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.     

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.     

Nutrient dynamics in crown tissues of cork-oak (Quercus suber L.)

The seasonal fluctuation of N, P, K, Ca and Mg in leaves, young stems and fruits of young and matureQuercus suber trees growing at the same site was studied. Nutrient dynamics within the tree appeared to be linked to phenological events. Leaves maximized their nutrient contents throughout summer whereas stems maintained rather constant values during most of the year. Before the spring growth flush they apparently acted as temporary reservoirs for some nutrients, translocated from leaves or other organs, so that such nutrients would be easily invested in the new tissues. A significant investment was made in sink organs like flowers and fruits. The tree probably relies not only on leaves but also on other nutrient reservoirs like older branches and roots for an adequate supply to those sinks and to the spring growth. The importance of the sampling dates and the basis of expression used for nutrient concentrations has also been emphasized. Nutrient contents and seasonal trends of the studied trees were similar to those of other evergreen oaks, although somewhat higher values for N, K, and Mg were found. Moreover, soil nutrient contents in the studied montado were much lower than in other Iberic oak stands. However the nutrient status and requirements of cork-oak suggest high (or at least adequate) nutrient availability to the trees. The mechanisms by which this could be achieved are discussed.     

Oxidative Stress Promotes the Regression of Fetal Liver Hemopoiesis

Although apoptosis is believed to play an important role in the ontogenetic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during the perinatal period is not known. Apoptosis is induced by many factors such as a decrease in growth factors and increased oxygen stress. Since hepatic gamma-glutamyl transferase (GT) levels change markedly during the perinatal period in rodents, the metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. Hemopoietic cells but not hepatocytes exhibit significant apoptosis in thiol-free medium and the hemopoietic apoptosis can be inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine, and GSH. The contribution of GSH levels in and around fetal liver cells in the triggering of apoptosis in hemopoietic cells is discussed.     

Functional IFNG polymorphism in intron 1 in association with an increased risk to promote sporadic breast cancer

Interferon (IFN)- is an important Th1 cytokine, which plays a role in immune surveillance and anti-tumor activity. A case-control study involving 54 sporadic breast cancer patients and 144 healthy controls was carried out to explore if the genotype variation of a proposed non-specific enhancer element with a dinucleotide (CA)_n repeat in intron 1 has a role in the susceptibility to promote sporadic breast cancer. Genotype analysis carried out by single-strand length polymorphism and confirmed by sequencing showed an increased frequency of (CA)₁₂ allele (P<0.001) and decreased frequencies of (CA)₁₅ (P<0.01) and (CA)_>15 (p<0.001) alleles in sporadic breast cancer patients as compared to controls. Further, in vitro reporter assays for (CA)₁₂ and (CA)₁₅ alleles suggested these to be associated with decreased and increased expressions, respectively, suggesting the (CA)₁₂/(CA)₁₂ background to act as one of the factors that could lead to low production of IFN-. The study concludes that such genetic background for a proposed non-specific enhancer element with (CA)_n repeat within intron 1 of the IFNG gene might put the individuals with this genotype at higher risk to promote the development of sporadic breast cancer due to a resultant compromised immune surveillance.Anjana Saha and Ashish Dhir contributed equally.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Influence of selenium on glutathione and some associated enzymes in rats with mammary tumor induced by 7,12-Dimethylbenz(a)anthracene

A recent finding in epidemiological and laboratory studies suggests that the ratio of selenium to glutathione is lower in breast cancer subjects than its control counterparts. Selenium, an antioxidant and anticarcinogen, can modify the status of glutathione and some associated enzymes by blocking peroxidation of lipids in membranes of cancer subjects. Studies were conducted using female albino rats of Wistar strain bearing mammary tumor induced by 7,12-dimethylbenz(a) anthracene to assess the biological role of selenium on some antioxidant enzymes associated with the maintenance of glutathione status. For induction of mammary tumor, 25 mg DMBA in a 1 ml emulsion of sunflower oil and physiological saline was injected subcutaneously to each rat. One group in each of control and tumor bearing rats, were fed 5 mg sodium selenite/kg diet from the day of tumor induction for 24 weeks. Increase in the reduced glutathione concentration was preceded by significant increase in the oxidized glutathione as well as in the activities of -glutamylcysteine synthetase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase by selenium administration in rats bearing tumor. However, selenium administration to rats bearing tumor decreased the activity of -glutamyl transpeptidase. These observations clearly demonstrate the influence of dietary selenium supplementation in correcting abnormal changes in glutathione turnover and some associated enzymes in tumor induced rats.     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.     

Modifications of the EEG Activity upon Lateralized Stimulation of the Visual Inputs to the Right and to the Left Brain Hemispheres by Light with Different Wavelengths

In 80 healthy humans, we studied changes in the frequency spectrum and values of the relative spectral coefficients for subsequent 1-Hz-wide frequency bands under conditions of simultaneous lateralized stimulation of the retinal zones, which form receptive fields for the right- and for the left-brain hemispheres; light of different colors was used for stimulation. We found that reactions of the right and left hemispheres to such stimulations demonstrated obvious specificity; spatial characteristics of these reactions were obtained. Changes in the values of most corresponding spectral coefficients in the hemispheres usually had similar directions and demonstrated similarity in their intensities, which is indicative of a complementary pattern of the interhemispheric interaction. Reciprocal changes in the corresponding spectral coefficients in the hemispheres were observed more rarely. Modifications of EEG upon red-green stimulations of different polarity were found to be similar, which can be a manifestation of the moderating influence of the right hemisphere on the left one.     

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.     

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     

Similarity Study on Peptide ?-turn Conformation Mimetics

The ability of a series of structures to mimic the geometric and electronic properties of an ideal –turn has been studied. Initially, an exhaustive conformational analysis was carried out using the molecular dynamics technique at high temperature followed by minimization. Additionally, each minimum was optimized with the semi–ab initio molecular orbital method SAM1. Then, the unique minima found have been superimposed with ideal –turns, classic and inverse, using the SEAL program which takes into account steric and electronic parameters for the superpositions and finally, three molecular similarity indices were determined for each superposition. These indices consider the general steric and electronic characteristics of the structures, as well as, the position of the carbon atoms that correspond to the Cⁱ and Cⁱ⁺² in the peptide chain.     

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     

A theoretical investigation into the lipid interactions of m‐calpain

The protease, mcalpain, has been implicated in a number of pathological conditions. The enzyme is a calciumdependent heterodimer whose activity appears to be modulated by membrane interaction involving a segment, TAMRIL, located in domain V of the protein's small subunit. Based on a sequence analysis of mcalpain, using DWIH and hydrophobic moment plot based methodologies, we have shown that this segment may contribute to a lipid interactive, oblique orientated, helical region. Our results could form a basis for future studies on the postulated lipid modulation of mcalpain activity.     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P₂, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5-[-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P₂. Activation of the GTPase activity of dynamin II by pure POPC was then shown.     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.     

The Presence of 3′–5′ Exonuclease Activity in Rat Brain Neurons and Its Role in Template-Driven Extension of 3′-Mismatched Primers by DNA-Polymerase β in Aging Neurons

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.