Calcium Dobesilate protects human peripheral blood mononuclear cells from oxidation and apoptosis

The antioxidant effects of Calcium Dobesilate (CD, Doxium ®) were investigated in relation to the oxidative status, apoptosis and in vitro proliferation of human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. CD alone did not modify cell growth in vitrountil 10 M. This molecule counteracted oxidative damages generated by the high reducing sugar dR and was shown to reduce apoptosis by delaying both membrane permeability changes and DNA fragmentation. CD 10 M affected in a time-dependent dynamics several parameters representative of the cellular oxidative status. In particular, CD significantly increased the activity of glutathione S-transferase (GST) after three days of treatment and also, but to a lower extent, the activity of -glutamyltransferase (-GT). Both enzymes are known to be involved in the glutathione (GSH) metabolic cycle. This enzymatic behaviour was reversed at seven days of treatment, with a significant GST decrease and a -GT activation. After seven days of CD exposure, the intracellular GSH content was enhanced and this resulted in a dramatic decrease in lipid peroxidation, underlining the powerful antioxidant properties of CD in human PBMC.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.     

Metabolism of leukotrienes

Summary The in vitro metabolism of leukotriene B₄ is initiated by -hydroxylation. This reaction is followed by oxidation of the -hydroxyl group to a carboxyl group. In vivo extensive -oxidation occurs and the main excreted products after administration of leukotriene B₄ are water and carbon dioxide.Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal y-glutamyl residue, catalyzed by the enzyme -glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E₄, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C₄. In rats, the two major fecal metabolities of leukotriene C₄4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E₄. These compounds are formed in reactions between leukotriene E₄ or 11-trans leukotriene E₄ and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Summary The distribution of -tubulin throughout cell division is studied in several taxa of higher plants. -Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. -Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, -tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. -Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants -tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to - and -tubulin.Abbreviations Mt microtubule - MTOC microtubule-organizing center - PPB preprophase band     

Changes in Thiol Content in Roots of Wheat Cultivars Exposed to Copper Stress

Wheat (Triticum aestivum L.) cultivars GK Tiszatáj, Yubileinaya, GK Öthalom and a landrace Kobomugi were grown for 18 d in hydroponic cultures containing 0 (control), 0.1, 1.0 or 10.0 µM Cu²⁺. On a dry mass basis, cvs. Tiszatáj and Kobomugi accumulated slightly more Cu²⁺ in the root tissues than did cvs. Yubileinaya and Öthalom, but their controls also contained higher amounts of Cu²⁺. As a result of perturbation in the plasma membrane functions the K⁺ content of roots was reduced at 10 µM Cu²⁺ in all cultivars, whereas the K⁺/Na⁺ ratio decreased significantly only in the roots of cv. Öthalom. In the sensitive cultivar, Öthalom, the dry mass of the roots decreased while the cysteine content, which is a limiting factor for glutathione synthesis, did not satisfactorily increase with increasing tissue Cu²⁺ content. This suggests that in cv. Öthalom the membrane damage of the root cells at 10 µM Cu²⁺ concentration may affect the sulphur availability or metabolism. Concentrations of glutathione and hydroxymethyl-glutathione, a tripeptide which may play a similar biochemical role to glutathione, were also lower in the sensitive cultivar. In the absence of glutathione the root tissues failed to cope with the oxidative stress caused by the excessive amount of Cu²⁺. A significant accumulation of iron in the roots of the sensitive cultivar at 10 µM Cu²⁺ supply enhanced the oxidative damage.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Effect of cytochrome P450 1A induction on oxidative damage in rat brain

Polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs) can enhance the generation of reactive oxygen species (ROS) by inducing cytochrome P450 1A (CYP 1A) in vivo and in vitro. While the brain is vulnerable to oxidative injury, whether or not CYP 1A induction in the brain can produce measurable levels of oxidative damage has not been reported. The objective of this study was to investigate the effect of this induction on oxidative damage to the CNS. Time course changes in rat brain CYP 1A activity were determined by measurement of ethoxyresorufin Odeethylase (EROD) activity in whole brain homogenates. Three days after exposure of rats to five daily injections of 3methylcholanthrene (3MC) an approximately sevenfold increase in EROD activity was observed. Hepatic levels were increased 60–100 fold. This increase in CYP 1A activity was not accompanied by increased protein or lipid oxidation as measured by tryptophan fluorescence and TBAR formation, or decreased glutamine synthetase (GS) activity. These findings indicate that if increased CYP 1A activity in the brain following 3MC treatment leads to increased ROS generation, the increase is insufficient to overwhelm the endogenous antioxidant defense system, produce detectable oxidative damage, and alter glutamate homeostasis.     

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicologicalin vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated -glutamyl transferase activity.Abbreviations EDTA ethylenediaminetetraacetic acid - GGT -glytamyl transferase (EC 2.3.2.2) - GSH reduced glutathione - HPLC high-performance liquid chromatography - PA perchloric acid - PBS Dulbecco's phosphate-buffered saline     

Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

Rat hepatocytes prepared by collagenase digestion or ED TA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll cen trifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (TI S) supplementation, while GSH levels in collagenaseprepared cells increased, thereafter decreased with time in culture and was dependent on TI S supplementation. Cells prepared with ED TA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. -Cystathionase (CNase) activity was retained at initial levels in ED TAprepared hepatocytes supplemented with TI S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, -glutamyl trans-peptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.Abbreviations CNase -cystathionase - EDTA ethylenediamine tetraacetic acid - GGT -glutamyl transpeptidase - GSH reduced glutathione - GSSG oxidized glutathione - HEPES N-2-hydroxyethylpiperizine-N-2-ethane-sulfonic acid - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine - T/S transferrin- and selenite-supplemented     

Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras

Interleukin-2 (IL-2)-activated killer cells, also referred to as lymphokine-activated killer (LAK) cells, are stimulated by tumor cells to express cytotoxic activity and to also secrete cytokines such as interferon (IFN) and tumor necrosis factor (TNF ). We previously reported that secretion of cytokines by IL-2-activated T cells (LAK-T cells) is dependent on the initial cross-linking of the T cell receptor (TCR)-CD3-molecular complex, but the cross-linking of accessory molecules, such as LFA-1, CD2, CD44 and CD45, on LAK-T cells can enhance this cytokine production. We have developed an approach involving interspecific gene transfer to define further the contributions of LFA-1 and CD2 to the activation of LAK-T cells. The genes for huICAM-1 (a ligand for LFA-1) and huLFA-3 (a ligand for CD2) were transfected singly and in combination into a null mouse melanoma background, and clonal populations of cells that stably express ICAM-1 and/or LFA-3 were derived. Expression of the introduced ICAM-1 and/or LFA-3 by transfected cells enhanced their ability to bind LAK-T cells; the LFA-1/ICAM-1-mediated binding was not further enhanced by activation with phorbol 12-myristate 13-acetate. ICAM-1- and/or LFA-3-transfected cells, in the presence of immobilized anti-CD3, exhibited a greater ability to stimulate IFN secretion by LAK-T cells compared to the untransfected parental lines. This experimental system, which allows ICAM-1/LFA-1 and CD2/LFA-3 interactions to occur on the LAK-T cell at a site distal from the anti-CD3 signal, extends our understanding of LAK-T cell activation by establishing that both LFA-1/ICAM-1 and CD2/LFA-3 can mediate co-stimulation via adhesion and signaling events.     

Effects of the γ-aminobutyrate transaminase inhibitors gabaculine and γ-vinyl GABA on γ-aminobutyric acid release from slices of rat cerebral cortex

The release of [³H]-aminobutyric acid (GABA) from pre-loaded slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitors gabaculine and -vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 M) substantially reduced the Ca²⁺-dependence of the release of [³H]GABA evoked by a 4 min 30 mM K⁺ pulse, whereas this was not appreciably reduced by the presence of -vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of [³H]GABA release were not identical in the presence and absence of either inhibitor.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

High levels of soluble IFN γ receptor α chain in the plasma of rheumatoid arthritis patients

Soluble receptors for hormones and cytokines have beendescribed. They can serve as natural blockers of theirrespective ligands. The natural soluble interferongamma receptor (sIFNR) has been isolated andcharacterized only in urine. Chromatography of human(hu) plasma from rheumatoid arthritis (RA) patientsand controls on immobilized hu IFN orantibodies against IFN R chainpermitted us to isolate the sIFNR. Thereceptor isolated from one control is a protein witha molecular weight between 60-67 kDa depending on thepresence of reducing agents. We detected asignificantly higher level of plasma sIFNR inpatients with rheumatoid arthritis than in apparentlyhealthy subjects.