients receiving non-T cell-depleted bone marrow. The efficacy of BU/CY in conjunction with a T cell depletion still requires exploration.


Late life schizophrenia and its treatment: pharmacologic issues in older schizophrenic patients.

Schizophrenia in late life includes both chronic early-onset schizophrenia and late-onset schizophrenia. Treatment with neuroleptics in relatively low doses is often useful in controlling psychotic symptoms in these patients. The value of neuroleptics is limited, however, by the risks of side effects such as tardive dyskinesia.


Self-injury resulting in amputation among vascular patients: a retrospective epidemiological study.

In the course of 24 months, 164 nontraumatic vascular amputees were accepted for prosthetic rehabilitation; 120 (73%) were diabetic and 44 (27%) non-diabetic. Subjects were divided into two groups, the first consisting of 114 with spontaneous foot infection or gangrene and the second of 50, of whom 44 were diabetic and six had peripheral arterial disease on its own. Sixty-five per cent of all amputees had fewer than nine years of formal education and 46% were in the low-income bracket, but no significant relationship was found between mechanism of self-injury and level of education and income. All patients in the second group were able to detail ways in which they had injured their feet. The relationship between the mechanism of self-injury and the presence of diabetes mellitus was found to be highly significant. By instructing in better self-care it may be possible to prevent or at least postpone amputation in these patients.


Isolation of clobazam-orosomucoid complexes from patients' sera.

Orosomucoid, a member of the lipocalin family, may function in the in vivo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-1-methyl-1,5-benzodiazepine-2,4-dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.


Neuroendocrine differentiation in carcinoma of the prostate. Diagnostic, prognostic, and therapeutic implications.

Endocrine-paracrine cells of the prostate (also known as APUD or neuroendocrine cells) constitute, in addition to the basal and exocrine secretory cells, a third population of highly specialized epithelial cells in the prostate gland. These endocrine-paracrine cells contain, and most likely secrete, serotonin and calcitonin, as well as variety of other peptides. Little is known of the functional role of these cells, but they probably subserve a paracrine or local regulatory role. They may also regulate via endocrine, lumencrine, or neurocrine mechanisms. These endocrine-paracrine cells probably play a significant role during prostatic growth and differentiation as well as regulating the secretory process of the mature gland. Neuroendocrine differentiation in prostatic carcinoma occurs in the form of the relatively rare small cell carcinoma and carcinoid or carcinoid-like tumor, but most commonly as focal neuroendocrine differentiation in a conventional prostatic adenocarcinoma that is a very frequent, if not ubiquitous phenomenon, and reflects tumor cell heterogeneity mimicking the normal differentiation process. The world's literature on neuroendocrine differentiation in prostatic carcinoma is reviewed. Neuroendocrine differentiation in all types of prostatic carcinoma appears to correlate with a poor prognosis. This correlation is probably multifactorial and may relate to a positive correlation with grade, a direct resistance to hormonal manipulation, and/or autocrine/paracrine growth factor activity due to the secretion of neuroendocrine products. Neuron-specific enolase and chromogranin, as well as other neuroendocrine products, may be useful as serum markers in patients with prostatic carcinoma with neuroendocrine differentiation. New therapeutic strategies need to be developed to treat these tumors. This includes the use of specialized protocols that have been effective against neuroendocrine carcinomas arising in other organ systems.


[Echo-Doppler evaluation of the internal mammary artery bypass on the left coronary artery: a 4-year follow-up].

In this study ultrasonographic techniques are suggested to monitor internal mammary artery bypass graft on the anterior descending coronary artery. One hundred and fourty patients were studied using 3 different ultrasonographic methods: zero-crossing continuous wave Doppler, fast Fourier transform (FFT) continuous wave Doppler and high resolution echo-Doppler. The patients underwent the ultrasonographic examinations 3 times a year for 4 years. By means of FFT Doppler analysis and echo-Doppler it was possible to perform the study in 138 patients and by zero-crossing system in 127 patients. Ultrasonographic techniques showed pathologies of the graft in 17 (13%) patients: 15 with obstructive pathologies and 2 with haemodetournament of the second intercostal artery.


Analysis of CD4 gene expression in human fetal brain and neuroblasts.

1. The expression of the gene codifying for CD4, the most important human immunodeficiency virus type 1 (HIV-1) receptor molecule, was analyzed in 11 fetal brains at various gestational ages and in 9 human neuroblastoma (NB) cell lines. CD4 gene expression in fetal and malignant neural cells was then compared with that observed in a hematopoietic cell line and adult hippocampus. 2. In addition, CD4 mRNA was evaluated in two NB cell lines induced to differentiate in vitro with retinoic acid (RA) or 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H7), a protein kinase C inhibitor. 3. All fetal brains and NB cell lines express a 1.8-kb signal when hybridized with pT4BcDNA probe, while a 3.0-kb signal such as observed in hematopoietic human cells was found in 1 of 11 fetal brains and in 0 of 9 NB cell lines. The 1.8-kb signal was lost in all analyzed poly(A)+ mRNA samples. 4. Moreover, CD4 gene expression was not induced in either RA- or H7-treated NB cells at any tested time and dose. The analysis of NB cells by polymerase chain reaction failed to demonstrate CD4 expression in either poly(A)+ or poly(A)- RNA. 5. In conclusion, the results show that the 1.8-kb signal observed in RNA extracted from fetal or transformed human neural cells is probably due to an aspecific hybridization. However, the gene codifying for CD4 can rarely be expressed by fetal brain cells early during gestation, in still unclear circumstances.


Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I).

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.


Vasoactive intestinal polypeptide and acetylcholine coexist with neuropeptide Y, dopamine-beta-hydroxylase, tyrosine hydroxylase, substance P or calcitonin gene-related peptide in neuronal subpopulations in cranial parasympathetic ganglia of rat.

Immunohistochemistry has been used to demonstrate that neuropeptide Y, dopamine-beta-hydroxylase, calcitonin gene-related peptide or substance P are colocalized with vasoactive intestinal polypeptide and choline acetyltransferase in subpopulations of neurons in cranial parasympathetic ganglia of rat. These comprise the ciliary, sphenopalatine, otic, glossopharyngeal-vagal and internal carotid ganglia. In the ciliary and glossopharyngeal-vagal ganglia tyrosine hydroxylase is also found in such neurons. The findings emphasize that the combined localization of dopamine-beta-hydroxylase and neuropeptide Y or the presence of tyrosine hydroxylase is not exclusively a marker for peripheral adrenergic neurons. Further, the co-localization of calcitonin gene-related peptide and substance P is not a decisive indication that a neuron is sensory in nature. It is discussed whether the presence of the enzymes and peptides other than vasoactive intestinal polypeptide is a remnant of a different expression during ontogenesis or indicates target-specific functions in the adult.


Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986.

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.


Differential ontogeny of functional dopamine and muscarinic receptors mediating presynaptic inhibition of neurotransmitter release and postsynaptic regulation of adenylate cyclase activity in rat striatum.

To study the ontogeny of functional striatal dopamine (DA) D2 and muscarinic receptors we determined the first appearance of the inhibitory effects of activation of autoreceptors on neurotransmitter release and that of postsynaptic receptors on adenylate cyclase activity in striatal slices of rat foetuses and pups. On embryonic day 17 (E17), activation of D2 receptors with LY 171555 (1 microM) resulted in a 50% inhibition of the electrically evoked release of [3H]DA from superfused striata, indicating that D2 autoreceptors are functional at that time. Stimulation of adenylate cyclase activity with the Da D1 agonist SK&F 38393 could also be determined in the striatum on E17. In contrast, inhibition of D1-stimulated adenylate cyclase activity through activation of postsynaptic D2 receptors did not occur until postnatal day 14 (P14), whereas activation of postsynaptic muscarinic receptors with oxotremorine (1 microM) resulted in 30% inhibition already on E17. Endogenous activation of muscarinic receptors with physostigmine (1 microM) was ineffective in the prenatal period, but its inhibitory effect on D1-stimulated adenylate cyclase increased strongly between P7 and P21. Inhibition of striatal [3H]acetylcholine (ACh) release by activation of muscarinic receptors could not be determined until P7, because the release of the neurotransmitter was not measurable before that day. But on P7, oxotremorine and physostigmine (as well as the D2 receptor agonist LY 171555) reduced the electrically evoked release of [3H]ACh from striatal slices. Taken together, these data show that there is a marked time difference between the coupling of D2 receptors and that of the D1 and muscarinic receptors to adenylate cyclase in the developing striatum.(ABSTRACT TRUNCATED AT 250 WORDS)


The relationship of HIV-1 viral sequences detected by the polymerase chain reaction in haemophilic patients to clinical and other markers of infection.

A nested primer polymerase chain reaction (PCR) of pol gene sequences of human immunodeficiency virus-1 (HIV-1) was applied to whole blood of 31 haemophiliacs who were, or had been, positive for HIV p24 antibody (HIVAb) by enzyme linked immunosorbent assay (ELISA) and samples from 22 persistently HIVAb negative haemophiliacs who had been at risk of contracting HIV from treatment. The results were compared with those of p24 HIV antigen determination, T4 cell counts beta 2 Microglobulin (beta 2M) levels and clinical evidence of progression of HIV disease. There was no discrepancy between the PCR results and past or present seropositivity for HIVAb. The qualitative PCR was more sensitive than the p24 antigen assay but the presence of the latter was predictive of progression of infection as determined clinically and by falling T4 cell counts and rising levels of beta 2M. The results of the PCR are reassuring for HIVAb negative haemophiliacs at risk from treatment and to HIVAb negative sexual contacts of HIVAb positive persons.


The role of prostaglandin H synthase in 3-methylindole-induced pneumotoxicity in goat.

1. Aspirin and indomethacin were used to investigate the role of prostaglandin H synthase (PHS) system in 3-methylindole (3MI)-induced pneumotoxicity. 2. A functional test was developed to detect the inhibitory effect of oral doses of aspirin and indomethacin on PHS activity based on thromboxane B2 (TXB2) production from thrombin-stimulated platelets in whole blood. 3. Goats which received oral doses of aspirin or indomethacin before administration of 3MI (0.1 g 3MI/kg body wt) showed a reduced severity in clinical signs and pathological lesions in the lung than those that received 3MI alone. 4. There was no difference in the severity of the disease between the control and the aspirin-treated animals if aspirin was given after 3MI administration. 5. The protective effect of inhibitors when administered before, but not after, 3MI dosing suggests it is the inhibition of PHS activity in activation of 3MI, not in production of prostanoids which prevented the disease process.


Response of Xenopus laevis to cadmium administration.

1. Levels of copper, zinc and cadmium have been determined in the plasma, liver and kidneys of Xenopus laevis. 2. Administration of cadmium by subcutaneous injection induces metallothionein synthesis in the liver of Xenopus laevis. Cadmium is found to accumulate in both liver and kidneys following injection. 3. Cadmium uptake, following injection, is biphasic, suggesting a saturable first-phase uptake mechanism, followed by a linear second phase. 4. Zinc is displaced from the liver by administration of cadmium in a dose-dependent manner.


Xenobiotic-metabolizing enzyme systems in test fish--IV. Comparative studies of liver microsomal and cytosolic hydrolases.

1. The initial slopes of the substrate-activity curves of several hydrolases were determined in the microsomal and cytosolic fractions of the liver of several fish recommended by OECD for the regulatory testing of chemicals. 2. Inter-species differences ranged within a factor of 7-17 for the esterases and reached a factor of 60 for the amidase. Guppy and carp appeared endowed with hydrolase activities which, overall, are much higher than zebra fish, trout and golden orfe. 3. The comparison with the rat liver microsomal hydrolases strongly suggests that fish are endowed with similar or higher levels of A-esterase and with much less B-esterase/amidase activities.


Effect of diethyl maleate on glutathione, hepatic and renal cortical perfusion, and portal 6-ketoPGF1 alpha and TxB2 levels in swine.

1. Effects of diethyl maleate (DEM) mediated glutathione (GSH) depletion on hepatic and renal cortical blood flow (perfusion), plasma GSH, and portal prostacyclin (6-ketoPGF1 alpha) and thromboxane (TxB2) were determined in anaesthetized swine. 2. Although DEM depleted hepatic GSH to 25% of control, plasma GSH increased 10-fold in comparison to controls. DEM caused a drop in blood pressure and renal cortical perfusion but had no effect on hepatic perfusion or portal 6-ketoPGF1 alpha or TxB2 levels. 3. Possibly, the unexpected rise in plasma GSH may have inhibited prostanoid synthesis, preventing any alterations in tissue perfusion that may have occurred following tissue GSH depletion.


Properties of cyclic AMP-dependent inward current in two identified neurons of the snail Lymnaea stagnalis.

1. Intracellular iontophoresis of cyclic AMP induces a slow, inward sodium current in identified neurons B1 and RPD1 of Lymnaea stagnalis, which is TTX-insensitive and voltage independent. 2. High extracellular Ca2+ or Co2+ ions cause a reduction in the amplitude of the current, whereas low Ca2+ enhances it. 3. Agents known to increase intracellular cyclic AMP lead to depolarization and increased activity in B1, but RPD1 is unaffected. 4. The properties of cyclic AMP-stimulated inward current in neurons of L. stagnalis are compared with those of similar currents in other gastropod species.


Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea).

1. The effects of temperature on the anesthetic potencies of halothane, enflurane and ethanol have been studied in the water flea Daphnia magna. 2. In the absence of anaesthetics, decreasing temperature resulted in decreased activity by the daphnids. 3. Potencies in the gas phase decreased with increasing temperature for all of the anaesthetics, while aqueous potency decreased for halothane and enflurane but increased for ethanol. 4. Enthalpy calculations suggest that the observed potency changes for the inhalational anaesthetics cannot be accounted for in terms of changing solubility in lipid bilayers but most likely reflect more specific interactions with animal target sites.


Effect of cyclosporine treatment on carnitine and myo-inositol in diabetic rats.

1. The effect of long-term (20 wk) treatment of cyclosporine A (CyA) was studied in urine, blood, liver, kidney and pancreatic concentrations of acid-soluble carnitine and free myo-inositol in streptozotocin diabetic rats. 2. Diabetic rats excreted significantly higher concentrations of carnitine and myo-inositol; CyA prevented the urinary loss of carnitine but not myo-inositol. 3. Blood carnitine levels were not different between normal and diabetic rats, however, CyA significantly decreased these levels. Conversely, blood myo-inositol concentrations were higher in diabetic than in normal rats; CyA prevented this increase. 4. Hepatic concentrations of both carnitine and myo-inositol were increased in diabetic rats; CyA treatment caused even further increase. 5. Pancreas from diabetic rats contained less carnitine and myo-inositol compared to normal pancreas. CyA treatment did not affect pancreatic carnitine, but it normalized myo-inositol in diabetic rats. 6. The kidney carnitine or myo-inositol levels were not influenced either by diabetes or by CyA treatment. 7. These results suggest that CyA treatment causes changes in carnitine and myo-inositol concentrations in biologic fluids and certain tissues.


Seasonal changes in subcellular distribution of zinc, copper, cadmium and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible essential role of cadmium and metallothionein in the hepatic metabolism of copper.

1. An increase in total Zn concentration in the liver of sexually mature bank voles in spring and summer was primarily accompanied by an increase in Zn levels in the nuclear and mitochondrial-lysosomal fractions, while in immature voles in an analogous situation, Zn tended to concentrate mainly in the post-mitochondrial fraction. 2. Seasonal changes in Cu concentration in all subcellular fractions followed closely those of total hepatic Cu; however, the mitochondrial-lysosomal Cu underwent the most dramatic changes. 3. Multiple regression analysis between the concentration of metallothionein (MT) in the postmitochondrial fraction and the levels of Zn, Cu and Cd in the particular subcellular fractions showed that MT was principally induced by small amounts of Cd concentration of which in the liver ranged from 0.019 microgram/g in winter to 1.1 micrograms/g wet wt in spring. 4. The analysis revealed further that the level of MT in the post-mitochondrial fraction concurrently decreased as total hepatic Cu, as well as mitochondrial-lysosomal Cu, increased. 5. The data indicate that MT, first induced by small amounts of Cd, is involved in the hepatic metabolism of Cu in bank voles; most probably the protein sequesters free Cu ions in cytoplasm and then transfer them into the mitochondrial-lysosomal fraction.


Helix aspersa neurons maintain vigorous electrical activity when co-cultured with intact H. aspersa ganglia.

1. Identified neurons from the right parietal lobe of the circumoesophageal ganglion of adult land snails, Helix aspersa, were isolated and placed in primary cell culture. 2. The individual neurons were removed from the right parietal lobe by microdissection without the aid of exogenous enzymes and plated on poly-L-lysine coated coverslip in normal Helix Ringer's solution conditioned with Helix circumoesphageal ganglia. The neurons become firmly attached to the substrate and begin to extend processes within 6 hr. 3. The cells show a dramatic increase in electrical activity when they are co-cultured with intact Helix circumoesophageal ganglia (1 ganglion/ml). Co-cultured neurons have resting potentials between -45 and -110 mV, comparable to in situ, show spontaneous action potential firing and respond to putative neurotransmitters. Heat-treated conditioned media was inactive. 4. When dopamine or serotonin (5-HT) are added, right parietal beating neurons, F14-F16 and F29-F32, (i) show a suppression of action potentials occurrence; (ii) a decrease in action potential amplitude and duration, and (iii) an increase in after-hyperpolarization. 5. Right parietal bursting neuron F1 in culture fire action potentials only in a beating mode. Dopamine addition to F1 suppresses action potential occurrence and causes an increase in action potential after-hyperpolarization, but there is only a small decrease in duration of action potentials and no significant change in action potential amplitude. 5-HT addition to F1 increases the occurrence of action potentials with little or no change in action potential shape. 6. This primary cell culture method is an efficient system for doing biochemical and electrophysiological studies on individual, identified neurons.


Effects of adenine nucleosides and nucleotides on the isolated heart of the snail Helix aspersa and the slug Arion ater.

1. Adenine nucleosides and nucleotides were examined for pharmacological activity in hearts isolated from the snail Helix aspersa and the slug Arion ater. 2. Adenosine, AMP, ADP and ATP (above 100 microM) produced either an excitation or an inhibition in the isolated hearts of the snail and slug. 3. 2-Chloroadenosine, alpha, beta-methylene ATP and 2-methylthio ATP were inactive at concentrations up to 1 mM. 4. Responses were not blocked by any commonly accepted vertebrate purinoceptor antagonists, indicating that these purinoceptors are dissimilar to vertebrate purinoceptors and cannot be classified according to accepted purinoceptor classifications. 5. Electrical field stimulation of the snail heart produced frequency-dependent responses: 1-4 Hz produced predominantly excitation, 8-32 Hz predominantly inhibition. These responses were unaffected by the purines up 3 mM.


Production of the cytostatic agent aeroplysinin by the sponge Verongia aerophoba in in vitro culture.

1. The marine sponge Verongia aerophoba contains two bioactive secondary metabolites from tyrosine, (+)-aeroplysinin-1 [3',5'-dibromo-1',2'-dihydroxy-4'- methoxycyclohexa-3',5'-dien-1'-yl-methyl-cyanide; abbreviated AP] and dibromoverongia-quinol [3',5'-dibromo-1'-hydroxy- 4'-oxocyclohexa-2',5'-dien-1'-yl-acetamide; abbreviated DV], which display strong cytostatic activity. 2. The concentrations causing 50% inhibition of cell growth are 0.47 microM (AP) and 1.21 microM (DV), resp. 3. Depending on depth regions from which the sponges were collected, differences in occurrence of metabolites were observed. 4. AP and DV were found to be present in sponges collected at a depth of 5-10 m, whereas only DV could be detected in material from deeper regions (20-30 m). 5. AP is present only in the surface layers (both the outer and oscular region) of the sponge, while in the centre of the sponge only DV is detected. 6. Cubes from sponges, collected at a depth of 30 m, were cultivated in seawater in vitro and were found to have the capacity (i) to synthesize AP, and (ii) to release this bioactive material into the medium under defined conditions. Under optimal conditions (light and aeration) 100 g of sponge synthesize and release 13.02 mg of AP during a 10-day incubation period. 7. In the dark and without aeration this synthesis was prevented. 8. These data show that also under in vitro conditions sponges retain the capability of producing bioactive compounds and can be induced to produce even substances which they did not secrete in their natural environment.


Amino acid levels in cerebrospinal fluid of rats after administration of pentylenetetrazol.

1. We studied the effect of pentylenetetrazol (PTZ)-induced myoclonic jerks and generalized clonic-tonic convulsions (GC) on the levels of neurotransmitter amino acids in the cisternal CSF of rats. 2. The levels of aspartate, glutamate, glycine, and taurine were elevated in the CSF during myoclonic jerks and more distinctly immediately after GC. 3. During the recovery period of postictal depression seen in EEG (5 min after GC), the CSF levels of transmitter amino acids were lower than in the control group. 4. PTZ-induced irritative activity in the EEG disappeared in 24 hr but the levels of amino acids remained abnormal. 5. Amino acid changes in the CSF following PTZ-induced convulsions might indicate that the release of amino acids into the extracellular space is increased before and during the propagation of PTZ-induced seizure and decreased during postictal depression.


Two DNA polymerases from Trypanosoma cruzi: biochemical characterization and effects of inhibitors.

1. Two DNA polymerases have been partially purified from Trypanosoma cruzi epimastigotes by DEAE-cellulose, phosphocellulose, and DNA agarose chromatography. 2. Both enzyme activities were characterized by several biochemical criteria. 3. They showed different sensitivity to KCl and displayed characteristic Mg2+ and Mn2+ requirements, although they exhibited almost identical primer-template utilization. 4. The preferred substrates were poly dC-oligo dG, activated calf thymus DNA, and poly dT-oligo rA. 5. Both enzyme fractions are not inhibited by aphidicolin while N-ethylmaleimide and phosphonacetic acid inhibited them to different extents. 6. ButylphenyldGTP strongly inhibited T. cruzi enzyme fraction I while it had no effect on enzyme fraction III. 7. This dGTP analog also inhibited the poly dT-directed polymerization of dAMP as described for other mammalian DNA polymerases. Kinetic studies indicated that butylphenyldGTP inhibited enzyme fraction I in a non-competitive fashion.


Philanthotoxins block glutamatergic transmission in rat hippocampus--I. Reduction of high affinity glutamate uptake demonstrated by light microscopy surface autoradiography.

1. Effects of structural philanthotoxin (PTX) analogues on the high affinity uptake of [3H]-L-glutamate by slices of the rat hippocampus were studied using light microscopy surface autoradiography. 2. Considering the various effects of the PTX-analogues it can be concluded that the presence of an aromatic nucleus in the molecule is an important moiety, while the number and distribution of positive charges in the polyamine chain seems to be of a still not understood importancy.


Philanthotoxins block glutamatergic transmission in rat hippocampus--II. Inhibition of synaptic transmission in the CA1 region.

1. Philanthotoxins decrease the amplitude of the population spike (PS), the field excitatory postsynaptic potential (f-EPSP), and the presynaptic volley (PV), as evoked by Shaffer-collateral-commisural inputs to the CA1 pyramidal cells in the rat hippocampus slice. 2. The effects are slow and often not completely reversible. 3. Dideaza-philanthotoxin-12 is, in all experiments, the most active antagonist showing a very poor recovery. 4. Using a twin pulse the percentual decreases of f-EPSP and PV amplitudes are almost identical for the first and second response. However, the first PS is much more affected than the second one, indicating a possible effect on the inhibiting circuit. 5. Philanthotoxins cause a non-competitive inhibition. 6. Besides a possible postsynaptic block and a distinct presynaptic effect (preceding paper) a non-postsynaptic effect (on the PV) is described.


Haematological results of vanadium intoxication in Wistar rats.

1. Two-month-old rats of the Wistar strain of both sexes received, as sole drinking liquid, an aqueous solution of ammonium metavanadate (AMV) of 0.15 mg V/cm3 concentration for a period of 4 weeks. 2. A small decrease in the amount of food consumed and a significant reduction of the water AMV solution drunk were observed, as compared with the food and water taken up by the control group. 3. In the peripheral blood a significant decrease in the erythrocyte count and haemoglobin level and increased percentage of reticulocytes and polychromatophilic erythrocytes were noted. 4. No changes in the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6PD) were found in the erythrocytes of the animals tested. 5. The increase in the neutrophilic granulocyte and lymphocyte count was significant. 6. An inhibitory influence of vanadium on the phagocytic activity of granulocytes was observed.


The localization of FMRFamide in the nervous and somatic tissues of Nereis virens and its effects upon the isolated esophagus.

1. The tetrapeptide FMRFamide which is present in extracts of Nereis virens was localized in various nereid tissues immunohistochemically. 2. Immunoreactive FMRFamidergic cells and fibers were found in the supraesophageal (brain) and subesophageal ganglia, as well as in the intersegmental ganglia of the ventral nerve cord. Immunoreactive fibers were present in the neuropile of, and the connectives between, the supraesophageal, subesophageal, and intersegmental ganglia. 3. In the periphery immunoreactive FMRFamidergic fibers and a few cell bodies were observed in the gut. Sparse fibers were also seen in the body wall, parapodia, and cephalic palps. When the antiserum was preabsorbed with FMRFamide, no specific immunoreactivity was detected. 4. The esophagus of Nereis, isolated and suspended in a tissue bath, responded to FMRFamide with a dose-dependent relaxation; threshold was between 30 and 300 nM, and the EC50 was 1.55 +/- 0.60 microM. Benzoquinonium did not modify this response. 5. Thus, FMRFamide seems to be a neurotransmitter in both the central and peripheral nervous system of Nereis virens, and may be involved in the control of esophageal motility.


The utility of spirometry in the diagnosis of reversible airways obstruction.

Patients with suspected reversible airways obstruction (RAO) sometimes report subjective benefit after bronchodilator treatment despite no objective spirometric improvement. One possible explanation for this is improvement in volume-related or plethysmographic parameters in the absence of spirometric improvement. One hundred patients with RAO were assessed before and after inhaled bronchodilator to determine the prevalence of improvement by plethysmographic parameters in the absence of improvement in spirometric parameters. Spirometry alone (FEV1, FVC, and FEF25-75%) identified reversibility of airflow limitation in 82 patients. Reversibility was identified by body plethysmography (specific conductance [SGaw], thoracic gas volume [TGV], and isovolume maximum expiratory flow rates [IVMEF]) in 15 of the remaining patients. The percent predicted FEF25-75% at baseline was higher in patients who required plethysmography to identify reversibility, but could not be used to predict the lack of a spirometric response for any individual patient. We conclude that spirometry alone fails to identify reversibility in approximately 15 percent of patients, and that most of these patients can be identified by additional plethysmographic measurements of volume-related parameters. At any one point in time, multiple tests must be used together to adequately identify the majority of patients with reversible airways obstruction. Improvement in volume-related parameters may explain why some patients with RAO improve subjectively with bronchodilators but show no spirometric improvement.


Esmolol in the treatment of multifocal atrial tachycardia.

Multifocal atrial tachycardia (MAT) is a supraventricular tachydysrhythmia precipitated by a number of pharmacologic and physiologic disturbances. Corrections of these disturbances should take precedence in the treatment of MAT.


Ibuprofen inhibits adhesion-dependent neutrophil response by a glycoprotein-unrelated mechanism.

The anti-inflammatory drug ibuprofen was found to inhibit neutrophil aggregation and chemotaxis, triggered by the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP). The drug did not modify the surface expression of the glycoprotein CD11b-CD18, required for both aggregation and chemotaxis. Consistent with this finding, ibuprofen did not affect the release of lactoferrin from secondary granules which contain CD11b-CD18 molecules in their membranes. As the drug failed to interfere with the neutrophil oxidant production and primary granule release, the results suggest that it acts primarily at the initial steps of the neutrophil response during inflammation, i.e. the cell recruitment at inflamed tissue sites. Moreover, the data prove that adhesion-dependent neutrophil responses required cell processes independent of CD11b-CD18 but controlled, at least in part, by ibuprofen-inhibitable pathways.


Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological.

Microglia and astrocytes produce several cytokines including interleukin 1 (IL1) and tumor necrosis factor alpha (TNF alpha), which have pleiotropic effects in the immune and nervous systems. Recent evidence has come to light that they play a role in damage in the central nervous system. This indeed may be the result of overproduction of these factors as the consequence of trauma or disease. At lower concentrations, however, they may in fact be involved in the normal development of the nervous system. A review of brain IL1 and TNF alpha during normal development, abnormal development, and pathology is presented here. We have speculated as to the association of these cytokines directly and indirectly with neural cell migration, proliferation, differentiation, and death.


Postnatal dietary fat influences mRNAS involved in myelination.

The synthesis and composition of myelin in the developing mouse central nervous system can be influenced by diet. Postnatal maternal fat intake altered nursing pup brain and liver fatty acid composition. Peak (day 21) proteolipid protein (PLP) and myelin basic protein (MBP) mRNA levels were reduced when pups were nursed by mothers fed a fat-free or 5% coconut oil diet. This effect was reversed by feeding a corn oil based diet. Oleic acid accounts for about 30% of myelin fatty acids. mRNA levels of stearoyl CoA desaturase (SCD), the rate-limiting step in oleic acid synthesis, increase in neonatal mouse brain. Postnatal maternal fat-free feeding reduced day 21 pup brain SCD and LDL receptor, but not apolipoprotein (Apo E) E mRNA levels. In contrast to brain, nursing pup hepatic SCD mRNA levels were induced, LDL receptor mRNA levels were unaffected and Apo E mRNA levels were reduced by postnatal maternal fat-free feeding. Myelin-specific mRNA levels are developmentally regulated and influenced by dietary fat. Neonatal brain SCD and LDL receptor mRNA levels are also altered by neonatal fat intake. The neonatal response to dietary fat is tissue-specific at the mRNA level.


Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.


Maternal tyrosinaemia II: management and successful outcome.

A 25-year-old woman with tyrosinaemia type II was treated from the 5th week of pregnancy with a protein-restricted diet supplemented with a tyrosine/phenylalanine-free amino acid mixture. Tyrosine concentrations were maintained in the range 100-200 mumol/l by restricting natural protein intake to 0.16 g/kg per 24 h in early pregnancy, with increases up to 0.38 g/kg per 24 h in the last trimester. This treatment maintained plasma phenylalanine concentrations in the range 20-40 mumol/l. Maternal weight gain and fetal growth were normal, and the mother remained asymptomatic throughout the pregnancy. A normal infant was born at term with length, weight and head circumference between the 25-50th per centiles.


Induction of high-affinity paf receptor expression during T cell activation.

Activated human T cells via the CD2 or the CD3 pathways exhibited a higher capacity than resting T lymphocytes to incorporate and metabolize [3H]pafacether (paf) at 37 degrees C. Resting T lymphocytes lacked specific binding capacity for paf, yet high-affinity paf receptors (paf-R) were induced on CD3- or CD2-dependent activation. This up-regulation in the number of paf-R became apparent by day 1 of culture, reached a maximum of about 25,000 sites cell by days 4 to 6 and subsequently declined. Interestingly, human recombinant interleukin-2 in a dose-dependent manner prevented the decrease of high-affinity paf-R expression on T cells. By contrast, the receptor affinity was constant throughout the culture period. Thus, paf-R at different stages of T cell activation were indistinguishable with respect to receptor-ligand interaction, and differed only in their number. Together, these data demonstrate that after activation human T cells develop membrane high-affinity paf-binding sites. They also suggest for the first time that expression of the paf-R are coupled to T cell activation and/or differentiation.


Engagement of class I major histocompatibility complex molecules by cell surface CD8 delivers an activation signal.

Recent evidence has demonstrated that cross-linking class I major histocompatibility complex (MHC) molecules on human T cells with monoclonal antibodies (mAb) triggers T cell activation. The only known natural ligand for MHC class I molecules is CD8. Therefore, the possibility that CD8+ T cells might provide activation signals to other T cells by engaging MHC class I molecules was examined by culturing CD4+ peripheral blood T cells with Chinese hamster ovary cells (CHO) cells that had been transfected with the alpha chain or alpha and beta chains of CD8 and assessing interleukin (IL)-2 production. CD4+ T cells did not secrete IL-2 when cultured alone, with control or CD8+ CHO cells. In contrast, CD4+ T cells produced IL-2 when cultured with CD8+ CHO cells and co-stimulated with phorbol myristate acetate (PMA) or mAb to CD3 or CD28. PMA stimulated substantially less IL-2 when control CHO cells were employed and the mAb to CD3 and CD28 did not stimulate IL-2 production in the presence of control CHO cells. The co-stimulatory activity of CD8+ CHO cells was completely eliminated by mAb to CD8 or MHC class I molecules. The data demonstrate that CD8 can interact with MHC class I molecules expressed on T cells and deliver a costimulatory signal that increases IL-2 production. Thus, engagement of MHC class I molecules by its natural ligand, CD8, provides an activation signal to T cells. Under some circumstances, such interactions may amplify the responses of T cells.


Changes in expression of CD45R during the development of Th1 and Th2 cell lines.

Previous studies using anti-CD45R monoclonal antibodies (mAb) have shown that normal CD4+ T cells can be separated into virgin and memory cells based on their level of expression of CD45R. CD45Rhi (virgin) T cells secrete interleukin (IL)-2 whereas CD45Rlo (memory) T cells secrete both IL-2 and IL-4. In contrast to these results, studies using cultured T cell lines have shown that IL-2-secreting T helper cell type 1 (Th1) cells are CD45Rlo and IL-4-secreting Th2 cells are CD45Rhi. To resolve this discrepancy, and to determine how the Th1 and Th2 cell lines relate to memory CD45Rlo and virgin CD45Rhi CD4+ T cells, we have isolated CD45Rlo cells from alloantigen-primed mice and developed allospecific Th1 and Th2 cell lines. The lymphokines secreted by these lines were then evaluated. Our results show that as CD45Rlo T cells develop into in vitro cell lines which secrete IL-4, they become CD45Rhi. In contrast, CD45Rlo cells that develop into lines which secrete IL-2 maintain their CD45Rlo phenotype. Thus, although both Th1 and Th2 cells arise initially from CD45Rlo cells, Th2 cells become CD45Rhi during prolonged in vitro culture but, Th1 cells do not.


Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects.

The cytokine secretion profiles of T cell lines (TCL) specific for purified protein derivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate (PMA) plus anti-CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)-2 and interferon (IFN)-gamma. The mean IL-2 production by PPD- or SK-specific TCL from both atopics and nonatopics was similar, whereas the mean IFN-gamma production by TCL derived from atopics was significantly lower. In addition, both PPD- and SK-specific TCL from atopics produced detectable amounts of IL-4 and IL-5, whereas the corresponding TCL derived from nonatopics did not. A total number of 107 and 99 PPD-specific CD4+ T cell clones (TCC) were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti-CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD-specific TCC from both atopic and nonatopic individuals produced IL-2 and IFN-gamma. In contrast, the great majority of PPD-specific TCC derived from nonatopic individuals did not produce IL-4 and IL-5, whereas high proportions of PPD-specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL-4 and IL-5 besides IL-2 and IFN-gamma. These data indicate that CD4+ T cells from atopic individuals are able to produce IL-4 and IL-5 in response to bacterial antigens, such as PPD and SK, that usually evoke responses with a restricted type-1 T helper (Th1)-like cytokine profile in nonatopic individuals. Aberrant IL-4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.


Sensitivity of T cells to anti-CD3-stimulated suicide is independent of functional phenotype.

We have examined mixed populations and cloned murine CD4+ T cell lines for their sensitivity to anti-CD3-stimulated suicide. The sensitivity of CD4+ clones to anti-CD3-stimulated suicide is independent of their interleukin-2/interleukin-4 or interferon-gamma secretion profile or their lytic efficiency in anti-CD3 redirected lytic assays.


Effects on ingestive behavior in rats of the alpha 1-adrenoceptor agonist cirazoline.

Microinjections of various alpha 1-adrenoceptor agonists including phenylephrine and phenylpropanolamine into the paravenricular hypothalamic nucleus (PVN) suppress food intake in rats, suggesting that this receptor type might act in opposition to previously identified facilatory PVN alpha 2-adrenoceptors in the modulation of feeding. In the present experiments, we examine the effects on food and water intake of intra-PVN as well as systemic injection of cirazoline, a highly potent alpha 1-adrenoceptor agonist. In Experiment 1, intra-PVN microinjection of cirazoline (0, 3, 6, 12 and 24 nmol) suppressed food intake (ED50 = 23.4 nmol) without significant effects on water intake. In Experiment 2, systemic injection of cirazoline (0, 0.05, 0.1, 0.2, 0.4 mg/kg) also markedly suppressed food intake (ED50 = 0.05 mg/kg i.p.), with a less potent action on water intake (ED50 = 0.22 mg/kg i.p.). The results of this study as well as our previous investigations strongly support the notion that alpha 1-adrenoceptors within rat PVN act to reliably suppress food intake.


B-HT 920 stimulates feeding and antagonizes anorexia induced by ACTH and immobilisation.

The influence of immobilisation and i.c.v. injection of ACTH on feeding in rats was examined using a new experimental model. An X-maze with alternate open and covered arms, each baited with standard laboratory chow was used, where individual rats were placed and observed for 5 min. Two essential aspects of the behaviour towards food were considered, namely, tasting and feeding. A number of parameters were applied to demonstrate the anorectic activity of ACTH and immobilisation, in accordance with data obtained using classical procedures for feeding analysis. ACTH at the dose used did not modify rat exploratory activity and grooming in the X-maze without food pellets. In the same X-maze feeding test, B-HT 920, a selective agonist of dopamine D2 receptors and alpha 2-adrenoceptors, shown earlier to have anxiolytic- and antidepressive-like properties in rats, enhanced appetite and exerted anxiolytic activity when injected i.p. Pretreatment with B-HT 920 counteracted the restraint- and ACTH-induced effects. The results are discussed in the light of the relation between control of feeding and affective disorders. B-HT 920 activity seems to be of particular interest in view of its antagonism towards the anorexia elicited by two different agents reputed to have in common a key role in the stress-related disturbances of food intake.


Spontaneous firing level distinguishes the effects of NMDA and non-NMDA receptor antagonists on the ganglion cells in the cat retina.

Different responses of retinal ganglion cells to iontophoretically applied NMDA receptor antagonists and non-NMDA receptor antagonists were studied in anaesthetized cats. Cells with normal range of spontaneous firing and those with abnormally high spontaneous firing levels showed a different response to these drugs. Both visually driven and spontaneous firing of cells with 'normal' spontaneous firing level were blocked by non-NMDA receptor antagonists, but not by NMDA receptor antagonists which often raised spontaneous firing. In contrast, the responses of cells with abnormally high spontaneous firing level were blocked effectively by NMDA antagonists including MK-801, an NMDA channel blocker, as well as by non-NMDA receptor antagonists. The results suggest that under normal physiological conditions, NMDA receptors which are not involved in synaptic transmission may play a role in reducing the resting discharge level of the retinal ganglion cells. NMDA receptors, however, appear to open ion channels in response to glutamate input when ganglion cells become abnormally depolarized.


Possible mechanisms of age-associated reduction of vascular relaxation caused by atrial natriuretic peptide.

We investigated the effect of aging on atrial natriuretic peptide (ANP)-induced relaxation and cyclic GMP (cGMP) formation in the rat thoracic aorta. In the aorta from young rats (4 weeks old), removal of the endothelium, and treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), the radical scavenger, hemoglobin (Hb), and the soluble guanylate cyclase inhibitor, methylene blue (MB), attenuated ANP-induced relaxation and considerably reduced ANP-stimulated cGMP formation. With increasing age of the rats, the ANP-induced relaxation and cGMP formation in endothelium-intact aorta decreased, and Hb, L-NAME and MB no longer inhibited the ANP-induced effects, irrespective of whether the endothelium was present or absent. In the arteries without endothelium, the age-associated reduction in ANP-induced relaxation was less than in arteries with endothelium. Aging also decreased the relaxation induced by the soluble guanylate cyclase activator, nitroprusside. Potentiation due to the cGMP-phosphodiesterase (cGMP-PDE) inhibitor, M&B 22948, of the ANP-induced relaxation was greater in aortas from old rats than in those from young rats, suggesting that the degradation of cGMP may be accelerated in old rats. These results suggest that the relaxant action of ANP on the thoracic aorta from young rats is in part modulated by endothelium-derived relaxing factor (EDRF/nitric oxide), which in turn activates soluble guanylate cyclase, thus elevating the cGMP level. Aging may decrease the ANP-induced relaxation and ANP-stimulated increase in cGMP level by decreasing the ability of endothelial cells to produce EDRF, by decreasing guanylate cyclase activity, and by enhancing cGMP-PDE activity.


In vivo labelling of the neuronal dopamine uptake complex in the mouse striatum by [3H]GBR 12783.

Various characteristics of the in vivo striatal binding of [3H]GBR 12783 (1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenyl-1[3H]-2-propenyl)pipera zine), a specific ligand of the neuronal dopamine uptake complex, were determined in mice. Increasing doses of the ligand revealed the saturability of the binding at a single site with half-maximal saturation at a dose of approximately 7 mumol/kg and an apparent maximal number of binding sites (Bmax) of 12.8 pmol/mg protein in striatum. Specific binding was prevented by various dopamine uptake blockers, pyrovalerone, GBR 13069, GBR 12783, N-[1-2-benzo(b)thiophenyl)cyclohexyl] piperidine, cocaine, methylphenidate and was inhibited in a stereoselective manner by the enantiomers of nomifensine. Other drugs which are not dopamine uptake blockers either did not modify [3H]GBR 12783 binding (the diphenylbutylpiperazine derivative flupenthixol) or increased it (the diphenylpiperazine derivative flunarizine or the chemically unrelated compounds fenfluramine and SKF 525A). A close correlation was found between occupancy of the striatal [3H]GBR 12783 binding site and the stimulant locomotor effect of the drug. A similar specific striatal binding of [3H]GBR 12783 was evidenced in both NMRI and CD1 strains. It was concluded that [3H]GBR 12783 administered in vivo provides a measure of the density of dopamine uptake sites in mouse striatum.


Effects of gentamicin on catecholamine levels in the striatum, hypothalamus, adrenal gland and vas deferens.

The effects of gentamicin, an aminoglycoside antibiotic, on changes in the catecholamine levels in the rat striatum, hypothalamus, adrenal medulla and vas deferens were studied. When rats were i.v. injected with gentamicin (1.0 mg/kg), the catecholamine content of all tissues increased 2-4 h after injection. The increases in catecholamine levels induced by gentamicin were about 1.4- to 2.3-fold those induced by saline. The effect of gentamicin was observed at 0.1 or 0.5 mg/kg, and was maximal at 2.0 mg/kg. The activity of tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis, was markedly increased in all four tissues of rats treated with gentamicin (1.0 mg/kg, 4 h). However, direct addition of gentamicin to the tyrosine hydroxylase assay medium did not affect tyrosine hydroxylase activity. These data indicate that gentamicin administration to rats increases the catecholamine content of both central and peripheral catecholamine-containing tissues. The results also suggest that the effect of gentamicin is due to an indirect activation of tyrosine hydroxylase.


A thromboxane A2 synthetase inhibitor retards hypertensive rat diabetic nephropathy.

Spontaneously hypertensive rats (SHR) were injected with streptozotocin (STZ-SHR) to induce diabetes. The effect of DP-1904, a thromboxane A2 synthetase inhibitor, on diabetic nephropathy was then studied by administering it for 5 months (1 or 10 mg/kg). DP-1904 did not affect renal 6-keto prostaglandin (PG)F1 alpha production in STZ-SHR, but markedly inhibited renal thromboxane (TX) B2 production, so that the 6-keto PGF1 alpha/TXB2 ratio was significantly increased (P less than 0.05). STZ-SHR showed significant uraemia and proteinuria, plus increases in urinary gamma-glutamyl-transpeptidase and urinary N-acetyl-beta-glucosaminidase. DP-1904 significantly decreased (P less than 0.01) the urinary changes. STZ-SHR also showed an increase in mesangial periodic acid-Schiff-positive substance and in relative renal weight, both of which were significantly inhibited by DP-1904 (P less than 0.05). Thus, DP-1904 inhibited both TXB2 production and the progression of renal damage in STZ-SHR.