A cdc2 gene of Petunia hybrida is differentially expressed in leaves, protoplasts and during various cell cycle phases.

Analysis of p34cdc2 kinase in higher eukaryotes has demonstrated that p34cdc2 function is conserved in all eukaryotic cells. The p34cdc2 kinase (the product of the cdc2 gene) is required during the G1 cell cycle phase at the initiation of DNA replication and also in G2-M phases for entry into mitosis. In this paper we report the isolation and characterization of a cdc2 Petunia hybrida PCR fragment (cdc2Pet). Using a DNA probe based on this fragment and a p34cdc2-specific antibody, cdc2Pet transcript and p34 protein levels were found to be constant both in 2C nuclei of highly proliferating mesophyll 2C cells derived from protoplasts and in 2C nuclei isolated directly from expanded petunia leaves. Both the cdc2Pet transcript and p34cdc2 protein levels were found to be higher in nuclei at 4C than in those at 2C, even when these 4C nuclei were from non-proliferating tissue. Thus cdc2Pet mRNA and protein levels measured in different tissues should not be interpreted to reflect exclusively the proliferative state of the tissue but also the frequency of G2 cells including those in the differentiated state.


Ovariectomy promotes the growth of altered hepatic foci after withdrawal and reintroduction of phenobarbital during hepatocarcinogenesis in rats.

Female F344/N rats were given 70% partial hepatectomies and intubated with diethyl-nitrosamine (DEN, 10 mg/kg) 24 hours later. They were fed a cereal-based diet, NIH-07 (NIH) + 0.05% phenobarbital (PB) for 6 months, at which time NIH + PB was withdrawn and the rats were ovariectomized (OV) or sham-operated (SH). Groups of 7-10 rats were fed a semipurified diet (AIN-76) for 1 or 2 months after withdrawal of NIH + PB, or NIH + PB for 2 months, or AIN-76 diet for 1 month and subsequently NIH + PB for 1 month. Placental glutathione S-transferase (PGST)- and gamma-glutamyltransferase (GGT)-positive (+) altered hepatic foci (AHF) were analysed by quantitative stereology. Ovariectomy stimulated growth of AHF after withdrawal and reintroduction of NIH + PB. AHF, especially PGST+ AHF, continued to regress throughout the PB withdrawal period in rats fed AIN-76 diet. In most studies of chemical hepatocarcinogenesis, females have been shown to develop a greater volume of AHF than males. In our study, however, ovariectomy stimulated the growth of AHF after withdrawal and reintroduction of PB. Because AHF occurring spontaneously in male rats develop more rapidly than in female rats, the greater rate of growth of AHF in OV female rats may reflect a similar mechanism.


Isolation and characterization of equinatoxins from the sea anemone Actinia equina L.

Equinatoxins were purified from the tentacles and bodies of the sea anemone Actinia equina by the use of acetone precipitation, as well as column chromatographies on Sephadex G-50 and CM-cellulose according to the modified method of Macek and Lebez (1988). The equinatoxins obtained, equinatoxin 1 and 2, were hemolytic glycoproteins with a relative molecular mass of 20000. Equinatoxin 2 was found to be rich in glycine, alanine and valine. The amino acid sequences of equinatoxins 1 and 2 were partially determined. A portion of the N-terminal amino acid sequence of equinatoxin 2 was similar to those of pyruvate kinase and sialidase.


Quantitative determination of CGS 18102A, a new anxiolytic, in human plasma using capillary gas chromatography/mass spectrometry.

CGS 18102A is a novel hexahydrobenzopyranopyridine that has a mixed pharmacological profile of 5-HT-1A agonist and 5-HT-2 antagonist properties. Based upon these mechanisms, the compound is predicted to have anxiolytic efficacy with possible efficacy in depression. Preclinical studies in the rat have shown the drug to be well absorbed and extensively metabolized. Because of the anticipated low plasma levels in humans a gas chromatography/mass spectrometry (GC/MS) analytical method has been developed and validated to determine plasma concentrations of CGS 18102A in early clinical studies. The method utilizes CGS 18416A as the internal standard. Samples (1 mL) were extracted with pentane:ethyl acetate (75:25, v:v). Extracts were then concentrated and analysed directly by GC/MS. Separation was accomplished on a methylsilicone capillary column (30 m x 0.32 mm i.d.). GC/MS was carried out under positive ion ammonia CI conditions, with selected ion monitoring of the [M + H]+ ions (m/z = 262 and 248) for CGS 18102A and CGS 18416A, respectively. The method was successively applied to the analysis of clinical samples from an ascending multidose safety and tolerability study conducted in six normal healthy male volunteers.


Fading responses in the evoked EMG after rocuronium in cats.

This study was performed to evaluate the inhibitory effect on motor nerve terminals by rocuronium using recovery curves of muscle compound action potentials (CAPs) and train-of-four ratios (TOFRs) in anaesthetized cats, and to compare the results with other relaxants reported previously. Recovery curves were derived from the amplitude of the CAP induced in the gastrocnemius muscle by the second of a paired stimulus (test response) to the sciatic nerve and compared with results evoked by the first component (conditioning response). The interval between the paired stimuli was increased stepwise from 7 to 1,000 msec, and the differences in amplitude of the test and conditioning responses were plotted on a graph by relating the changes in paired intervals. The recovery curve after rocuronium was less inhibited than after pancuronium, (100.4 +/- 5.9%, 82.3 +/- 6.7% and 68.5 +/- 6.7% at 60, 100 and 500 msec intervals, compared with 70.3 +/- 3.3%, 59.0 +/- 4.7% and 46.7 +/- 4.3% after pancuronium (P < 0.05). The recovery curves with d-tubocurarine were more depressed than with pancuronium; however, the RC with vecuronium was similar to that of rocuronium. The degree of fade in TOF by rocuronium was also less than those seen with d-tubocurarine and pancuronium. The results obtained suggest that rocuronium has less inhibitory effect on motor nerve terminals than do d-tubocurarine and pancuronium, and has a similar effect to that of vecuronium.


Cross-resistance of drug-resistant murine P388 leukemias to taxol in vivo.

The antimicrotubule agent taxol (NSC 125973) has shown clinical antitumor activity against several classically refractory tumors. We developed a drug-resistance profile for taxol using ten drug-resistant P388 leukemias to identify potentially useful guides for patient selection for further clinical trials of taxol and possible non-cross-resistant drug combinations with taxol. Multidrug-resistant P388 leukemias exhibited either clear (leukemia resistant to amsacrine) or marginal cross-resistance (leukemias resistant to doxorubicin, actinomycin D, and mitoxantrone) to taxol. Leukemias resistant to vincristine (non-multidrug-resistant leukemia), camptothecin, melphalan, cisplatin, 1-beta-D-arabinofuranosylcytosine, and methotrexate were not cross-resistant to taxol. The data suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with amsacrine, doxorubicin, actinomycin D, or mitoxantrone and (2) a combination of one of the non-cross-resistant drugs and taxol might exhibit therapeutic synergism.


Glutamate in the inferior colliculus plays a critical role in audiogenic seizure initiation.

Alterations of excitant amino acid (EAA) action are implicated in seizure susceptibility in the genetically epilepsy-prone rat (GEPR). The inferior colliculus (IC) is critical for audiogenic seizure (AGS) initiation in the GEPR. The present study observed that bilateral microinjection into the IC of L-canaline, a glutamate synthesis inhibitor, decreased AGS severity in the GEPR and also decreased potassium-evoked release of glutamate from IC slices. Bilateral microinjection of NMDA receptor antagonists, 2-amino-7-phosphonoheptanoate (AP7) or 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) into IC blocked AGS, and an antagonist at non-NMDA EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), also blocked AGS. NMDA receptor antagonists were 5-200 times more effective than CNQX. Microinjection of a non-competitive NMDA receptor antagonist, dizocilpine (MK-801), into IC had little effect except with very high doses. Microinjection of CPP or AP7 into the IC blocked AGS at considerably lower doses as compared to pontine reticular formation (PRF). However, MK-801 attenuated AGS when microinjected into PRF at doses that were ineffective in IC. Systemically administered CPP blocked AGS and significantly reduced IC neuronal firing in the behaving GEPR, suggesting an important action of systemically administered NMDA receptor antagonists on brainstem auditory nuclei critical to AGS. The present results support a critical role for glutamate acting, in part, through NMDA receptors in IC in initiation of AGS.


GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.

GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.


A novel homo-oligomeric protein responsible for an MPF-dependent microtubule-severing activity.

An activity that severs stable microtubules has previously been detected in M phase extracts, but not in interphase extracts, of Xenopus eggs. We show that incubation of interphase extracts with purified MPF rapidly increases the microtubule-severing activity. We then report the identification and purification of a novel protein factor responsible for this MPF-dependent microtubule-severing activity. The purified microtubule-severing factor is a homo-oligomeric protein composed of 56 kDa polypeptide subunits. These subunits appear to assemble into a pentagonal loop, forming a doughnut-shaped molecule whose overall contours resemble a flattened ball. The microtubule-severing activity of the purified factor does not require ATP or divalent cations, and is inhibited by monomeric tubulin. The purified factor is capable of binding to both monomeric tubulin and microtubules. This factor is thus a novel kind of microtubule-binding protein in both structure and function, and may play an important role in the cell cycle-dependent change in microtubule organization.


Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili.


Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.


Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.


Regulation of the mec-3 gene by the C.elegans homeoproteins UNC-86 and MEC-3.

The mec-3 gene encodes a homeodomain protein with LIM repeats that is required for the specification of touch cell fate in Caenorhabditis elegans. Previous experiments suggested that mec-3 expression requires the product of the unc-86 gene, a POU-type homeoprotein, and mec-3 itself. We have analyzed the control of mec-3 expression by identifying potential cis regulatory elements in the mec-3 gene (by conservation in a related nematode and by DNase I footprinting using unc-86 and mec-3 proteins) and testing their importance by transforming C.elegans with mec-3lacZ fusions in which these sites have been mutagenized in vitro. Both unc-86 and mec-3 proteins bind specifically to the promoter of the mec-3 gene, suggesting that both proteins may be directly involved in the regulation of the mec-3 gene. In addition, the footprint pattern with mec-3 protein is altered in the presence of unc-86 protein. In vivo transformation experiments reveal that some of the binding regions of the two proteins are needed for general positive control and maintenance of mec-3 expression while others have no detectable, unique function. Interestingly, the unc-86 gene appears to be required not only to initiate mec-3 expression but also to maintain it.


The DNA binding specificity of the bipartite POU domain and its subdomains.

The POU domain is a conserved DNA binding region of approximately 160 amino acids present in a family of eukaryotic transcription factors that play regulatory roles in development. The POU domain consists of two subdomains, the POU-specific (POUS) domain and a POU-type homeodomain (POUHD). We show here that, like the POUHD, the Oct-1 POUS domain can bind autonomously to DNA but with low affinity. DNA binding studies and in vitro binding site selection revealed that the POU subdomains each have a different sequence specificity. The binding consensus of the POUS domain [gAATAT(G/T)CA] and POUHD (RTAATNA) respectively overlap the 'left half' and right half' of the POU domain recognition sequence [a(a/t)TATGC(A/T) AAT(t/a)t]. In addition to the core sequence, which is very similar to the octamer motif (ATGCAAAT), the flanking bases make a significant contribution to the binding affinity of the POU domain. Interestingly, at some positions the sequence preferences of the isolated POU subdomains are distinct from those of the POU domain, suggesting that the POU domain binding site is more than a simple juxtaposition of the POUS and POUHD target sequences. In addition, analysis of the binding kinetics of the POU domain and POUHD indicates that the POUS domain enhances the binding affinity by reducing the dissociation rate. Our results show that the POU domain proteins have DNA binding properties distinct from those of classic homeodomain proteins. We suggest a model for the way in which an additional conserved domain adds further specificity to DNA recognition by homeodomain proteins.


Electrophysiological and electropharmacological studies in pre-excitation syndromes: results with propafenone therapy and isoproterenol infusion testing.

To study the electrophysiological effects of oral propafenone on accessory pathways and determine the potential for catecholamine-mediated reversal of these effects, comprehensive electrophysiology studies (EPS) were conducted in 11 patients with manifest (n = 9) or concealed (n = 2) pre-excitation syndrome. EPS were performed at baseline (in the drug-free state), after oral propafenone loading, and with isoproterenol infusion during propafenone therapy. The study group included 10 men and 1 woman with a mean age of 39 +/- 13 years, who presented with symptoms of palpitations (n = 6), presyncope (n = 3) and syncope (n = 2). The clinical arrhythmia was atrioventricular reciprocating tachycardia (n = 6), atrial flutter/fibrillation (n = 3), or both (n = 2). During the baseline EPS the accessory pathway location was identified as left (n = 6) or septal (n = 5). The mean anterograde effective refractory period was 265 +/- 42 ms, the shortest pre-excited RR interval 259 +/- 20 ms and the retrograde refractory period 258 +/- 39 ms. Orthodromic atrioventricular reciprocating tachycardia was induced in 10 patients (mean cycle length = 324 +/- 31 ms). Antidromic reciprocating tachycardia was induced in one patient (cycle length = 340 ms). In all the 11 patients EPS were repeated after 4 days of oral propafenone loading (668 +/- 226 mg daily) when drug steady state was expected to have been achieved. One additional patient had baseline EPS but developed clinical arrhythmia recurrences after propafenone loading and thus he was excluded from the study; follow-up EPS were conducted on procainamide.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA.

Five monoclonal antibodies raised against rat PCNA cross-reacted with a similar protein in the fission yeast Schizosaccharomyces pombe. One of these was used to screen an S.pombe cDNA expression library. An incomplete cDNA was isolated and used to screen a genomic library, identifying a single gene, designated pcn1+ (proliferating cell nuclear antigen). The gene encodes a protein of 260 amino acids, with a deduced sequence 52% identical to human and rat PCNAs, which are 98.5% identical to each other. The budding yeast PCNA homologue POL30 is only 35% identical to the human and rat proteins. Pcn1 has a region near the C-terminus of particularly high homology to higher eukaryotic PCNA proteins. pcn1+ is essential for viability and delta pcn1 cells undergo aberrant DNA replication before cell cycle arrest. Overproduction of the protein leads to cell cycle delay in G2. Disruption of pcn1+ is complemented by the human PCNA gene, demonstrating that these genes are functional homologues.


Differentiation of beta-blocker effects on serum lipids and apolipoproteins in hypertensive patients with normolipidaemic or dyslipidaemic profiles.

To evaluate the differential effects of beta-blockers on serum lipids and apolipoproteins in normolipidaemic and dyslipidaemic hypertensives, 330 patients with mild to moderate essential hypertension were studied 1 month after placebo therapy and 6 months after monotherapy with propranolol (n = 53), atenolol (n = 66), metoprolol (n = 58), pindolol (n = 53), or celiprolol (n = 100). Serum total cholesterol, triglycerides, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and apolipoproteins (Apo) A1 and B were measured at baseline and study end. A total of 136 (41.2%) patients were considered normolipidaemic (pretreatment LDL-C < 160 mg.dl-1) and 194 (58.8%) were considered dyslipidaemic (LDL-C > 160 mg.dl-1). Changes in total cholesterol differed between normolipidaemics and dyslipidaemics with propranolol (+13% in normolipidaemics vs -0.5% in dyslipidaemics, P < 0.001), atenolol (+7% vs -2%, P = 0.01), metoprolol (+9% vs -4%, P0.0006), pindolol (+8% vs -9%, P < 0.001), and celiprolol (-1% vs -13%, P = 0.002). HDL-C differed less, with propranolol (-18% vs -13%), atenolol (-6% vs -2%), metoprolol (-2% vs -6%), pindolol (+4% vs +1%), and celiprolol (+9% vs +4%); none of these changes between normolipidaemic and dyslipidaemic patients were statistically significant. LDL-C changes differed the most, with propranolol (+35% vs -1%, P < 0.0001), atenolol (+15% vs -4%, P = 0.001), metoprolol (+12% vs -6%, P = 0.004), pindolol (+12% vs -13%, P < 0.0001), and celiprolol (+3% vs -16%, P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)


[A novel function of tetrahydrobiopterin].

6R-L-erythro-5, 6, 7, 8-Tetrahydrobiopterin (6R-BH4) is known as a cofactor for the hydroxylases of phenylalanine, tyrosine and tryptophan and also as a cofactor for nitric oxide synthase. Recently, a novel function of 6R-BH4 has been found: that is, 6R-BH4 acts on specific membrane receptors to directly stimulate the release of monamine neurotransmitters such as dopamine and serotonin, independently of its cofactor activity. In addition, it indirectly stimulates the release of non-monoamine neurotransmitters such as acetylcholine and glutamate, through activation of monoaminergic systems. In this paper, we briefly review recent experimental data, which provide new insights into the role of 6R-BH4 as a regulator of neuronal function. We also discuss the possibility of treatment by 6R-BH4 of neuropsychiatric diseases such as Parkinson's disease, Alzheimer's disease, depression and infantile autism.


[Effects of M6434, an orally active alpha 1-adrenoceptor agonist, on experimental postural hypotension in rabbits and dogs].

We examined the effects of M6434 on mean blood pressure and heart rate in conscious rabbits and dogs and on experimental models of postural hypotension in conscious rabbits and anesthetized dogs. M6434, given orally, elevated the mean blood pressure in conscious rabbits and dogs. The pressor effect of M6434 was more potent than that of midodrine, but the bradycardiac action of M6434 was weaker than that of midodrine. M6434 (1.0 and 3.0 mg/kg, p.o.) prevented the head-up tilt-induced reductions of mean blood pressure and cerebral tissue blood flow in conscious rabbits, and these effects of M6434 were about 3 times more potent than those of midodrine. In the postural hypotension of anesthetized dogs, M6434 at the doses more than 10 micrograms/kg, i.v. also produced the preventive effects on mean blood pressure and cerebral tissue blood flow. These effects of M6434 were about 10-30 times more potent than those of midodrine. These results show that M6434 possesses a potent hypertensive effect with a weaker bradycardiac action and suggest that M6434 may be a potential candidate for an anti-hypotensive agent that can prevent the deterioration of hemodynamics in postural hypotension.


Comparative biochemical, morphological, and immunocytochemical studies between C-6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres.

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.


[The electromotor unit of the upper urinary tract].

The muscular architecture of the upper urinary tract constitutes a functional but no anatomical syncytium, where the cells are coupled by nexus and crosslinking with low electrical resistance. The action potentials are evoked by spontaneous depolarisation of muscle cells. The sites of peak depolarisation could be termed pace-maker cells. They are localized in the calices. Recent studies have, however, described myofilaments capable of contraction in the proximal tubuli already. Volume and pressure loads on the hollow system have been identified as the regulation mechanism. Modulatory function is directly related to evidence of alpha- and beta-adrenergic receptors, of receptor subtypes, of the adenylate cyclase system, of histamine- and possibly prostaglandin- and serotonin-receptors as well.


Intense sound increases the level of an unidentified amine found in perilymph.

The hypothesis tested was that intense sound increases the levels of a substance such as glutamate, a putative neurotransmitter and neurotoxic substance, in the perilymph compartment of the cochlea. Artificial perilymph was perfused through the perilymphatic compartment of the guinea pig cochlea and the effluent collected during successive 10-min periods. The effects of perfusing an artificial perilymph containing normal levels of Na+ (NARP) were compared to the effects of perfusing an artificial perilymph containing very low concentrations of Na+ (VLNa). The effluent was collected during ambient noise and during increasing intensities of broad-band noise (10 min at 106, 112, 118 and 124 dB SPL). Levels of amines in the effluent were measured by HPLC utilizing precolumn o-phthalaldehyde (OPA) derivatization and fluorescence detection. VLNa increased the levels of glutamate and several other amines in effluent from the cochlea compared to levels obtained in NARP. Compared with its level during ambient room noise, the concentration of an unidentified amine labeled Unk 2.5 increased during intense noise (124 dB SPL). Intense noise induced no detectable changes in the concentrations of glutamate and fifteen other amines. The chemical identity and role of Unk 2.5 remain to be determined.


Evidence for L-glutamate release in frog vestibular organs.

The present study was devised in order to ascertain whether L-glutamate (Glu) is the neurotransmitter at the primary afferent synapse in frog vestibular organs. To this end different groups of frog isolated semicircular canals were stimulated by means of solutions slightly enriched in K+ (5 mM K(+)-rich solutions are sufficient to produce a strong, long-lasting, transmitter release from the basal pole of sensory cells) both in normal conditions and after low-Ca(2+)-high-Mg2+ impairment of the synaptic transmission. The concentration of Glu in the surrounding medium, determined by means of a bioluminescence-enzymatic method, was evaluated in two different experimental conditions: a) when the canals (5 canals placed inside little net bags) were immersed in a 5 mM K(+)-stimulating solution; b) during the superfusion of the canals (25 canals placed into a little perfusion chamber) with a 5 mM K(+)-stimulating solution. The net bag experiments demonstrated that K(+)-rich solutions can provoke an outflow of Glu from canal organs only if the crista ampullaris is present and functioning. Glu fluctuations were in fact suppressed by employing canals deprived of the ampulla or after low-Ca2(+)-high-Mg2+ synaptic blockade. The superfusion experiments demonstrated that the time course of 5 mM K(+)-induced release of Glu from the sensory organ strictly parallels the time course of 5 mM K(+)-induced EPSPs and spike discharge in afferent axons. These results strongly support the hypothesis that Glu is, or is released with, the afferent transmitter in frog inner ear sensory organs.


Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.

An IgG1 monoclonal antibody (mAb 54G8) which binds to both Bordetella pertussis chaperonin-60 (cpn60) and Escherichia coli cpn60 (GroEL) was produced. mAb 54G8 as well as Fab fragments prepared from this antibody were found to abolish the ability of chaperonin-10 (cpn10, GroES) to inhibit the ATPase activity of both B. pertussis cpn60 and E. coli cpn60. Electron microscopy was used to localize the binding site of the monoclonal antibody on the B. pertussis cpn60 molecule. In the absence of the antibody, the B. pertussis molecule exhibited the tetradecameric structure typical of cpn60. Both end views (showing 7-fold symmetry of the face of the molecule) and side views were evident. When mAb 54G8 was bound, B. pertussis cpn60 molecules appeared to be cross-linked so that they formed long chains. Only side views of the molecules were seen in these long chains. When B. pertussis cpn60 complexed with Fab fragments of mAb 54G8 was examined, chains were no longer observed. Instead, side views of B. pertussis cpn60 were often seen with Fab fragments extending from the ends of the molecule. These data indicate that mAb 54G8 appears to bind at or near the end of the B. pertussis cpn60 molecule and that binding of mAb 54G8 at this location affects the ability of cpn10 to productively interact with cpn60, most likely either by sterically blocking the binding of cpn10, by affecting the conformation of cpn60 in such a way that it no longer binds cpn10, or by inhibiting proper transduction of the effects of cpn10 binding.


An involucrin-like protein in hepatocytes serves as a substrate for tissue transglutaminase during apoptosis.

Cornified envelopes and apoptotic bodies are transglutaminase-cross-linked end-products of physiological cell death pathways. The two structures have similar amino acid composition. Involucrin has been considered as a cornified envelope precursor protein expressed specifically in terminally differentiating keratinocytes and squamous epithelia. We report the presence in hepatocytes of an involucrin-like protein which could be purified from dog liver with procedures characteristic to involucrins. When compared to purified dog esophagus involucrin, the liver protein also reacts with anti-involucrin antibodies, has the same relative molecular mass, possesses similar amino acid composition, and shows almost identical peptide mapping pattern. The involucrin-like protein is detectable by immunohistochemistry in normal and apoptotic hepatocytes, is a substrate of tissue transglutaminase, and is incorporated into cross-linked apoptotic bodies. These results suggest that there are overlapping molecular components in the two characteristic forms (cornification and apoptosis) of naturally occurring cell death.


A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.


Effect of introducing different carboxylate-containing side chains at position 85 on chromophore formation and proton transport in bacteriorhodopsin.

During the initial stages of the bacteriorhodopsin photocycle, a proton is transferred from the Schiff base to the deprotonated carboxylate of Asp85. Earlier studies have shown that replacement of Asp85 by Asn completely abolishes proton transport activity, whereas extension of the side chain by an additional carbon-carbon bond (Asp85-->Glu) results in a functional proton pump. Here we show that extension of the Asp85 side chain by two additional bond lengths also results in a functional proton pump as long as the terminal group is a carboxylate moiety. These side chains were created by modification of the cysteine residue in the Asp85-->Cys mutant with either iodoacetic acid or iodoacetamide. In vitro chromophore formation studies show that the rate of Schiff base protonation in mutants that contain a carboxylate at residue 85 is invariably faster than in mutants that contain neutral substitutions at this position. We conclude that in bacteriorhodopsin, there is considerable tolerance in the volume of the side chain that can be accommodated at position 85 and that the presence of a carboxylate at residue 85 is important both for proton pumping and for stabilizing the protonated Schiff base.


Dopamine D2 receptor stimulation of Na+/H+ exchange assessed by quantification of extracellular acidification.

A microphysiometer was used to quantify the rate of extracellular acidification by C6 glioma cells and L fibroblasts expressing recombinant dopamine D2 receptors. The dopamine D2 receptor agonist, quinpirole, accelerated the rate of acidification of the medium by C6 cells expressing either the short or long form of D2 receptors, D2(415) and D2(444), but not by wild-type cells that were not transfected with a D2 receptor cDNA. The rate of acidification increased with increasing concentrations of quinpirole up to 100 nM. Inhibition of the response by the dopamine D2 antagonist, spiperone, provided additional evidence that the enhanced extracellular acidification resulted from stimulation of D2 receptors. To test the hypothesis that D2 receptor-stimulated extracellular acidification was due to transport of protons by a Na+/H+ antiporter and reflected intracellular alkalinization, the effect of two inhibitors of Na+/H+ exchange, amiloride and methyl-isobutyl-amiloride, was determined. Both compounds inhibited quinpirole-induced extracellular acidification at concentrations that did not alter D2 receptor-mediated inhibition of adenylylcyclase or radioligand binding to D2 receptors. In addition, quinpirole-induced extracellular acidification was greatly inhibited by removal of sodium from the extracellular medium, confirming the participation of Na+/H+ exchange in the extrusion of acid. Quinpirole (100 nM) also increased the rate of extracellular acidification by L cells expressing D2(415), LZR1 cells. Treatment with pertussis toxin (100 ng/ml for 18 h) had no effect on the quinpirole-induced acid extrusion by C6D2(415) and LZR1 cells, although the same pertussis toxin treatment regimen completely prevented inhibition of adenylylcyclase. We conclude that recombinant D2 receptors accelerate Na+/H+ exchange in C6 cells and L fibroblasts by a pathway that does not involve inhibition of adenylylcyclase or pertussis toxin-sensitive G proteins.


Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.


Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein.

Ninety percent of variant medium-chain acyl-CoA dehydrogenase (MCAD) alleles in patients with MCAD deficiency carry a 985 A-->G transition which causes glutamate substitution for lysine 329 in precursor (p) MCAD (K-304 in mature MCAD). We have used site-directed mutagenesis to produce three variant cDNAs encoding variant pMCAD with glutamate (Kp329E2), aspartate (Kp329D), or arginine (Kp329R) substitution for Kp329. We carried out in vitro expression of cDNAs, and incubated the translation products with isolated rat liver mitochondria. Kp329E was imported into mitochondria and processed into the mature subunit as efficiently as wild-type. Gel filtration analysis of the mitochondria revealed that at 10 min after import, markedly more K304E eluted as a monomer than did wild-type, and the amount of K304E tetramer formed was distinctly less than wild-type at any point up to 60 min after import, indicating that the assembly of K304E is defective. After further incubation, K304E decayed more rapidly than did wild-type, indicating a reduced stability. In similar studies, K304R behaved like the wild-type, while K304D closely resembled K304E, indicating that the presence of a basic residue at 304 is essential for tetramer formation and intramitochondrial stability of mature MCAD.


Molecular cloning of a homeobox transcription factor from adult aortic smooth muscle.

We report here the cloning of a cDNA encoding a homeobox transcription factor from vascular smooth muscle and describe its unique pattern of mRNA expression at different stages in development. The cDNA isolated is 1576 base pairs in length not including the poly(A) tail and contains an open reading frame coding for a predicted 372-amino acid homeobox protein. During early embryogenesis, expression was detected in the neural tube with a sharp expression boundary occurring at an anterior position, in the myelencephalon, in the third and fourth branchial arches, and in vessels leading from the heart. In adults, however, transcripts were only detected in aortic smooth muscle and lung but were undetectable in cardiac or skeletal muscle, visceral smooth muscle, and many other tissues including brain. In neonates, expression was detected in the outflow tracts of the heart as well as in the cardiomyocytes. The expression pattern of this gene suggests that, although it likely has multiple roles during development, in the adult, it may participate in the control of vascular smooth muscle differentiation and proliferation.


Issues and advances in the pharmacotherapy of asthma.

Evidence is accumulating that inflammation of the airways is directly responsible for the increased bronchial hyperresponsiveness (BHR) and lung function obstruction in asthma. Bronchoprovocation with non-specific, direct bronchoconstrictors (methacholine and/or histamine) can be used as an indirect measurement of inflammation. Thus bronchoprovocation is a useful method for evaluating the long-term benefits of various therapies in asthma. The focus of asthma therapy research is now on the development of anti-inflammatory agents. Inhaled corticosteroids are currently the most potent anti-inflammatory agents in the treatment of asthma and so are generally the most effective in reducing BHR with long-term use. Non-corticosteroid anti-inflammatory agents that are currently available are reviewed. Recent studies have suggested that regular use of inhaled bronchodilators may actually be detrimental in asthma. At this time the data is still inconclusive but certainly warrants the attention of practitioners and requires further research, particularly in relation to the long-acting beta 2-agonists, formoterol and salmeterol.


Thyroid stimulatory autoantibodies in different patients with autoimmune thyroid disease do not all recognize the same components of the human thyrotropin receptor: selective role of receptor amino acids Ser25-Glu30.

To study the interaction between TSH, autoantibodies and the amino-terminal half of the TSH receptor extracellular region (amino acids 1-260; domains ABC), we constructed 20 LH/CG chimeric receptor cDNAs. The prototypic receptor for modification contained domains ABC of the TSH receptor and domains DE of the LH/CG receptor. Segments (6-13 amino acids) within the ABC domains were replaced with the corresponding amino acids of the rat LH/CG receptor. Fifteen of the 20 chimeric receptors could be expressed functionally in Chinese hamster ovary cells. Twelve retained both high affinity TSH binding and normal signal transduction. These 12 receptors were tested with a panel of 10 patients' immunoglobulin G (IgG) samples containing potent TSH receptor stimulatory activity. With 11 of the receptor variants, the cAMP responses were similar to those with the prototype receptor (TSH-LHR-6). However, the -A1 variant of TSH-LHR-6 (Ser25-Glu30) responded poorly to 6 of 10 IgGs. The same pattern was observed when the IgGs were tested for their ability to inhibit [125I] TSH binding to the receptor variants, suggesting qualitative differences between the different stimulatory TSH receptor autoantibodies. Therefore, we examined the dose-response relationship of 2 IgGs that were approximately equipotent when tested with TSH-LHR-6 and its -A1 variant and another 2 IgGs that displayed greatly diminished potency with respect to the -A1 variant. Despite dilution to nearly undetectable levels, the relative potencies of the 4 IgG samples for both types of receptors remained similar. These data demonstrate directly that stimulatory TSH receptor autoantibodies do not all recognize the same components of the TSH receptor. The segment of the TSH receptor discriminated by these autoantibodies is between amino acids Ser25-Glu30.


Binding assay for thyrotropin receptor autoantibodies using the recombinant receptor protein.

We have characterized a transfected Chinese hamster ovary cell line, JP09, which expresses high levels of the human TSH receptor (TSH-R). Based on a theoretical biological activity for TSH of 40 IU/mg, JP09 has approximately 90,000 receptors per cell, having a dissociation constant of 1.64 x 10(3) mU/L or 1.47 x 10(-9) mol/L. We have used JP09 to prepare solubilized TSH-Rs which have formed the basis of a binding assay for thyroid-binding inhibiting immunoglobulins in unfractionated sera. We have compared the JP09 assay with the TRAK assay (which is based on solubilized porcine TSH-R) and found a highly positive correlation between the two assays, r = 0.83 P < 0.0001, in 55 sera from patients with autoimmune thyroid disease. JP09 can be adapted to growth in suspension culture, permitting large scale production. The tracer in the assay is bovine [125I]TSH; surprisingly, despite the use of a hTSH-R, hTSH had no effect on the binding of the tracer up to 10(3) mU/L and only a minor effect at 10(4) mU/L.


Alertness and attention: basic science and electrophysiologic correlates.

There has been much research in the electrophysiologic correlates of alertness and attention, but it is fragmented into many subfields. This article integrates current knowledge across multiple disciplines and methodologies to provide a broad overview of alertness and attention. First, terms that are related to alertness and attention are clarified. Then, there is a discussion of basic neuroscience, human neurophysiology, and clinical fields that impact on alertness and attention. Areas discussed include thalamic and neurotransmitter-specific ascending pathways. EEG, event-related potentials, and both physiologic and pathologic states of decreased alertness or attention.


Familial apolipoprotein E deficiency and type III hyperlipoproteinemia due to a premature stop codon in the apolipoprotein E gene.

A kindred with apolipoprotein E deficiency and a truncated lower molecular weight apoE mutant, designated apoE-3Washington, has been identified. Gel electrophoresis demonstrated complete absence of the normal apoE isoproteins and the presence of a small quantity of a lower molecular weight apoE. Plasma apoE levels in the proband were approximately 4% of normal. This marked deficiency of apoE resulted in delayed uptake of chylomicron and very low density lipoprotein (VLDL) remnants by the liver, elevated plasma cholesterol levels, mild hypertriglyceridemia, and the development of type III hyperlipoproteinemia. Sequence analysis of the patient's apoE gene revealed a single nucleotide substitution of an A for a G, which converted amino acid 210 of the mature protein, tryptophan (TGG), to a premature chain termination codon (TAG), thus leading to the synthesis of a truncated E apolipoprotein of 209 amino acids with a molecular mass of 23.88 kDa. Northern blot analysis of differentiated monocyte-derived macrophages demonstrated a mutant mRNA indistinguishable in size from normal apoE mRNA. The nucleotide substitution also resulted in the formation of a new restriction site for Mae I. Using this enzyme we were able to establish that the proband is a homozygote and that her two offsprings are heterozygous for the epsilon-3Washington allele. These data demonstrate that the striking deficiency of apoE-3Washington results in a moderate form of type III hyperlipoproteinemia. The clinical presentation also suggests a dispensable role of apoE in the nervous system and in immunoregulation.


Cellular bases of neocortical activation: modulation of neural oscillations by the nucleus basalis and endogenous acetylcholine.

In the mammalian neocortex, the EEG reflects the state of behavioral arousal. The EEG undergoes a transformation, known as activation, during the transition from sleep to waking. Abundant evidence indicates the involvement of the neurotransmitter acetylcholine (ACh) in EEG activation; however, the cellular basis of this involvement remains unclear. We have used electrophysiological techniques with in vivo and in vitro preparations to demonstrate actions of endogenous ACh on neurons in auditory neocortex. In vivo stimulation of the nucleus basalis (NB), a primary source of neocortical ACh, (1) elicited EEG activation via cortical muscarinic receptors, (2) depolarized cortical neurons, and (3) produced a change in subthreshold membrane potential fluctuations from large-amplitude, slow (1-5 Hz) oscillations to low-amplitude, fast (20-40 Hz) oscillations. The NB-mediated change in pattern of membrane potential fluctuations resulted in a shift of spike discharge pattern from phasic to tonic. Stimulation of afferents in the in vitro neocortex elicited cholinergic actions on putative layer 5 pyramidal neurons. Acting via muscarinic receptors, endogenous ACh (1) reduced slow, rhythmic burst discharge and facilitated higher-frequency, single-spike discharge in burst-generating neurons, and (2) facilitated the appearance and magnitude of intrinsic membrane potential oscillations. These in vivo and in vitro observations suggest that neocortical activation results from muscarinic modulation of intrinsic neural oscillations and firing modes. Rhythmic-bursting pyramidal neurons in layer 5 may act as cortical pacemakers; if so, then modifying their discharge characteristics could alter local cortical networks. Larger, intercortical networks could also be modified, due to the widespread projections of NB neurons. Thus, NB cholinergic neurons may play a critical role in producing different states of neocortical function.


Spontaneous synchronous synaptic calcium transients in cultured cortical neurons.

The firing pattern displayed by neuronal aggregates is thought to play a key role in cortical development and physiology. In this study, we have employed optical recording of intracellular calcium to monitor activity of multiple neurons simultaneously in primary cortical cultures. With this approach, we have observed spontaneous synchronous calcium transients among adjacent cortical neurons. These transients appear to be mediated by prominent spontaneous synaptic excitation, as they are enhanced by picrotoxin, a blocker of inhibitory GABAergic transmission, and reduced by antagonism of glutamate receptors or addition of TTX. After picrotoxin treatment, the calcium transients exhibit regular frequency and amplitude, and occur in synchrony with bursts of excitatory synaptic potentials every 10-20 sec. Using electrical stimulation, we have identified a relative refractory period, extending up to 5 sec after a synchronous burst, that may play a role in cell synchronization. NMDA receptor antagonists or reduced extracellular calcium levels lower the amplitude of the calcium transients yet fail to alter their frequency, suggesting that intracellular calcium levels may not be a major determinant of burst frequency. In contrast, mild depolarization with kainic acid (0.5-1 microM) increased burst frequency up to fivefold, suggesting a critical dependence of rhythmic activity on membrane potential. Chronic blockade of electrical activity with TTX beginning a few days after plating of cultures dampens the amplitude and significantly increases the frequency of calcium transients in mature cultures. These studies demonstrate that aggregates of cultured cortical neurons express synchronous firing activity in vitro and that this network activity is dependent in part on neuronal firing during development.


Effect on cardiac sympathetic nerve activity of phenylephrine microinjected into the cat intermediolateral cell column.

1. In anaesthetized cats the effect of the alpha 1-adrenoceptor agonist phenylephrine, microinjected into the left intermediolateral cell column of the spinal cord at the third thoracic level, was studied on left inferior cardiac nerve activity. 2. Microinjection of 100 nl of 10 or 40 mM-phenylephrine caused increases in inferior cardiac nerve activity in fifteen out of seventeen experiments. 3. The microinjection of the alpha 1-adrenoceptor antagonist alfuzosin (100 nl of 10 mM) into the intermediolateral cell column antagonized the excitatory response elicited by phenylephrine. 4. Increases in inferior cardiac nerve activity produced by glutamate and 5-hydroxytryptamine microinjected into the intermediolateral cell column were not antagonized by alfuzosin. 5. It is concluded that activation of alpha 1-adrenoceptors in the region of the intermediolateral cell column can cause an increase in the firing rate of sympathetic preganglionic neurones which innervate postganglionic neurones projecting into the inferior cardiac nerve.


Induction of haemodynamic oscillations in the perfused rat liver by K+ channel blockers.

1. Exposure of the isolated perfused (constant flow) rat liver to the K+ channel blockers 4-aminopyridine (4-AP) or Cs+ causes the appearance of oscillations in portal pressure and oxygen uptake. The oscillations have a mean frequency of 0.035 Hz (2.1 cycles/min) and are fully reversible upon perfusion with blocker-free saline. Tetraethylammonium (0.17-24.7 mM) does not induce oscillatory behaviour. 2. Reversible block of the 4-AP-induced oscillations is caused by 2 mM-EGTA, or verapamil, chlorpheniramine, phentolamine or propranolol with IC50 values of 0.42, 13.5, 15 or 11.5 microM respectively. The oscillations are transiently blocked by atropine (IC50 = 8.3 microM at peak inhibition) and are not affected by 2.7 microM-tetrodotoxin. 3. Endothelium-dependent vasorelaxants, Kupffer cell activity modifiers, retrograde perfusion, or removal of the portal vein from the circuit do not modify the oscillation parameters. 4. Oscillations are also caused by infusion of physiological concentrations of adrenaline or phenylephrine, but not isoprenaline. 5. The results provide new evidence for the existence of intrahepatic voltage-sensitive Ca2+, and 4-AP- and Cs(+)-sensitive K+ channels. We propose that the K+ channel blockers reveal an intrinsic oscillator in the liver, and that phasic vasoactivity may involve a minor contribution from neurotransmitter and/or hormonal substances.


DNA analysis of HLA-DR, DQ, and DP alleles in children with polyarticular juvenile rheumatoid arthritis.

HLA-DR, DQ and DP alleles were determined by restriction fragment length polymorphism analysis and oligonucleotide probe hybridization of polymerase chain reaction amplified genomic DNA in 94 Caucasian children with polyarticular juvenile rheumatoid arthritis (JRA) [13 rheumatoid factor (RF)+ and 81 RF-] and 100 healthy controls. HLA-DRw8, DQw4, DQA1*0401, DQB1*0402 were increased in frequency in those patients with RF seronegative disease, with highest frequencies seen in patients with young age at onset (< 5 years of age). These findings were similar to what we observed in children with pauciarticular JRA, especially those with young age at onset. DPB1*0301 was also found in increased frequency in the RF- group, and in particular those seronegative for antinuclear antibody. In contrast to what is observed in patients with pauciarticular JRA, the frequency of DPB1*0201 was not increased in any polyarticular JRA patient group. These data suggest that polyarticular JRA shares many genetic features with pauciarticular JRA.


Retroperitoneal polyarteritis nodosa presenting as ureteral obstruction.

Polyarteritis nodosa (PAN) may be systemic or isolated in distribution and may involve virtually any organ or tissue in the body. Retroperitoneal PAN with ureteral obstruction as the first manifestation of the disease, to our knowledge, has not been previously reported. Two such unusual cases are described.


Polyarteritis nodosa with aortic dissection: necrotizing vasculitis of the vasa vasorum.

A 59-year-old woman was admitted to our hospital with acute onset of chest pain. She had experienced high fever, weight loss, polyarthralgia, myalgia, polyneuropathy and hallucinations for 3 years before admission. The diagnosis of polyarteritis nodosa with hepatitis B surface antigenemia was made by muscle biopsy and serological examinations, and administration of prednisolone induced remission of all the manifestations. After developing the acute attack of severe chest pain, she died suddenly. At autopsy, a DeBakey type 1 aortic dissection was found and the immediate cause of death was found to be cardiac tamponade secondary to rupture of the aortic dissection. Microscopically, necrotizing inflammatory lesions were present in the medium sized vascular arteries throughout her body. Furthermore, necrotizing vasculitis was also found in the vasa vasorum of the adventitia and media of the thoracic aorta. The dissecting lesion was seen in the outer layer of the media. Our results suggest that spontaneous dissection of the aorta may be attributed to necrotizing vasculitis of the vasa vasorum.


A metabolic assessment of the beta 1 selectivity of bisoprolol.

Twelve healthy volunteers were given single oral doses of bisoprolol 5 mg, 10 mg and 20 mg and atenolol 50 mg and 100 mg in a randomised, placebo-controlled study. The effects of these drugs on beta 2-stimulated hypokalaemia and hyperglycaemia (produced by intravenous terbutaline infusion) were studied. Comparable beta-blockade was achieved with bisoprolol 20 mg, and atenolol 50 mg and 100 mg as measured by attenuation of exercise heart rate. Measurements of areas under or over the curve (AUC and AOC) of hypokalaemic or hyperglycaemic response to terbutaline infusion showed that bisoprolol (10 mg and 20 mg) and atenolol (50 mg and 100 mg) were significantly less beta 1 selective than 5 mg bisoprolol. Furthermore, there was a trend towards decreasing beta 1 selectivity with increasing doses of bisoprolol. Bisoprolol, an effective once daily antihypertensive and antianginal treatment, has comparable beta 1 selectivity to atenolol as measured by metabolic response. At a dose of 5 mg, bisoprolol has a measurable impact on beta 1 receptors but minimal effect on beta 2 receptors.


Early ambulation and discharge in 100 patients with burns of the foot treated by grafts.

Traditional treatment after grafting of foot, ankle, and lower leg burns is bedrest, limb elevation, and gradual ambulation only after 5 to 10 days. In 1982 we suggested that aggressive surgical treatment and early ambulation could shorten hospital stay and decrease morbidity. Our treatment of these burns is excision and grafting, application of an Unna (dome paste) boot immediately in the operating room or the next morning, with normal ambulation 4 hours later and discharge of the patient if there are no other reasons for continued hospitalization. This paper reports the continuation of this plan in 100 patients treated since 1982 with a mean age of 28.8 +/- 16.9 (SD) years and burn size of 3.7% +/- 4.4%. Sheet grafts were applied to 64% with a 96% take and narrowly meshed grafts to 36% with a 97% take. Results were excellent in 85 patients, satisfactory in ten, and poor in three who required another graft. Return to work was in 4.7 +/- 3 weeks. Unna boot application permits immediate ambulation, avoids frequent dressing changes, permits a brief or no hospital stay, and provides excellent graft take with prompt return to work.


Endotoxin-induced uveitis in mice. 1. Induction of uveitis and role of T lymphocytes.

Endotoxin-induced uveitis (EIU) with a high frequency of posterior iris synechiae was induced by the systemic injection of 200 micrograms of endotoxin into C3H/HeN mice, an endotoxin-responsive strain. The cell number and the protein concentration within the aqueous humor began to increase 6 hours after the injection, achieving a peak at 24 hours, and decreased gradually thereafter. Inflammatory cells were observed in the anterior chamber, the vitreous body and near the iris-ciliary body histologically. Most of the inflammatory cells were polymorphonuclear cells. On the other hand, C3H/HeJ mice, an endotoxin-unresponsive strain, showed no increase in either cell number or protein concentration in the aqueous humor after endotoxin administration. Pretreatment of C3H/HeN mice with anti-Thy-1.2 antibody significantly decreased both the cell number and the protein concentration in the aqueous humor and the incidence of the posterior synechiae, as compared with the control group. Anti-CD4 antibody also significantly reduced the severity of EIU, while anti-CD8 antibody had no influence on the disease. Anti-IFN-gamma antibody increased the cell number in the aqueous humor. These observations indicate that T lymphocytes, especially CD4+ T lymphocytes, have an extremely important role in the development of EIU in mice.


The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells.

The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50 concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.


The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis.

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.


A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider.

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N-methyl-D-aspartate antagonist; responses to applications of N-methyl-D-aspartate or L-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 microM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10 microM. The actions of agatoxin-489 against responses to N-methyl-D-aspartate or L-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from -100 to -30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N-methyl-D-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20 mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N-methyl-D-aspartate antagonist which preferentially interacts with activated N-methyl-D-aspartate receptors and/or open N-methyl-D-aspartate-activated ionic channels.


Is the neuronal ATP release from guinea-pig vas deferens subject to alpha 2-adrenoceptor-mediated modulation?

The effects of a variety of alpha 2-adrenoceptor agonists and antagonists were studied on stimulation-evoked release of endogenous ATP, measured by the luciferin-luciferase assay, and on the release of [3H]noradrenaline from the guinea-pig vas deferens. The biphasic mechanical contraction of the guinea-pig smooth muscle was recorded concomitantly. The alpha 2-adrenoceptor agonist, xylazine (1 microM) inhibited the field stimulation-evoked (8 Hz, 0.1 ms, 480 shocks) release of ATP and [3H]noradrenaline, and both phases of the contraction. The inhibitory effect of xylazine on the release of ATP, noradrenaline and muscle contraction was prevented by the selective alpha 2-adrenoceptor antagonist, CH 38083 [7,8-(methylenedioxi)-14 alpha-alloberbanol, 1 microM]. In the presence of prazosin (0.1-1 microM) or WB 4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl- 1,4-benzodioxane hydrochloride, 0.1-1 microM], i.e. under the condition when the effect of noradrenaline on postjunctional alpha 1-adrenoceptors was excluded, the stimulation-evoked release of [3H]noradrenaline was significantly enhanced, however, the release of endogenous ATP and also both phases of contraction were reduced. In the presence of prazosin, xylazine was able to inhibit the stimulation-evoked release of ATP. In vas deferens dissected from reserpine pretreated (2 x 5 mg/kg, i.p.) guinea-pigs, the content of noradrenaline was 0.5% of control and there was no detectable evoked release of noradrenaline. Under this condition, the release of ATP evoked by electrical stimulation was still detectable, but the amount of ATP was much smaller than that measured from control animals. Xylazine did not reduce the release of ATP. Oxymetazoline, a relatively selective alpha 2-adrenoceptor agonist failed to inhibit the release of [3H]noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)


Synaptic activation of rat adrenal medulla examined with a large photodiode array in combination with a voltage-sensitive dye.

The adrenal medulla is innervated by sympathetic preganglionic nerve fibers in the splanchnic nerve. Synaptic activation of the adrenal medulla causes catecholamine secretion which is known to be modified by various neuropeptides and other factors. To understand the neuronal control mechanism of catecholamine secretion, it is necessary to know the transfer function at the synapse and how it is affected by such factors. By using a large photodiode array in combination with a voltage-sensitive dye, membrane potential changes in a slice of the rat adrenal gland were recorded upon brief local electrical stimulation. Electrical signals were recorded only on the portion of the diode array corresponding to the medulla. In a typical record, a spike and an underlying slow potential were observed following a small deflection due to a presynaptic nerve action potential. Both the spike and slow potential were blocked in Ca(2+)-free solution or by hexamethonium, a nicotinic antagonist, but were not affected by atropine, a muscarinic antagonist. The slow potential was interpreted as a nicotinic synaptic potential in the chromaffin cells and the spike as a population action potential. A double pulse experiment revealed that the chromaffin cell action potential began to fail only when the stimulus interval was less than 50 ms (20 Hz). When the stimulus intensity was reduced, the minimal response was found to behave in an all-or-none fashion. This suggested that one nerve fiber is innervating a cluster of chromaffin cells, which may correspond to a previously histologically identified "complex" of cells [Hillarp (1946) Acta. anat. 4, Suppl. 1]. Each complex was innervated by approximately four nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)


Somatostatin release is enhanced in the hippocampus of partially and fully kindled rats.

The release of somatostatin (somatostatin-like immunoreactivity) from hippocampal slices during the development of hippocampal kindling in rats was measured under resting and depolarizing conditions. Preliminary experiments in naive rats showed that the spontaneous efflux of somatostatin (4.0 +/- 0.3 fmol/ml every 10 min) was independent of external Ca2+ but was reduced to 71.5 +/- 6% of baseline (P < 0.05) during 20 min incubation with 5 microM tetrodotoxin. Neuronal depolarization with 25, 50 and 100 mM KCl induced a Ca(2+)-dependent somatostatin release, respectively 4.3 +/- 0.4, 16.7 +/- 1.6 and 22.0 +/- 1.3 times baseline (P < 0.01). Veratridine caused a dose-dependent Ca2+ and tetrodotoxin (5 microM) sensitive release ranging from 6.5 +/- 0.1 to 13.0 +/- 1.4 times baseline at 1.4 microM and 50 microM respectively (P < 0.01). One week after the last of three consecutive stage 5 seizures (full seizure expression) or 48 h after the last stage 2 stimulation (preconvulsive stage), 50 mM KCl-induced somatostatin release was significantly higher (1.8 +/- 0.1, P < 0.01) than in shams (animals implanted with electrodes but not stimulated) in the stimulated and contralateral hippocampus. Somatostatin release measured under resting conditions was increased by 1.5 times in the stimulated hippocampus at stage 2 (P < 0.05) and by 2.2 and 1.7 times in both hippocampi at stage 5 (P < 0.01). Forty-eight hours after the induction of a single afterdischarge no significant changes were found in either spontaneous or 50 mM KCl-induced release of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)


Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity.

It has been suggested that Hox genes play an important part in the patterning of limbs, vertebrae and craniofacial structures by providing an ordered molecular system of positional values, termed the Hox code. Little is known about the nature of the signals that govern the establishment and regulation of Hox genes, but retinoic acid can affect the expression of these genes in cell lines and in embryonic tissues. On the basis of experimental and clinical evidence, the hindbrain and branchial region of the head are particularly sensitive to the effects of retinoic acid but the phenotypes are complex and hard to interpret, and how and if they relate to Hox expression has not been clear. Here we follow the changes induced by retinoic acid to hindbrain segmentation and the branchial arches using transgenic mice which contain lacZ reporter genes that reveal the endogenous segment-restricted expression of the Hox-B1 (Hox-2.9), Hox-B2(Hox-2.8) and Krox-20 genes. Our results show that these genes rapidly respond to exposure to retinoic acid at preheadfold stages and undergo a progressive series of changes in segmental expression that are associated with specific phenotypes in hindbrain of first branchial arch. Together the molecular and anatomical alterations indicate that retinoic acid has induced changes in the hindbrain Hox code which result in the homeotic transformation of rhombomeres (r) 2/3 to an r4/5 identity. A main feature of this rhombomeric phenotype is that the trigeminal motor nerve is transformed to a facial identity. Furthermore, in support of this change in rhombomeric identity, neural crest cells derived from r2/3 also express posterior Hox markers suggesting that the retinoic acid-induced transformation extends to multiple components of the first branchial arch.


Control of 40-Hz firing of reticular thalamic cells by neurotransmitters.

This study bears on the control exerted by neurotransmitters on the expression of a 40-Hz pacemaker activity observed in reticular thalamic cells. Experiments were conducted in urethane-anaesthetized rats using extracellular recordings and local applications of antagonists against the neurotransmitters involved in the modulation of reticular thalamic cells. All drugs were dissolved in a Ringer's solution (pH 7.4) and were applied in small quantities (25-150 nl) by pressure through one barrel of a micropipette assembly. Forty-Hertz firing was abolished by local application of the alpha 1 antagonist prazosin and by bilateral lesion of the locus coeruleus. Local applications of glutamate antagonists reduced the rate of discharges by 30-50% as did cortical cooling or complete transection of the internal capsule. Conversely, scopolamine exerted a permissive action on the expression of 40-Hz activities; many spontaneously bursting units started firing at 40 Hz under the influence of this muscarinic antagonist. Since reticular thalamic cells are GABAergic and synaptically coupled via axonal collaterals, we investigated how GABAergic drugs affected the regular firing of these cells. Local applications of bicuculline produced a transient increase of the firing rates while the application of GABA induced intermittent pauses on a background of regular discharges. The application of piperidine-4-sulphonic acid, a GABAA receptor agonist, produced a similar effect. The length of pauses generated by piperidine was statistically analysed. It was found that the duration of short pauses was a multiple integer of the mean interspike interval of surrounding discharges. The preservation of the period and phase of the rhythm across the pauses implies that a subthreshold oscillation was presented into the cells during the arrests of discharges. Given the mode of action of noradrenaline and acetylcholine on reticular thalamic neurons, and considering a possible metabotropic action of glutamate, the above results suggest that deactivation of a leaky K conductance is critically involved in the regular firing of these cells in urethane-anaesthetized rats. Alternatively, because reticular cells are coupled via inhibitory synapses, it is proposed that the 40-Hz firing frequency reflects, in the frequency domain, a point of equilibrium in the reticular thalamic network when the leaky K conductance is fully deactivated by the metabotropic effects of monoamines and/or excitatory amino acids.


An experimental arthritis in rats: dorsal horn aspartate and glutamate increases.

Amino acid release in the dorsal horn of awake rats was examined by microdialysis during the development of arthritis induced by injection of 3% kaolin and 3% carrageenan into the knee joint. The following amino acids were measured by HPLC at baseline and for the first 8 h of arthritis: Asp, Glu, Asn, Gln, Ser, Gly and Tau. An initial increase in all amino acids examined was observed on injection of the knee joint with kaolin and carrageenan. Subsequently, there was a peak increase in Asp (184%), Glu (188%) and Gln (146%) during a prolonged release phase which began at 3.5 h and persisted at least 8 h. While Asn showed no changes from baseline, extracellular fluid concentrations of Ser, Gly and Tau were variable. This data indicates that the induction of arthritis is accompanied by an increased release of excitatory amino acids Asp and Glu which may be important in the generation of acute arthritis.


Neurotransmitter modulation of calcium channels is dependent on the charge carrier used in the recording of currents.

Currents through calcium channels were recorded using calcium, barium and strontium as charge carriers in NG108-15 cells. The mean normalised peak current amplitude at 0 mV was not significantly different between the charge carriers; however, the sustained component (measured at the end of the 500 ms command step) was ca. 3 times larger in barium and strontium. Further, the inhibition by acetylcholine or noradrenaline, although the same at the peak of the current envelope, was significantly greater on the sustained portion of the current for barium and strontium. Increasing internal calcium-buffering (to reduce calcium-dependent inactivation with calcium as the charge carrier) did not increase the amount of inhibition of the sustained portion of current. These results suggest a cautious approach to analysis of neurotransmitter modulation of calcium currents using other charge carriers than calcium.


Is forskolin stimulation of rat striatal tyrosine hydroxylase dependent on adenylate cyclase activation?

Because some responses to forskolin are adenosine 3',5'-cyclic phosphate (cyclic AMP) independent, we investigated the involvement of adenylate cyclase in the stimulatory effect of forskolin on synaptosomal tyrosine hydroxylase (TH) activity of rat striatum. The forskolin analogue, 1,9-dideoxyforskolin, which mimics the cyclic AMP-independent effects of forskolin, was a very weak activator of both striatal adenylate cyclase and tyrosine hydroxylase activities, whereas forskolin stimulated the two enzymes effectively and with similar potencies. Moreover, exposure of synaptosomes to the specific cyclic AMP antagonist Rp-adenosine 3',5'-cyclic phosphorothioate reduced basal TH activity and counteracted the stimulatory effect on the enzyme activity by submaximal concentrations of forskolin. These results provide circumstantial evidence that in striatal dopaminergic nerve terminals a presynaptic adenylate cyclase mediates the stimulation of TH activity by forskolin.


Increased plasma concentrations of aspartate, glutamate and glycine in Parkinson's disease.

We measured fasting plasma amino acids in 20 patients with Parkinson's disease (PD) and 20 controls matched for age and sex. PD patients had significant elevations in plasma levels of aspartate, glutamate and glycine. The levels of other amino acids were not significantly different from those found in controls. No correlation was noted between PD severity and the degree of abnormality of plasma amino acids. We conclude that excitatory amino acids may be altered in patients with PD, and raise the possibility that neuroexcitotoxic mechanisms may be involved in the neurodegeneration of PD.


Endogenous extracellular glutamate accumulation in rat neocortical cultures by reversal of the transmembrane sodium gradient.

Glutamate excites receptors located on neurons that cause calcium and sodium influx involved in excitatory synaptic transmission. During ischemia, excess glutamate is present in the extracellular space of brain tissue, leading to abnormal levels of calcium influx and eventually to cell death. In mixed neuronal/glial cell cultures we have found that reduction of extracellular sodium concentration below approximately 10 mM causes marked increases in glutamate and aspartate in medium collected 10 min after changing to low sodium. Various data indicate that the accumulated glutamate comes from reversal of normal cellular glutamate uptake, a process also thought to occur during ischemia.


Neuroprotective synergism of 2-amino-3-phosphonoproprionate (D,L-AP3) and MK-801 against ibotenate induced brain injury.

The neuroprotective characteristics of the functional antagonist of metabotropic stimulated phosphoinositide hydrolysis, 2-amino-3-phosphonoproprionate (D,L-AP3), were examined alone and in combination with the non-competitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, against ibotenate induced brain injury. Postnatal day (PND) 7 rats received unilateral stereotaxic intrastriatal injections of 10 nmol ibotenate and treated with either D,L-AP3 (600 nmol i.c.), MK-801 (1 mg/kg i.p.) or both. The severity of brain injury was assessed on PND 12 by comparison of the weights of injected and contralateral cerebral hemispheres. Ibotenate induced injury was partially reduced by treatment with MK-801 (34.0 +/- 4.4% protection, P < 0.05 vs. PBS treated, independent t-test) but not D,L-AP3. However, combined treatment with both MK-801 and D,L-AP3 produced marked synergistic neuroprotection (83.5 +/- 7.6% protection, P < 0.001 vs. PBS treated, independent t-test). The data suggest that metabotropic stimulated phosphoinositide hydrolysis contributes to excitotoxic neuronal injury in the presence of concurrent ionotropic receptor activation.


Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum.

The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumen was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SLI from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about 1 during basal release but increased to approximately 2 during carbachol stimulation.


[Pharmacology aspects of glaucoma].

The aim of all treatment for glaucoma is to lower intra-ocular pressure. During the past decade, the short-action cholinergic agonists, eserine and pilocarpine, that have been used for over a hundred years have been replaced as the drugs of choice by beta-blockers, in particular the non-selective beta-blocker, timolol maleate. Of the series of drugs developed in recent years for the treatment of glaucoma, two types--carbonic anhydrase inhibitors and prostaglandin analogues--are undergoing phase III trials. The article provides an account of the pharmacological basis of glaucoma treatment today and in the immediate future.


[Use of inhaled beclomethasone dipropionate in adult asthma].

Beclomethasone dipropionate has now been used for more than 10 years during which our knowledge of how to use inhaled corticosteroids has gradually improved: high dose initial treatment followed by progressive reduction down to the minimum effective dosage; administration in 2 daily doses when the asthma is stable and 4 daily doses in case of instability; mild and transient undesirable effects, often minimized by a correct use of the inhaler; effectiveness assessed from bronchial hyper-reactivity and respiratory function tests, reduction or avoidance of oral corticosteroid therapy, or results of association with other treatments, and in particular bronchodilators. When exactly should inhaled corticosteroid therapy should be started and how long should it be pursued are controversial points, but an early and prolonged treatment must probably be recommended.


Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.


Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker.

In this report, 8- and 2-azidoadenosine 5'-[gamma-32P]triphosphate were used to examine cerebrospinal fluid (CSF) samples for the presence of an ATP binding protein unique to individuals with Alzheimer disease (AD). A 42-kDa ATP binding protein was found in the CSF of AD patients that is not observed in CSF from normal patients or other neurological controls. The photolabeling is saturated with 30 microM 2-azidoadenosine 5'-[gamma-32P]triphosphate. Photoinsertion can be totally prevented by the addition of 25 microM ATP. Photoinsertion of 2-azidoadenosine 5'-triphosphate into the protein is only weakly protected by other nucleotides such as ADP and GTP, indicating that this is a specific ATP binding protein. A total of 83 CSF samples were examined in a blind manner. The 42-kDa protein was detected in 38 of 39 AD CSF samples and in only 1 of 44 control samples. This protein was identified as glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] based on similar nucleotide binding properties, comigration on two-dimensional gels, reaction with a polyclonal anti-GS antibody, and the presence of significant GS enzyme activity in AD CSF. In brain, GS plays a key role in elimination of free ammonia and also converts the neurotransmitter and excitotoxic amino acid glutamate to glutamine, which is not neurotoxic. The involvement of GS, if any, in the onset of AD is unknown. However, the presence of GS in the CSF of terminal AD patients suggests that this enzyme may be a useful diagnostic marker and that further study is warranted to determine any possible role for glutamate metabolism in the pathology of AD.


Recycling and phosphorylation of eukaryotic initiation factor 2 on 60S subunits of 80S initiation complexes and polysomes.

Phosphorylation of the alpha-subunit (38 kDa) of eukaryotic initiation factor 2 (eIF-2 alpha) regulates initiation of protein synthesis in eukaryotic cells. This phosphorylation is enhanced in cycloheximide-treated heme-deficient reticulocyte lysates in which polysomes are maintained. In early heme deficiency prior to polysome disaggregation, eIF-2(alpha P) accumulates primarily on the 60S subunits of polysomes. Further, isolated polysomes contain eIF-2 alpha that is efficiently phosphorylated in vitro by heme-regulated inhibitor (HRI). Immunoblot analysis of eIF-2 distribution in sucrose gradients of actively protein-synthesizing lysates indicates that eIF-2 is distributed at low levels throughout the polysome profiles. These findings suggest that polysome-bound eIF-2 alpha is a target of HRI under physiological conditions. The presence of eIF-2 on the 60S subunits of polysomes is incompatible with the conventional model in which eIF-2 is recycled during the joining of the 48S preinitiation complex and the 60S subunit to form the 80S initiation complex. A modified model is presented with emphasis on the translocation of eIF-2 from the 40S ribosomal subunit of the 48S preinitiation complex (eIF-2.GTP.Met-tRNA(f).40S.mRNA) to the 60S subunit of the 80S initiation complex.


Activity of the Hsp70 chaperone complex--DnaK, DnaJ, and GrpE--in initiating phage lambda DNA replication by sequestering and releasing lambda P protein.

Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.


Effects of the phthalazinone azelastine on epidermal metabolism after mechanical skin irritation.

In female NMRI mice, the phthalazinone azelastine was administered orally once daily over 7 days. The drug influenced the epidermal thymidine triphosphate and amino acid incorporation rates at doses between 1 and 5 mg/kg. In control mice, an epidermal hyperproliferation induced by abrasion of superficial epidermal layers was characterized by enhanced prostaglandin and leukotriene concentrations in epidermal homogenate, an increase in thymidine triphosphate and amino acid incorporation and an increase in epidermal thickness. In mice treated with 1 mg/kg azelastine HCl, this epidermal reaction was changed. Compared to controls, the increase in leukotriene concentration was diminished, and that of prostaglandins was enhanced. The incorporation of thymidine triphosphate and of amino acids as well as the epidermal thickness and the ratio cell count/epidermal thickness were increased in irritated skin of azelastine-treated mice. In conclusion, azelastine influences the epidermal metabolism in irritated and unirritated skin. Therefore, a beneficial role of this phthalazinone in the treatment of psoriasis and related skin disorders seems to be possible.


The role of imidazoline receptors in blood pressure regulation.

Using the ligands [3H] clonidine and [3H] idazoxan, nonadrenergic imidazoline preferring binding sites have been identified in a range of tissues from several species including man. These sites may represent a new family of receptors. An endogenous ligand and potential clonidine displacing substance has been identified. There is strong evidence for an involvement of the nonadrenergic imidazoline [3H] clonidine labelled sites in the nucleus reticularis lateralis in blood pressure regulation, and some evidence for a role in sodium regulation in the kidney for the [3H] idazoxan labelled sites. Some drugs which were previously thought to act via alpha 2-adrenoceptors, may mediate their effects in part via these imidazoline sites.


Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents.

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.


Trend towards decreased survival in patients infected with HIV resistant to zidovudine.

The survival of 35 patients with AIDS or advanced HIV infection on treatment with zidovudine was related to the viral sensitivity to the drug and to the CD4+ cell count. 14 patients died, the survivors were followed up for an average of 804 days. In a univariate Cox model, survival was strongly related to log IC90 (p = 0.0003) and to the CD4+ count (p = 0.0002). In a bivariate model, log IC90 and the CD4+ count contributed to the prediction of survival (p = 0.12 and 0.06, respectively). Large studies of combination or alternation therapy with several anti-HIV drugs should be given high priority.


Absence of an association between enteric parasites in the manifestations and pathogenesis of HIV enteropathy in gay men. The GI/HIV Study Group.

49 gay men confirmed to be infected with the human immunodeficiency virus (HIV) and 9 HIV seronegative gay men participated in a pilot study comparing clinical status and enteric parasite load with gastrointestinal structure, function and symptomatology. Cases included 16/49 (33%) men who were CDC stage II, 7/49 (14%) who were CDC stage III, and 26/49 (53%) who were CDC stage IV. The mean CD4-lymphocyte count was 476 +/- 199 (SD)/microliter. The prevalence of enteric parasitic flora was similar in HIV seropositive patients and controls. Seven cases had enteric infection with pathogenic agents including 3 patients with Entamoeba histolytica, and 4 patients with Giardia lamblia, one of whom also had cryptosporidiosis. Other cases were most frequently colonized with Blastocystis hominis (44%) and Endolimax nana (41%) regardless of the HIV clinical status. HIV seropositive patients with enteric parasitic colonization tended to have lower mean levels of serum IgA than cases without parasites. Duodenal morphometric mucosal changes demonstrated a significant decrease in the mean villous height (p < 0.01) with no elongation of the crypt depth in HIV-infected patients with and without diarrhea compared to controls. Despite gastrointestinal symptoms including diarrhea and weight loss being more prevalent in HIV infected individuals than controls, no correlations were found between the presence of particular enteric parasites, gastrointestinal symptomatology, the clinical HIV status of the CD4-lymphocyte count, the malabsorption of D-xylose or morphometric changes in the duodenum.(ABSTRACT TRUNCATED AT 250 WORDS)


[Developments in the treatment of myocardial infarction in Switzerland 1986-1990: results of a population survey].

The Swiss cantons of Vaud and Fribourg participate in the international MONICA project (MONI-toring of trends and determinants in CArdiovascular disease). Within this context, drug therapies and procedures were recorded during two separate years (1986 and 1990) for all hospitalizations of men aged 25 to 64 with the diagnosis of myocardial infarction. The medical files were reviewed to classify this diagnosis as possible or definite on the basis of the symptoms, the ECG results and the enzymatic tests. The two study populations (n = 318 in 1986 and n = 332 in 1990) are comparable with respect to age, history of ischemic heart disease and initial care. In 1990, half of the patients arrived at hospital in less than 3 hours, the median time delay being 4 hours for those first attending a general practitioner and 2 hours for those transferred directly. The frequency of treatments between 1986 and 1990 is compared only for cases with a definite diagnosis of myocardial infarction (respectively n = 217 and n = 223). The proportion of patients given thrombolytic therapy rose from 9% to 44% (p < 0.005) and from 51% to 95% (p < 0.005) for those treated with antiplatelet drugs, whereas the proportion fell from 72% to 55% (p < 0.005) for calcium blockers and from 33% to 24% (p < 0.05) for inotropic drugs. The use of anticoagulants (in 98% of patients) and of beta blockers (in 57%) remained stable across time.(ABSTRACT TRUNCATED AT 250 WORDS)