Biomedical subjects

R Wagner

Publications and source records attributed to R Wagner.

At least 19 recordsLinked to original sources

PubMed1992

Inhibition of chloroplast ATPase by the K+ channel blocker alpha-dendrotoxin.

Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.

Adenosine Triphosphatases↗

PubMed1992

Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter.

The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA P1 promoter in transcription activation was studied. This sequence region had been shown to activate transcription from P1 in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB P1 promoter activation were studied in vivo in fis+ and fis- backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis- strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB P1 promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.

Base Sequence↗

PubMed1992

Distinct cytoplasmic islet cell antibodies with different risks for type 1 (insulin-dependent) diabetes mellitus.

The cytoplasmic islet cell antibody patterns of sera from islet cell antibody positive non-diabetic and diabetic endocrine autoimmune patients, and newly-diagnosed Type 1 (insulin-dependent) diabetic patients were characterised using four layer immunofluorescence with monoclonal anti-proinsulin or anti-glucagon antibodies. Two distinct islet cell antibody types were identified. One gave a diffuse cytoplasmic staining in both Beta and Alpha cells ('whole' islet pattern), and was not affected by pre-incubation with rat brain homogenate. The other had a granular appearance with staining restricted predominantly to Beta cells ('selective' islet pattern) and was completely inhibited by pre-incubation with rat brain homogenate. Some sera appeared to have a 'mixed' islet pattern, in which glucagon-positive cells gave a weaker cytoplasmic staining than proinsulin-positive cells. The granular 'selective' pattern was found in sera from 19 (79%) of 24 non-diabetic endocrine autoimmune patients, in two (22%) endocrine autoimmune patients who developed Type 1 diabetes (p less than 0.0001 vs non-diabetic endocrine autoimmune patients), and in none of 19 newly-diagnosed diabetic patients. The 'whole' islet pattern was found only in sera from patients who had, or who subsequently progressed to, Type 1 diabetes. This study has identified a novel islet cell antibody specificity and demonstrates that in islet cell antibody positive endocrine autoimmune patients, only islet cell antibodies which stain both Beta and Alpha cells are associated with progression to Type 1 diabetes.

Adult↗

PubMed1992

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

AIDS Vaccines↗

PubMed1992

Immunological reactivity of a human immunodeficiency virus type I derived peptide representing a consensus sequence of the GP120 major neutralizing region V3.

To reduce the opportunities for human immunodeficiency virus type 1 (HIV-1) to evade vaccine induced immunity, the development of subunit vaccines must focus on the characterization of immunogenic epitopes, which are major targets for the immune system. The most dominant site for elicitation of neutralising immune response is located on the external envelope glycoprotein gp120 within the third variable domain (V3). To overcome virus type specificity of antibodies directed to the V3-domain we designed a 36 amino acids long gp120/V3-consensus peptide (V3-C36) based on published biological data and sequence comparisons of various HIV-1 virus isolates. This peptide contains a conserved core sequence which is suggested to form a surface-exposed beta-turn. This peptide also includes T-cell epitopes defined in mice and humans, an ADCC-epitope and two highly conserved cysteine residues which were oxidized to form a cystine derivate, thus allowing correct peptide folding. In ELISA-tests, this peptide reacts with at least 90% of randomly selected sera of European and African patients infected with HIV-1 and is recognized by three different HIV-1/V3 "type-specific" antisera (MN, RF, IIIB-strain). Using this peptide as immunogen in rabbits, antisera could be raised with highly cross-reactive and HIV-1/IIIB strain neutralizing properties. Moreover, HTLV/HIV-1/IIIB specific cytotoxic T-lymphocytes (CTLs) of BALB/c mice infected with a gp120 recombinant vaccinia virus recognized the central 16- and 12-mer peptides of the V3-C36 consensus peptide in cytolytic assays, indicating perfect compatibility of the consensus peptide with the IIIB-primed CTLs. The DNA-sequence encoding the V3-consensus loop region might be an important component in newly designed recombinant subunit vaccines. In addition, due to its broad serological reactivity, the V3-consensus peptide might play an important role in special diagnostic purposes.

AIDS Vaccines↗

PubMed1992

Positron emission tomography in degenerative disorders of the dopaminergic system.

21 patients who had Parkinson's disease (PD), PD plus dementia of Alzheimer type (PDAT) or progressive supranuclear palsy (PSP), were studied with positron emission tomography (PET) using (18F)-2-fluoro-2-deoxy-D-glucose (FDG). In one patient with strictly unilateral PD side differences in striatal dopa uptake were studied with 6-(18F)fluoro-L-dopa (F-dopa). In patients with PD PET with FDG did not show any significant change in regional cerebral metabolic rates for glucose (rCMR(Glu)). In PDAT glucose metabolism was generally reduced, the most severe decrease was found in parietal cortex. The metabolic pattern was similar to that typically found in patients with Alzheimer's disease (AD). In the patient with strictly unilateral PD rCMR(Glu) was normal, F-dopa PET, however, revealed a distinct reduction of dopa uptake in the contralateral putamen. In PSP glucose metabolism was significantly decreased in subcortical regions (caudatum, putamen and brainstem) and in frontal cortex. Thus PET demonstrated a clear difference of metabolic pattern between PDAT and PSP.

Adult↗

PubMed1992

Production of 6-[18F]fluoro-L-dopa and its metabolism in vivo--a critical review.

This report critically appraises methods for the synthesis of 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA) that are based on labelling by non-regioselective electrophilic fluorination, regioselective fluorodemetalation or nucleophilic substitution. Recommendations for the standardization of labelling procedures, the optimization of radiochemical yield and the assurance of product quality and safety are given. Studies of the metabolism of 6-FDOPA in vivo are also reviewed to emphasize the importance of the biochemical component of the development of this tracer for positron emission tomography (PET).

Animals↗

PubMed1992

Progressive derangement of periinfarct viable tissue in ischemic stroke.

Sixteen patients were studied by multitracer positron emission tomography (PET) within 6-48 (mean of 23) h of onset of a hemispheric ischemic stroke and again 13-25 (mean of 15.6) days later. Cerebral blood flow (CBF), cerebral blood volume (CBV), cerebral metabolic rate of oxygen (CMRO2), oxygen extraction fraction (OEF), and cerebral metabolic rate of glucose (CMRglc) were measured each time by standard methods, and the sets of brain slices obtained at the two studies were matched using a three-dimensional alignment procedure. On matched brain slices, regions of interest (ROIs) for infarct and peri-infarct tissue, contralateral mirror regions, and major brain structures were outlined. In the core of infarction, blood flow and metabolism were significantly lower than in the corresponding contralateral regions at the first study, and did not change during the observation period. In the peri-infarct tissue, CMRO2 was moderately decreased at the first measurement; over time, the CMRO2 deteriorated progressively while flow did not change. When peri-infarct regions were selected on the basis of increased OEF (25 +/- 29.8% above corresponding contralateral regions) on the early scans, the CBF was significantly decreased (23 +/- 6.6%) while the CMRO2 showed only a slight difference from the mirror region. Within the observation period, the CBF improved but the CMRO2, OEF, and CMRglc deteriorated. Only in a few regions with increased OEF and slightly impaired CMRO2 was metabolism preserved close to normal values. These data from repeat PET studies in reproducibly defined tissue compartments furnish evidence of viable tissue in the border zone of ischemia up to 48 h after stroke. While this viable peri-infarct tissue exhibits some potential for effective treatment of ischemic stroke, therapeutic routines available today cannot prevent subsequent metabolic derangement and progression to necrosis. Multitracer PET studies identifying viable tissue could be of value in the development of effective treatment of ischemic stroke.

Adolescent↗

PubMed1992

Binding of unopsonized Cryptococcus neoformans by human bronchoalveolar macrophages: inhibition by a large-molecular-size serum component.

Infection with Cryptococcus neoformans usually begins after inhalation of airborne organisms. Since levels of opsonins in the alveolar space may be low, the ability of human bronchoalveolar macrophages to bind C. neoformans in the presence and absence of opsonins was studied. Bronchoalveolar macrophages bound unopsonized C. neoformans. Surprisingly, component(s) in pooled human serum (PHS) inhibited binding, as evidenced by 26% and 71% inhibition of binding when 20% PHS and heat-inactivated PHS (HI-PHS), respectively, were added to the system. Separation of PHS by molecular size revealed that the inhibitory component had an apparent molecular weight greater than 10(6) and was inhibitory at nanomolar concentrations. PHS stimulated and HI-PHS had no effect on binding of acapsular C. neoformans and zymosan particles to bronchoalveolar macrophages. These data demonstrate that bronchoalveolar macrophages can bind unopsonized, encapsulated C. neoformans, but that serum component(s) inhibits binding.

Bacterial Adhesion↗

PubMed1992

Frequency of intra-abdominal injury in cases of blunt trauma to the cervical spinal cord.

Historically, early management of the blunt trauma victim with hemodynamic instability and cervical spinal cord injury has been hampered by the physician's inability to perform an accurate physical examination. Invasive and time-consuming diagnostic tests are often run to check for the presence of occult intra-abdominal injuries. For this reason, we decided to study these patients by reviewing a clinical registry to assess the frequency of intra-abdominal injuries in cases of cervical spinal cord trauma. We hypothesized that intra-abdominal injury would occur infrequently in cases of blunt trauma to the cervical spinal cord. In fact, data from the Maryland Institute for Emergency Medical Services Systems revealed that blunt trauma victims with cervical cord injury rarely (2.6%) sustained intra-abdominal wounds. Further analysis of this population revealed that specific mechanisms of trauma and the presence of hemodynamic instability and other major injuries were factors strongly associated with occult intra-abdominal injury. In light of these findings, we have outlined a protocol for management of these patients that is geared toward more rapid stabilization of the injured spinal column.

Abdominal Injuries↗

PubMed1992

Evaluation of three template matching algorithms for registering images of the eye.

The purpose of this paper is to evaluate the ability of three similarity measures to register a template in a sequence of near-infrared eye images. Three measures are evaluated: the normalized correlation coefficient (rho), the sum of absolute valued differences (SAVD), and a relatively new technique based on a sign change criterion, called the stochastic sign change criterion (SSC). Performance is measured in terms of the method's ability to track the pupil center derived from fitting a general 2-D ellipse to the pupil contour. Experiments using static eye images indicate that rho is not a reliable similarity measure. Conversely, both SAVD and SSC show enough potential to merit an investigation into their performance in tracking dynamic eye movements. It is anticipated that SAVD and SSC can accommodate a range of eye movements spanning at least 60 degrees horizontally and 40 degrees vertically.

Algorithms↗

PubMed1992

Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region.

Transcription elongation catalysed by DNA-dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid-encoded ribosomal RNA operon. Plasmid templates with different superhelical densities (sigma between +0.0017 and -0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.

Bacterial Proteins↗

PubMed1992

Structure activity studies of tryptophan30 modified analogs of Ac-CCK-7.

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7[Ac-Tyr(SO3H)-Met-Gly-Trp30-Met-Asp-Phe-NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal30-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the 1-naphthylalanine30 (19) and (benzo[b]thien-2-yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp30 replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.

Amino Acid Sequence↗

PubMed1992

Human neutrophil mobilization during open heart surgery.

Phagocyte released reactive oxygen species are often discussed in connection with ischemic and reperfusion injuries to the myocardium. The kinetics of the accumulation and oxidative burst of human blood phagocytes was studied by chemiluminescence during open heart surgery in the myocardium of human patients. Direct evidence is presented for an accumulation of neutrophils along with their markedly increased metabolic activity (oxygen radical formation), especially following the reperfusion of the ischemic myocardium. Leukocyte numbers and activity remained significantly elevated even in the venous blood obtained 24 h after the operation.

Cardiac Surgical Procedures↗

PubMed1992

Opposite effects of enantiomers of clofibric acid derivative on rat skeletal muscle chloride conductance: antagonism studies and theoretical modeling of two different receptor site interactions.

The R-(+) enantiomer of 2-(p-chlorophenoxy) propionic acid (CPP) produces a biphasic effect on chloride channel conductance (GCl) of rat skeletal muscle, increasing GCl at low concentrations and decreasing it at concentrations greater than 10 microM; on the other hand, the S-(-) isomer mostly blocks GCl in a concentration-dependent manner. To explain the peculiar behavior of these compounds, a theoretical model based on the presence of two opposing receptor populations controlling chloride channel conductance has been used to fit the experimental data of the concentration-response curves of both S-(-) and R-(+) CPP. An analysis performed by means of the algebraic summation of two logistic terms suggests a reasonable merit of the proposed model and explains the resultant effect of each optical form as follows: S-(-) acts as a full agonist on an inhibitory sites, whereas R-(+) acts as a full agonist at both the inhibitory and excitatory sites. Antagonism studies appear to be consistent with the proposed model. Dose-response curves in which the block of GCl by the S-(-) isomer was evaluated in the presence of the R-(+) isomer (3-10 microM) clearly showed an antagonistic interaction between the two enantiomers, with an increase in the S-(-) concentration for half-maximal block. The antagonism was overcome by high concentrations of S-(-), and this might be consistent with the hypothesis that the block of GCl is modulated by an inhibitory site at which the two enantiomers compete.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed1992

[Results of surgical therapy of bronchial cancers with reference to adjuvant therapeutic measures in an advanced stage].

UNLABELLED: The prognosis of bronchial carcinoma especially in advanced tumor stage is poor despite different methods of treatment. The 5-year survival rate after radical operation is about 25-40% for all stages, and between 17-20% in stage III. Without therapy it is 3.5-9.5%. T3-tumors and N2-lymphnodes represent the limit of surgical therapy. From 1980 to 1988 we operated 456 patients because of bronchial carcinoma, 80% men (mean age 59.2 y) and 20% women (mean age 56.9 y). 83.1% had been smokers, 16.9% had been exposed to contaminants. At admission to the hospital 36% were in stage I, 13% in stage II, 47% in stage III, and 4% in stage IV. HISTOLOGY: squamous cell carcinoma 40.9%, adenocarcinoma 26.1%, large cell carcinoma 21.9%, and small cell carcinoma 11.1%. Looking at all cases we mostly performed a lobectomy, in stage III tumors the most common operation was pneumonectomy. In this group only 57.5% of the patients were resectable. In 77 potentially curative operated patients in stage III with non-small cell carcinoma we performed an adjuvant radio-, chemo- or combined therapy. The cumulative survival rates for all patients in the 1st year were 85% in stage I, 67% in stage II, and 36% in stage III and IV. In the 3rd year 57%, 14% and 10%, and in the 5th year 41%, 7%, and 5%. In stage III the survival rates of the potentially curative operated vs. the exploratively operated patients were in the 1st year 50%/36%, in the 3rd year 16%/10%, and in the 5th year 11%/5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Non-Small-Cell Lung↗

PubMed1992

[Protection of the tunica intima during preservation of venous bypass grafts from harvesting to implantation].

The patency-rate of venous bypasses depends--beside other important factors--upon the integrity of the transplanted vessel, especially on the condition of the tunica intima. We investigated two different methods of venous storage and their on the intima. We examined 24 segments of healthy human saphenous vein and 65 segments of the inferior vena cava in the rat which had been harvested using a minimal touch technique. The veins were stored in the patients own arterial heparinized blood vs. Euro Collins solution at 4 degrees C, 20 degrees C and 37 degrees C over 15, 30, 60, and 90 minutes. As a reference we examined 11 segments of human saphenous vein which had been fixed immediately after harvesting. The stainings were performed with PAS, HE, Domagk-Elastica, and Toluidine-blue. We examined 1,181 slices by light-microscopy and used a damage score table for the documentation of the vein-trauma. No significant differences were found between human and animals veins. In the control-group the endothelium, the subendothelial layer, and the intern elastic membrane were intact in 57%, 78%, and 38%. At 4 degrees C the endothelium was intact in 31% having been stored in blood, and in 44% at Euro Collins solution. For the subendothelial layer the ratio was 48%/66%, and for the intern elastic membrane 26%/42%. At 20 degrees C we found a ratio of 55%/47% vs. 65%/58% vs. 37%/39%, and at 37 degrees C 56%/37% vs. 64%/51%/32%/38%. The factor time could be neglected up to 90 minutes. The results show that veins should be used immediately after harvesting.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells.

An improved method including extraction procedures is presented for the analysis of nucleotides in suspension-cultivated animal cells. Quantification was performed by ion-pair high-performance liquid chromatography after perchloric acid extraction. It was found that the amount of perchloric acid taken for extraction influenced the yield and that cell washing procedures caused deterioration of the analysis results for triphosphates. More than thirty nucleotides and sugar-nucleotides were separated within 25 min using a Supelcosil reversed-phase column (3 microns) with tetrabutylammonium hydrogensulphate as pairing agent and methanol-pH gradient elution. Cultivated hybridoma cells showed variations in intracellular nucleotide concentrations as well as relative amounts during different growth phases, which could reflect the physiological state of a cell culture.

Adenosine Triphosphate↗