Purification from human plasma of a tetrapeptide that potentiates insulin-like growth factor-I activity in chick embryo cartilage.

Human plasma has been shown to contain a low molecular weight factor that potentiates human IGF-I stimulation of glycosaminoglycan synthesis in chick embryo cartilage. The peptide was purified and characterized by Edman degradation and electrospray mass spectrometry. The primary structure determined was: Trp-Gly-His-Glu. A homologous synthetic peptide similarly promoted matrix biosynthesis in cartilage exposed to IGF-I.

Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture.

Following hypoxia/reoxygenation (6h/96h), cultured neurons from the embryonic rat forebrain undergo delayed apoptosis. To evaluate the participation of oxidative stress and defense mechanisms, temporal evolution of intraneuronal free radical generation was monitored by flow cytometry using dihydrorhodamine 123, in parallel with the study of transcriptional, translational, and activity changes of the detoxifying enzymes Cu/Zn-SOD and Mn-SOD. Two distinct peaks of radical generation were depicted, at the time of reoxygenation (+ 27%) and 48 h later (+ 25%), respectively. Radical production was unaffected by caspase inhibitors YVAD-CHO or DEVD-CHO, which prevented neuronal damage, suggesting that caspase activation is not an upstream initiator of radicals in this model. Cell treatment by vitamin E (100 microM) displayed significant neuroprotection, whereas the superoxide generating system xanthine/xanthine oxidase induced apoptosis. Transcript and protein levels of both SODs were reduced 1 h after the onset of hypoxia, but activities were transiently stimulated. Reoxygenation was associated with an increased expression (139%), but a decreased activity (21%) of the inducible Mn-SOD, whereas Cu/Zn-SOD protein and activity were low and progressively increased until 48 h post-hypoxia, when the second rise in radicals occurred. In spite of a temporal regulation of SODs, which parallels radical formation, oxidative stress might account for neurotoxicity induced by hypoxia.

Stimulation of translation by reactive oxygen species in a cell-free system.

Wheat germ lysate was used as a model system for in vitro translation. We show that an increase of the exchange surface between the reaction mixture and the atmosphere enhanced the amount of incorporated cysteine, indicating that early arrest of protein synthesis previously observed in such a system was due to oxygen starvation in the reaction mixture. This hypothesis was confirmed since the amount of proteins synthesized and the rate of translation increased when oxygen was added. We show that an addition of hydrogen peroxide to the translation mixture had the same effect as oxygen, allowing us to postulate that stimulation could be due to a common property between both molecules: the oxidizing behaviour. Free radicals in in vitro translation were believed to be involved since the utilization of iron chelating agents inhibited translation. This hypothesis was emphasized by the positive effect of a free radical generating system and the negative effect of free radical scavengers. These experiments suggest that the superoxide radical plays an important role in in vitro translation.

Improved long-term storage of hybridomas at -80 degrees C using a bovine milk derivative.

A medium comprising 40% bovine milk fraction and 10% DMSO (medium A) was used for the long-term storage of hybridomas at -80 degrees C. The viability of the cells, their growth recovery and ability to secrete antibody were studied and the results were compared to those obtained after storage in a medium containing 40% fetal calf serum and 10% of DMSO (medium B). Hybridomas have been kept for 2 years in medium A; the viability of such cells was 75%, the cells were healthy (electron microscopy), they rapidly proliferated when they were cultured in RPMI supplemented with 10% FCS or with 9% milk fraction + 1% FCS and they released measurable levels of antibody. In contrast, hybridomas stored under the same conditions but in medium B died after 6 months.

Screening of new antioxidant molecules using flow cytometry.

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.

Comparison of norepinephrine and dobutamine to epinephrine for hemodynamics, lactate metabolism, and gastric tonometric variables in septic shock: a prospective, randomized study.

OBJECTIVES: To compare the effects of norepinephrine and dobutamine to epinephrine on hemodynamics, lactate metabolism, and gastric tonometric variables in hyperdynamic dopamine-resistant septic shock. DESIGN: A prospective, intervention, randomized clinical trial. SETTING: Adult medical/surgical intensive care unit in a university hospital. PATIENTS: 30 patients with a cardiac index (CI) > 3.51 x min(-1) x m(-2) and a mean arterial pressure (MAP) < or = 60 mmHg after volume loading and dopamine 20 microg/kg per min and either oliguria or hyperlactatemia. INTERVENTIONS: Patients were randomized to receive an infusion of either norepinephrine-dobutamine or epinephrine titrated to obtain an MAP greater than 80 mmHg with a stable or increased CI. MEASUREMENTS AND MAIN RESULTS: Baseline measurements included: hemodynamic and tonometric parameters, arterial and mixed venous gases, and lactate and pyruvate blood levels. These measurements were repeated after 1, 6, 12, and 24 h. All the patients fulfilled the therapeutic goals. No statistical difference was found between epinephrine and norepinephrine-dobutamine for systemic hemodynamic measurements. Considering metabolic and tonometric measurements and compared to baseline values, after 6 h, epinephrine infusion was associated with an increase in lactate levels (from 3.1 +/- 1.5 to 5.9 +/- 1.0 mmol/l;p < 0.01), while lactate levels decreased in the norepinephrine-dobutamine group (from 3.1 +/- 1.5 to 2.7 +/- 1.0 mmol/l). The lactate/pyruvate ratio increased in the epinephrine group (from 15.5 +/- 5.4 to 21 +/- 5.8; p < 0.01) and did not change in the norepinephrine-dobutamine group (13.8 +/- 5 to 14 +/- 5.0). Gastric mucosal pH (pHi) decreased (from 7.29 +/- 0.11 to 7.16 +/- 0.07; p < 0.01) and the partial pressure of carbon dioxide (PCO2) gap (tonometer PCO2-arterial PCO2) increased (from 10 +/- 2.7 to 14 +/- 2.7 mmHg; p < 0.01) in the epinephrine group. In the norepinephrine-dobutamine group pHi (from 7.30 +/- 0.11 to 7.35 +/- 0.07) and the PCO2 gap (from 10 +/- 3.0 to 4 +/- 2.0 mmHg) were normalized within 6 h (p < 0.01). The decrease in pHi and the increase in the lactate/pyruvate ratio in the epinephrine group was transient, since it returned to normal within 24 h. CONCLUSIONS: Considering the global hemodynamic effects, epinephrine is as effective as norepinephrine-dobutamine. Nevertheless, gastric mucosal acidosis and global metabolic changes observed in epinephrine-treated patients are consistent with a markedly inadequate, although transient, splanchnic oxygen utilization. The metabolic and splanchnic effects of the combination of norepinephrine and dobutamine in hyperdynamic dopamine-resistant septic shock appeared to be more predictable and more appropriate to the current goals of septic shock therapy than those of epinephrine alone.

Effect of L-carnitine and acylcarnitine derivatives on the proliferation and monoclonal antibody production of mouse hybridoma cells in culture.

The effect of L-carnitine (Cn) on cell growth metabolism and antibody production rates was investigated using the murine hybridoma cell line, Mark3, in batch and fed harvest cultures. Two acylcarnitine derivatives were also tested: palmitoyl L-Cn and acetyl-DL-Cn. The addition of 20 microM L-Cn to cultures of Mark3 hybridoma cells that had been adapted to L-Cn significantly stimulated monoclonal antibody (mAb) production without affecting cell growth. In contrast, mAb secretion slightly decreased when L-Cn was added to culture of cells that had not been adapted to L-Cn. Palmitoyl L-Cn also stimulated mAb production by adapted cells, whereas the acetyl-DL-Cn, reduced mAb secretion. The presence of L-Cn in the medium did not affect glucose consumption or lactate production, but the metabolism of some amino acids was altered. The medium concentrations of valine, leucine, isoleucine and lysine were enhanced from 22% to 41% according to amino acids, whereas those of alanine, glycine and proline decreased. The mechanism by which L-carnitine affects mAb production and the metabolism of some amino acids is unknown. This effect is likely to be indirect, since there was no net entry of L-[3H]carnitine into the cells.

Insulin utilization and kinetic effect on hybridoma metabolism in batch and continuous cultures.

This paper discusses the insulin utilization kinetics and the effect of its concentration during batch and continuous mass cultures of the murine VO208 hybridoma cells, using a home-made serum-free medium. Our results show that insulin is utilized by the cells, with a specific rate of 1 relative units (RU) per 10(9) cells per h in batch culture. In continuous reactor running at different insulin levels, this consumption rate is observed to vary from 0.13 to 0.55 RU per 10(9) cells per h when the insulin activity increases from 0.3 to 35 RU l-1 and then to stabilize for higher insulin levels until 110 RU l-1. A low insulin amount in the medium around 0.01 RU l-1, which is near physiological levels, is found sufficient to promote the cell growth. Interestingly, we observe that too high insulin levels, above 25 RU l-1, induce a reduction of the cell density due to an inhibitory effect on the maximal specific cell growth rate. Furthermore, the specific rate of MAb production is found to be independent of the insulin amount in the medium.

Expression of secreted recombinant human insulin-like growth factor-II (IGF-II) in Chinese hamster ovary cells.

Chinese hamster ovary (CHO-KI) cells were cotransfected with a plasmid pcDNAI containing the human preproinsulin-like growth factor II cDNA linked downstream to the human cytomegalovirus promoter and with a plasmid containing the neomycin resistance gene (pMAM-neo). CHO neo+ were selected by growth in medium supplemented with G418 geneticin. After amplification, the neomycin-resistant clones were screened for IGF-II production. IGF-II produced was identified by dot blot and quantified by ELISA. The clones C24, C40 and C94 secreted IGF-II at about 350-400 ng per 10(6) cells per day. DNA analysis of C24 and C40 CHO cells by PCR demonstrated the presence of the IGF-II construct in the transfected cells, presumably integrated into the chromosomal DNA. IGF-II produced by CHO cells and purified by RP-HPLC was a mitogen for MCF-7 stimulating mitosis 2-fold.

Methods for reducing the ammonia in hybridoma cell cultures.

The factors which limit the proliferation of eukaryotic cells in vitro are still not well known. Ammonia is believed to be toxic for mammalian cell proliferation and secretion. We have tried two approaches to reducing the ammonia in the medium. We first limited the ammonia produced by the cells by replacing glutamine by glutamate. Then, we used two chemical engineering methods to eliminate accumulated ammonia. In one the used medium was passed through a natural cation exchanger: the clinoptilolite. In the other, the culture medium was passed through a hydrophobic microporous hollow fiber module. Replacing the glutamine by glutamate reduced the medium ammonia concentration. The physicochemical removal of ammonia induced a better cell growth, but not a better specific antibody secretion.

Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM.

This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pHi) of cultured cells, and monitors the changes in pHi in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pHi was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pHi of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pHi increased during the exponential growth phase and dropped towards cell death. The pHi of the chick fibroblasts in monolayer culture was 7.30.

Search for cell proliferation markers suitable for cell count in continuous immobilized cell bioreactors.

The control of hybridoma cell cultures in bioreactors requires the use of convenient indicators to monitor the proliferation of the biomass. In order to select appropriate indications, we followed the variations of several compounds including tumoral markers, polyamines, sialic acids, purine and pyrimidine bases, enzymes and metabolites such as glucose, lactate and amino acids, and the variations of cell density during batch culture. Significant correlations were found between the number of viable cells and alkaline phosphatase, beta-glucuronidase, glucose and lactate measured in the culture medium of hybridoma strains. The correlation calculated from alkaline phosphatase and beta-glucuronidase concentrations in culture medium underestimated cell number. The correlation established with glucose and lactate gave the best indication of cell proliferation in continuous culture with an immobilized cell bioreactor. Finally, the exact quantification of the biomass in these culture conditions can be obtained using the mean of glucose and lactate correlations.

Bovine beta-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry.

Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).

Telithromycin (HMR 3647) achieves high and sustained concentrations in tonsils of patients undergoing tonsillectomy.

Telithromycin, the first ketolide antimicrobial to be developed for clinical use, has potent activity against group A beta-haemolytic streptococci (GABHS), including macrolide-resistant strains. The penetration of telithromycin into tonsils was assessed in 22 adults undergoing tonsillectomy at 3, 12 or 24 h after the fourth dose of oral telithromycin 800 mg once daily. Telithromycin rapidly penetrated tonsillar tissues, achieving a mean concentration of 3.95 mg/kg at 3 h post dose, 3.4 times greater than the corresponding plasma concentration (1.22 mg/l. The mean tonsil:plasma concentration ratio increased to 13.1 at 24 h post dose, indicating slower elimination from tonsils than plasma. Tonsillar and plasma concentrations exceeded the MIC(50) for GABHS throughout the 24-h dosing period. These findings suggest that telithromycin may be an effective new alternative treatment for GABHS tonsillopharyngitis.

Determination of oxalates in plasma and urine using gas chromatography.

A gas-chromatographic procedure for the analysis of oxalic acid is described. The procedure requires relatively small quantities of urine (1 ml) or plasma (5 ml). The procedure consists of three steps: (1) extraction of exalic acid by acidified diethyl ether; (2) esterification by isopropanol; and (3) final analysis by gas chromatography. The oxalic acid was found to have an elution temperature of 107 degrees C and a retention time of 14 min. The standard curve is linear up to 800 nmol. In the described condition the lower limit of detection is 20 nmol. The mean normal plasma and urine oxalate levels (mean +/- 2 S.D.) were found to be 20 mumol/l +/- 17.5 and 275 mumol/24 h +/- 200 respectively.

Assay of a seric human hexapeptide (HWESAS) using a monoclonal antibody and ELISA.

Human serum contains low-molecular-weight growth factors potentiating some in vitro biological effects of IGF-I and IGF-II and recently two peptides were mainly identified: HWESAS and WGHE. In order to determine seric HWESAS concentration, a specific monoclonal antibody against HWESAS was prepared. Its specificity was studied by inhibition tests: this antibody cross-reacts with Y-HWESAS, Cys-HWESAS. It does not react with HWESAS when its COOH is blocked, or with HWE, WGHE and tryptophan or with C3f (SSKITHRIHWESASLLR) which is a fragment of human complement containing HWESAS motif. Its affinity was measured by non competitive enzyme immunoassay (3.89+/-2.44.10(8) M(-1)). Then, this antibody was used in enzyme-linked immunosorbent assay (ELISA) and the preliminary assays were performed to detect HWESAS in serum. In contrast to healthy subjects, patients with chronic renal failure exhibited undetectable concentration of hexapeptide while after successful renal transplantation values increased to reach levels found in healthy subjects and varying according to post-operative evolution. These data are a strong hint that the kidney plays an important role in the production of this hexapeptide and underly the clinical interest of HWESAS detection in renal pathology.

A seric low molecular weight factor modulates IGFs effects on chick cartilage metabolism.

The effects of recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) alone and in combination with a partially purified serum low molecular weight growth factor (LMW-GF) were studied by measuring proteoglycan (PG) and total protein synthesis by chick embryo cartilage. rhIGF-I alone did not increase the incorporation of L[3H]serine or [35S]Na2SO4 into whole cartilages. LMW-GF + rhIGF-I markedly increased the incorporation of these precursors. rhIGF-I alone stimulated D[3H]glucosamine uptake, and LMW-GF increased the effect of rhIGF-I. These results for whole cartilage were reproduced with glycosaminoglycans (GAG) extracted from cartilage. LMW-GF acted in synergy with rhIGF-I to stimulate PG core protein synthesis, xylosyl transferase activity and sulfation. Total protein synthesis, as measured by [35S]methionine uptake, was not altered by rhIGF-I. LMW-GF plus rhIGF-I increased the incorporation of this precursor into whole cartilage. The effect of rhIGF-II on PG synthesis was different from that of rhIGF-I. rhIGF-II alone stimulated GAG chain lengthening and sulfation. LMW-GF did not modify the effect of rhIGF-II on these steps. In contrast, rhIGF-II did not stimulate the synthesis of core protein. LMW-GF plus rhIGF-II increased the [3H]serine incorporation into the whole cartilage, but this combination did not stimulate the uptake of [3H]serine into extracted GAG. rhIGF-II plus LMW-GF were also without effect on xylosyl transferase activity. The combination of these factors increased the [35S]methionine incorporation into cartilage total proteins. These results suggest that rhIGF-II does not regulate the PG core protein synthesis, but in combination with LMW-GF, stimulates the synthesis of proteins other than proteoglycan core protein.

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma.

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.

Comparison of metabolism of hybridoma cells cultured in media supplemented with whey or fetal calf serum.

The effects of industrial whey on cell physiology were studied in a repeated fed-harvest mode using free-suspended murine hybridoma cells. After several days of culture in medium containing 9% whey and 1% fetal calf serum (FCS), cell growth and viability, carbohydrate, amino acid, energy metabolism, and antibody production rates were investigated. Differences were found between cells cultured in whey medium and those cultured in conventional FCS medium. The cell growth obtained in medium supplemented with fresh whey was similar to that obtained in FCS medium. The viability showed an increase for hybridoma cells cultured in whey medium. Glucose consumption rates were similar, whereas the lactate production rate was higher in whey medium. The metabolic uptake rates of glutamine and ammonia increased in whey medium. More alanine, glutamate, glycine, and proline were produced; their production partly came from glutamine and lactate. The consumption rates of branched amino acids changed little; their utilization was higher in whey medium. Finally, antibody productivity was increased about 20% for cells cultured in medium containing whey.

Development and validation of a methodology for intracellular pH measurements of hybridoma cells under bioreactor culture conditions.

The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes. It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment. We developed and validated therefore a methodology that permits a reproducible and reliable pH(i) measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM (BCECF = 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), and analyzed by flow cytometry. Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured. Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.