Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

Cystoisospora belli: in vitro multiplication in mammalian cells

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.     

Absorption and enterohepatic circulation of baicalin in rats

Pharmacokinetics of baicalin, in form of its parent drug (BG) and conjugated metabolites (BGM), were studied following intravenous and oral administration of baicalin to intact rats. The enterohepatic circulation of BG and BGM was also assessed in a linked-rat model. Multiple plasma and urine samples were collected, and concentrations of BG and BGM were determined using a liquid chromatography/tandem mass spectrometry method. The concentration of BGM was assayed in the form of baicalein after treatment with beta-glucuronidase/sulfatase. After i.v. administration, plasma concentration of BG rapidly declined with the elimination half-life (T1/2) of 0.1 till 4 h post dose, followed by slight increase from 4-8 h in plasma concentrations after drug administration. These plasma concentrations resulted in a significant prolongation of the terminal elimination half-life of BG (T1/2 TER, 9.7 h). BG also displayed slight increase in plasma concentrations (12-24 h) after oral administration, with T1/2 TER of 12.1 h. Based on the AUC of BG and BGM, the absolute bioavailability of baicalin was 2.2+/-0.2% and 27.8+/-5.6%, respectively. The exposure of baicalin to the systemic circulation was approximately 118-fold lower than that of BGM after oral administration (AUC0-t, 4.43 versus 523.97 nmol.h/mL). The high extent of glucuronidation suggested the possible presence of enterohepatic circulation, which was confirmed in the linked-rat model since plasma concentrations of BG and BGM were observed in bile-recipient rats at 4 to 36 h. The extent of enterohepatic circulation after intravenous administration of baicalin was 4.8% and 13.3% for BG and BGM, respectively. It was determined that 18.7% and 19.3% of the administered baicalin were subjected to enterohepatic circulation for BG and BGM, respectively, after oral administration. These results confirm that BG undergoes extensive first-pass glucuronidation and that enterohepatic circulation contributes significantly to the exposure of BG and BGM in rats.     

Effect of butyrate on the expression of the human preprourokinase gene introduced into Chinese hamster ovary cells

A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K1 cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase production. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the other hand, was inhibited by butyrate concentrations greater than 2.5 mM. The response of cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the intracellular level of urokinase.     

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?10⁶ cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH₄ ⁺ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.     

Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.

VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.     

Evidence for a synergistic interaction between cadmium and endotoxin toxicity and for nitric oxide and cadmium displacement of metals in the kidney.

This study was undertaken to examine changes in Zn and Cu homeostasis in the liver and kidney of rats caused by cadmium (Cd) or lipopolysaccharide (LPS) administration. Twenty-five male, 7- to 8-week-old Wistar rats were divided into five groups: saline only treatment, saline treatment and food deprivation, exposure to a single dose of Cd, exposure to LPS alone, and exposure to Cd + LPS. Changes in plasma nitrate concentrations and hepatic and renal Zn and Cu contents were measured together with urinary excretion rates for the metals and nitrate on 3 consecutive days: 24 h before treatment and 24 and 48 h after treatments. Cd exposure alone for 48 h caused a nearly 2-fold increase in plasma nitrate levels with no changes in urinary nitrate excretion whereas LPS treatment caused plasma nitrate levels to increase by 10-fold and urinary nitrate excretion to increase by 4-fold. Administration of LPS 24 h after Cd exposure caused a 10-fold increase in plasma nitrate concentrations and a 100-fold increase in urinary nitrate excretion compared to the rates prior to LPS administration. These results indicate a synergistic interaction between Cd and LPS toxicity. Cd exposure also caused a marked increase in hepatic Zn levels, but LPS did not cause any changes in hepatic Zn or Cu content. In sharp contrast, both Zn and Cu contents were decreased in the kidneys by 16 and 36% in animals exposed to Cd or LPS. A correlation analysis of measured variables reveals that renal Cu contents were inversely associated with plasma nitrate concentrations while urinary Cu excretion on day 3 showed a strong positive correlation with both urinary nitrate and Cd excretions on the same day. A linear regression analysis shows 20% of the variation in urinary Cu excretion was associated with urinary Cd excretion on the same day. It is concluded that reductions in renal Cu contents caused by Cd or LPS administration may be a result of Cd and NO displacement of Cu previously bound to metallothionein.     

Morphologic and functional correlates of plasma membrane injury during oxidant exposure

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.     

Nuclear pore complex oxalate binding protein p62: its expression on oxalate exposure to VERO cells

Oxalate rich stones are the most common among the various stones. Oxalate binding protein plays a vital role in the transport of oxalate. Nuclear pore complex (NPC) contains a protein of molecular weight 62 kDa and it has maximum oxalate binding activity. The physiological significance of the presence of oxalate binding protein in the NPC is not well understood. In order to study its function, the expression of this protein during oxalate stress condition and the morphological changes on oxalate exposure to synchronized VERO cells have been determined. VERO cells were synchronized at different stages of cell cycle using cell cycle blockers and expression of the NPC p62 was assessed using enzyme linked immunosorbent assay (ELISA) technique with p62 antibody (MAb 414). Expression of NPC p62 was more pronounced in 1.0 mM oxalate concentration in mitotic phase than in S phase, suggesting cell cycle dependency. During oxalate exposure there is cell aggregation and complete degeneration of cell morphology occurs, which in turn lead to the expression of certain genes, including the NPC oxalate binding protein p62. Thus, oxalate induces degeneration of cells (may be due to the lipid peroxidation) and leads to the expression of NPC oxalate binding protein and the expression is of cell cycle dependent manner.     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996     

Cytotoxicity of ferric chloride used as an adjuvant of organic acid precipitation]

The objective of this study was the amelioration of the organic acid precipitation technique currently used as a secondary stage for viruses concentration using ferric chloride (FeCl3) as an adjuvant. To carry out this work successfully, we have studied the cytotoxicity effect of FeCl3 towards cell lines usually used for the investigation of enteric viruses. Two kinds of african green monkey kidney cells, MA104 and BGM, were used as a cellular model. The results showed that optimum concentration of FeCl3 for either BGM or MA104 cell survival and organic acid precipitation varies from 0.5 to 1 mM. The cytotoxicity test used in this work was simple, easy to realize and gives an appreciable information about the cytotoxicity dose of a given biological and chemical product.     

病毒疫苗制备用不同核型VERO细胞系在裸鼠体内致癌/致瘤性的系统实验研究

生产疫苗用细胞系可能具有致瘤性,一些常用的细胞系需要检查不同代次有无致癌性。在建立传代细胞种子库与工作库基础上,对研制生产病毒活疫苗所用8株VERO细胞系在219只裸鼠进行了致癌(瘤)实验。本研究结果表明,VERO细胞染色体核型可发生变异,亚四倍体JA株与超二倍体KA株具有强的致癌性,不能用于致弱活病毒疫苗制备,但可替代HeLa细胞系用作恶性肿瘤阳性对照细胞。筛出无致瘤性的YB、dC、M和JB株亚二倍体VERO细胞系,可替代BHK-21细胞用于狂犬病减毒活疫苗制备。VERO细胞系染色体遗传相对稳定。不同代次变化不大。研究发现细胞染色体遗传特征决定致瘤性质并具有种属特异性,不同核型细胞致瘤性不同,细胞染色体数目变异大小和致癌性成正相关,通过体内外交替选育可在裸鼠体内快速选育成功高变异率肿瘤细胞系。高变异率HeLa或VERO细胞系移植于裸鼠可能产生恶性横纹肌样瘤。因此,应当强调疫苗生产用细胞系致瘤性评价的重要性。     

Comparative development and merozoite production of two isolates of Sarcocystis neurona and Sarcocystis falcatula in cultured cells

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.     

Recombinant human insulin: X. Simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing of Cys residues. The inclusion bodies were dissolved in 8 M urea with addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

A comparison between acute exposures to ethanol and acetaldehyde on neurotoxicity, nitric oxide production and NMDA-induced excitotoxicity in primary cultures of cortical neurons

Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated processes, such as nitric oxide (NO) formation and excitotoxicity. In the present study, we compared the effects of acute ethanol and acetaldehyde on NMDA receptor-mediated excitotoxicity and NO production in primary cultures of rat cortical neurons. The delayed cell death induced by NMDA (300 mM, 25 min) was evaluated by morphological examination and by measuring the release of the cytotoxic indicator, lactate dehydrogenase, in the culture media 24 hours after the NMDA exposure. The accumulation of nitrite, as an index of NO production, was also measured 24 hours after NMDA treatment. NMDA caused a dose-dependent cell death and nitrite accumulation, both effects were blocked by pretreatment of MK-801 (100 microM). Acute exposure to ethanol (1-1000 mM) or acetaldehyde (0.1-1 mM) for 35 minutes did not affect neuronal viability in the following 24-hr period. However, acute exposure to acetaldehyde (> or =10 mM) was neurotoxic. Neither ethanol nor acetaldehyde changed basal nitrite levels in the culture media. Acute ethanol (50-400 mM, 10 min) given before the NMDA treatment (25 min) resulted in a concentration-dependent suppression of the delayed cell death. The NMDA-induced NO production was, however, not affected by ethanol. Neither the NMDA excitotoxicity nor NO production was affected by acute ethanol given after NMDA treatment. Acute acetaldehyde (0.01-0.5 mM, 10 min) given before or after NMDA treatment had no effect on delayed NMDA neurotoxicity and NO production. Our data suggest that acute exposure to ethanol is not neurotoxic and is even protective against delayed NMDA-excitotoxicity when given before but not after NMDA treatment. Neither NO nor metabolism of ethanol to acetaldehyde is required for ethanol-mediated suppression of NMDA excititoxicity. Acetaldehyde, on the other hand, is toxic by itself at low concentrations (> or =10 mM). Furthermore, acute exposure to non-toxic concentrations of acetaldehyde could not protect cortical neurons against NMDA-induced excitotoxicity.     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Effect of lead acetate on Sertoli cell lactate production and protein synthesis in vitro

The effects of lead acetate on protein synthesis and lactate production by cultures of rat Sertoli cells in vitro were studied. Sertoli cell cultures prepared from 20 day old Sprague-Dawley rats were exposed to 0.01, 0.05 and 0.10 mM lead acetate. Lactate production was significantly elevated by all concentrations of lead after 3, 6, 9 and 12 hours of exposure. Protein biosynthesis as measured by [³H]-leucine incorporation was significantly depressed by 0.05 and 0.10 mM lead acetate after 2 hours of exposure. These results support the hypothesis that lead acetate may inhibit spermatogenesis by a disturbance of the metabolic activities of the Sertoli cells.Abbreviations MEM minimal essential medium - TCA trichloroacetic acid     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Summary Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.     

Karyokinetic reactions in rat kidney proximal convoluted segment epithelium on long-term lead supply with drinking water

In the rat kidney proximal convoluted segment epithelium the development of intranuclear inclusion bodies was observed after long-term lead administration with the drinking water. The inclusion bodies were PAS-positive. By electron microscopy they were identified as composed of filaments and granules lacking any kind of surrounding membrane. During the experiment, the nuclear volumes increased commensurately with the duration of lead exposure. The nuclear volume increase was considered a physiological reaction since it began long before the first appearance of intranuclear inclusion bodies.