Biomedical subjects

C Hebert

Publications and source records attributed to C Hebert.

At least 19 recordsLinked to original sources

PubMed2005

Temporal changes in whole-blood and plasma glutathione in ICU patients with multiple organ failure.

OBJECTIVE: This study investigated the temporal changes in whole-blood and plasma glutathione in ICU patients with multiple organ failure. DESIGN AND SETTING: Prospective and descriptive pilot study performed in an ICU with eight beds at a university hospital. PATIENTS: Critically ill patients (n=11) with multiple organ failure and ICU stay of at least 6 days were consecutively included. Patients with chronic obstructive pulmonary disease (n=21) and healthy volunteers (n=10) were used as reference groups. MEASUREMENTS AND RESULTS: Whole-blood and plasma glutathione were measured every 72 h. Total glutathione and the reduced fraction were determined in whole blood. The oxidized fraction and the redox status were calculated from these values. In plasma only the total concentration was determined. Patients were studied for 6-15 days. Nutrition was supplied according to routines supplying basal needs including glutamine. Both total and reduced glutathione was found to be depleted in whole blood compared to the reference groups. Redox status indicated continuing oxidative stress. Plasma glutathione showed higher values in total concentrations than the reference groups. CONCLUSIONS: This study demonstrates that glutathione remains depleted in whole blood. This contrasts to what has previously been shown in skeletal muscle where a restitution of glutathione concentration is seen.

Adult↗

PubMed2004

[Isolated familial corpus callosum agenesis prognosis].

Agenesia of corpus callosum belongs to a group of cerebral malformations whose prognosis is uncertain. In such cases, assessment of prognosis may benefit from eventual associated fetal, obstetrical or familial features. We report a patient with an isolated corpus callosum agenesia that led to the discovery of a similar malformation in her father. This observation demonstrates that some forms of isolated and familial corpus callosum agenesia could have a favorable outcome. However, the difficulty of the assessment of prognosis in isolated corpus callosum agenesia is emphasized and the question of parental RMI exploration in such a peculiar context is raised.

Adult↗

PubMed2003

Phenotypic switching of VEGF and collagen XVIII during hypoxia in head and neck squamous carcinoma cells.

The present study sought to determine the potential role of stress activated MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating phenotypic switching between angiogenic and angiostatic elements among squamous cell carcinoma (SCC) cell lines. In particular, we investigated the effects of hypoxia and those of cobalt chloride (CoCl(2)), which mimics the hypoxic response including the production of reactive oxygen species, on such phenotypic shifts. The expression and production of collagen XVIII, and CBP2/Hsp47 provided a measure of an angiostatic phenotype, while vascular endothelial growth factor (VEGF) expression was used to assess potential angiogenic states. These studies revealed that hypoxia produced a slight up-regulation of collagen XVIII and CBP2/Hsp47 that was inhibited by the stress kinase inhibitor SB203580 but was unaffected by N-acetylcysteine (NAC). In addition, VEGF expression was increased following hypoxia and this effect was reversed with inhibition of by SB203580. Conversely, CoCl(2) significantly diminished the expression of both collagen XVIII and CBP2/Hsp47 and enhanced VEGF expression. These changes were reversed by the PI3K inhibitor wortmannin and by treating cells with NAC. These studies show that phenotypic switching between collagen XVIII and VEGF is controlled by stress activated kinases under hypoxia, and PI3K signaling pathways as well as reactive oxygen species (ROS) following CoCl(2) treatment. Furthermore, modulation of the angiogenic switch is most profound during Akt activation than during activation of stress activated kinases.

Acetylcysteine↗

PubMed2003

Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates.

In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.

Antibodies, Monoclonal↗

PubMed2002

15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been linked to induction of differentiation, cell growth inhibition and apoptosis in several types of human cancer. However, the possible effects of PPARgamma agonists on human oral squamous cell carcinoma have not yet been reported. In this study, treatment with 15-deoxy-Delta(12,14)-PGJ(2) (15-PGJ(2)), a natural PPARgamma ligand, induced a significant reduction of oral squamous cell carcinoma cell growth, which was mainly attributed to upregulation of apoptosis. Interestingly, rosiglitazone and ciglitazone, two members of the thiazolidinedione family of PPARgamma activators, did not exert a growth inhibitory effect. Given the critical role that the oncogene signal transducer and activator of transcription 3 (Stat3) plays in head and neck carcinogenesis, its potential regulation by PPARgamma ligands was also examined. Treatment of oral squamous cell carcinoma cells with 15-PGJ(2) induced an initial reduction and eventual elimination of both phosphorylated and unphosphorylated Stat3 protein levels. In contrast, other PPARgamma did not induce similar effects. Our results provide the first evidence of significant antineoplastic effects of 15-PGJ(2) on human oral squamous cell carcinoma cells, which may be related to downmodulation of Stat3 and are at least partly mediated through PPARgamma-independent events.

Apoptosis↗

PubMed2002

Glutathione synthetase deficiency: is gamma-glutamylcysteine accumulation a way to cope with oxidative stress in cells with insufficient levels of glutathione?

Glutathione (GSH) plays a major role in the cellular defence against oxidative stress and other vital cellular functions. It therefore seems inevitable that patients with severe depletion of GSH will not survive. However, at least some with glutathione synthetase (GS) deficiency do. This study was done to determine whether these patients have a mechanism to compensate for their GSH deficiency. Cell-free extracts of cultured fibroblasts from 9 patients with GS deficiency and 9 control subjects were analysed by HPLC for low-molecular-weight thiol compounds. The patients' cells contained 7.4 nmol of GSH per mg of protein (median; range 2.8-25.2) compared to 33.0 nmol in control fibroblasts (range 26.7-51.4) (p < 0.01). On the other hand, the patients' cells accumulated 18.1 nmol of gamma-glutamylcysteine (gamma-GC) per mg of protein (median; range 6.9-71.7), whereas the control cells contained 0.1 nmol (range 0.05-0.16) (p < 0.01). The cysteine concentrations in the patients' cells were 20.7 nmol/mg protein (median; range 9.4-52.9) compared to 8.9 nmol in control cells (range 3.0-12.4) (p < 0.01). Cultured fibroblasts from patients with GS deficiency have low levels of GSH, but instead accumulate gamma-GC. We suggest that gamma-GC, which contains both reactive groups of GSH (i.e. the sulphydryl and gamma-glutamyl groups), can compensate for GSH in the cellular defence against oxidative stress. Thus, gamma-GC may alleviate, but only partly prevent, serious consequences of insufficient GSH levels in affected patients. Since the sum of the levels of GSH and gamma-GC in GS-deficient cells (median 31.5 nmol/mg protein, range 16.2-79.0) was similar to the level of GSH alone in control cells (33.0 nmol/mg protein, range 26.7-51.4), we propose that the cultured fibroblasts may have a mechanism to regulate in a coordinated way the levels of GSH and gamma-GC; for instance, by both compounds acting as feedback inhibitors of gamma-GC synthetase.

Adolescent↗

PubMed2001

Chronic exposure of human mesangial cells to high glucose environments activates the p38 MAPK pathway.

BACKGROUND: High glucose (HG) environments activate several protein kinase pathways in mesangial cells, including the mitogen-activated protein kinase (MAPK) pathway, ERK. The p38 MAPK pathway is activated by events that occur in the setting of diabetes, such as protein kinase C (PKC) up-regulation and cellular stresses (osmotic stress and redox changes). Substrates of activated p38 MAPK include transcription factors that are involved in the microvascular complications of diabetes. This current study investigated the mechanisms of HG-mediated activation of p38 MAPK in cultured human mesangial cells (HMCs) and the effects of p38 MAPK activation on the transcription factor activator protein-1 (AP-1). METHODS: HMCs were cultured in 5 mmol/L D-glucose [normal glucose (NG)] or 30 mmol/L D-glucose (HG) for seven days. Cells were also treated with HG for brief periods of time (0.5 to 4 hours) to assess the acute effects of HG on p38 MAPK. Using Western blotting of HMC lysates, changes in the tyrosine and threonine phosphorylation of p38 MAPK were measured. The kinase activity of immunoprecipitated p38 MAPK was determined by an in vitro assay that measured the phosphorylation and activation of MAPKAP kinase-2, an intermediary signaling protein downstream of p38 MAPK. To investigate the role of osmotic stress in HG activation of p38 MAPK, cells were acutely treated with mannitol (25 to 250 mOsm/L x 5 to 60 min) or were grown seven days in media supplemented with mannitol at concentrations iso-osmotic to HG media. To investigate the role of PKC in HG-mediated p38 MAPK activation, HMCs were treated with the PKC inhibitors GF 109203X, Ro 32-0432, or rottlerin during the last several hours of HG treatment. HG conditioned cells were also treated with the antioxidants L-N-acetylcysteine (L-NAC) or diphenyliodonium (DPI) prior to harvest. To determine a functional significance of HG-mediated p38 MAPK activation, the DNA binding of the transcription factor complex AP-1 was measured by electrophoretic mobility shift assay. RESULTS: The p38 MAPK pathway was not activated by the acute addition of HG to the HMCs. However, activation of p38 MAPK in HMCs grown seven days in HG was demonstrated by increased tyrosine and threonine phosphorylation of p38 MAPK proteins and increased kinase activity of immunoprecipitated p38 MAPK. As assessed by a kinase assay, p38 MAPK activity in cells grown in HG for seven days exceeded that of NG cells by more than 250%. This difference was not due to differences in the amount of p38 MAPK protein between the treatment groups. Acute osmotic activation of p38 MAPK occurred at extremely high mannitol concentrations (250 mOsm/L) that exceeded the osmotic stress of acute HG. Furthermore, in cells grown for seven days in mannitol at concentrations similar to HG, p38 MAPK activity was similar to control values. Phorbol ester (PMA) treatment stimulated a twofold increase in p38 MAPK activity. The addition of GFX or Ro 32-0432 to HG cells, at concentrations that inhibited PMA activation of p38 MAPK, did not inhibit the glucose-mediated p38 MAPK activation. Rottlerin, a PKC delta inhibitor, also failed to reverse the HG-mediated p38 MAPK activation. Treatment of HG cells with L-NAC or DPI inhibited the HG-mediated p38 MAPK phosphorylation. As we have previously shown, DNA binding of the transcription factor complex AP-1 was increased in HG cells. This binding was reversed by treatment of the HG cells with the p38 MAPK inhibitor SB 203580. CONCLUSIONS: Chronic exposure of HMC to HG concentrations activates the p38 MAPK pathway. This activation appears to be independent of changes in the amount of total p38 MAPK produced by the cells, independent of chronic osmotic stress and independent of PKC activation. The reversal of p38 MAPK by L-NAC and DPI suggests the glucose-mediated p38 MAPK activation may occur via reactive oxygen species. The activity of AP-1, a transcription factor complex that regulates several genes involved in diabetic nephropathy, is reversed when the p38 MAPK pathway is inhibited. These findings suggest the p38 MAPK pathway may be an important pathway involved in diabetic complications.

Antioxidants↗

PubMed2000

Binding motifs of CBP2 a potential cell surface target for carcinoma cells.

Previously we have shown (Hebert et al. [1999] J. Cell Biochem. 73:248-258) that among many cell lines the CBP2 gene product, Hsp47, eludes its retention receptor, erd2P, resulting in the appearance of Hsp47 on the cell surface associated with the tetraspanin protein CD9. Since Hsp47 possesses a highly restricted binding cleft, random peptide display libraries were used to characterize peptides binding to Hsp47 and then to target this protein on carcinoma cell lines in vitro. Comparison of the clones obtained from panning revealed little specific homology based on sequence alone. To determine whether carcinoma cells expressing Hsp47 could selectively take up the selected bacteriophages, traditional immunofluorescence and confocal microscopy were employed. These studies revealed that phage-displaying Hsp47 binding peptides bound to cell lines expressing Hsp47 and that the peptides were rapidly taken up to a location coincident with Hsp47 staining. These observations were confirmed by cytometric analyses. These data indicate that CBP2 product may provide a molecular target for chemotherapy and/or imaging of malignancies.

Amino Acid Motifs↗

PubMed1999

Spatial and temporal trends of contaminants in Canadian Arctic freshwater and terrestrial ecosystems: a review.

The state of knowledge of contaminants in Canadian Arctic biota of the freshwater and terrestrial ecosystems has advanced enormously since the publication of the first major reviews by Lockhart et al. and Thomas et al. in The Science of the Total Environment in 1992. The most significant gains are new knowledge of spatial trends of organochlorines and heavy metal contaminants in terrestrial animals, such as caribou and mink, and in waterfowl, where no information was previously available. Spatial trends in freshwater fish have been broadened, especially in the Yukon, where contaminant measurements of, for example, organochlorines were previously non-existent. A review of contaminants data for fish from the Northwest Territories, Yukon and northern Quebec showed mercury as the one contaminant which consistently exceeds guideline limits for subsistence consumption or commercial sale. Lake trout and northern pike in the Canadian Shield lakes of the Northwest Territories and northern Quebec generally had the most elevated levels. Levels of other heavy metals were generally not elevated in fish. Toxaphene was the major organochlorine contaminant in all fish analyzed. The concentrations of organochlorine contaminants in fish appear to be a function not only of trophic level but of other aspects of the lake ecosystem. Among Arctic terrestrial mammals, PCBs and cadmium were the most prominent contaminants in the species analyzed. Relatively high levels (10-60 micrograms g-1) of cadmium were observed in kidney and liver of caribou from the Yukon, the Northwest Territories and northern Quebec, with concentrations in western herds being higher than in those from the east. For the organochlorine contaminants, a west to east increase in zigma PCBs, HCB and zigma HCH was found in caribou, probably as a result of the predominant west to east/north-east atmospheric circulation pattern which delivers these contaminants from industrialized regions of central and eastern North America to the Arctic via long-range atmospheric transport. Radiocesium contamination of lichens and caribou has continued to decrease. Significant contamination by PCBs and lead of soils and vascular plants was observed in the immediate vicinity and within a 20-km radius of DEW line sites in the Canadian Arctic. There was also evidence for transfer of PCBs from plants to lemmings. There was no evidence, however, that large mammals such as caribou living in the general area of the DEW line sites had elevated levels of PCBs. There is very limited temporal trend information for most contaminants in biota of Arctic terrestrial and freshwater environments.

Animals↗

PubMed1999

Cell surface colligin/Hsp47 associates with tetraspanin protein CD9 in epidermoid carcinoma cell lines.

Hsps expressed on the cell surface have been associated with tumor invasiveness and used as targets for molecular surveillance. The present study utilized four human oral squamous cell carcinoma cells lines, SCC-4, SCC-9, SCC-15, SCC-25, the murine epidermoid carcinoma cell line LL/2, and primary cultures of human gingival fibroblasts to assess the cell surface expression of colligin/Hsp47, a proposed marker for malignancy. Immunoprecipitation studies following protein crosslinking revealed that Hsp47 was associated with a number of membrane proteins including the tetraspanin CD9. Cytometric analyses were performed to determine the distribution of cell surface colligin/Hsp47 during the phases of the cell cycle. These studies showed that colligin/Hsp47 was not limited to any phase of the cell cycle in epidermoid carcinoma cells. Boyden chamber tumor invasion assays and colloidal gold migration assays utilizing a reconstituted basement membrane (Matrigel), collagen type I, and laminin-5 substrates revealed that cell lines expressing constitutive high levels of colligin/Hsp47 manifested the lowest invasion and migration indices. The incorporation of antibodies against Hsps into the migration and invasion assays, likewise, increased the invasion indices and the phagokinetic migration indices. These data indicate that colligin/Hsp47 is anchored to the cell membrane in a complex with CD9 where it moderates tumor cell invasion and motility possibly by acting as a serpin protein inhibitor or as a receptor for collagen.

Animals↗

PubMed1998

Hsp47 binds to the KDEL receptor and cell surface expression is modulated by cytoplasmic and endosomal pH.

Hsp47 is a novel glycoprotein that binds specifically to procollagen and is retained in the ER by its COOH-terminus RDEL peptide sequence (Satoh, M. et al. Jol. Cell Biol. 1996; 133: 469-83). In this paper, we report that erd2P, the KDEL receptor, is distributed, coprecipitates with, and binds to Hsp47. Also, under stress conditions and lowering of pHi, the cytoplasmic epitope of erd2P is not recognized by erd2P antibodies unless the cells are pretreated with NEM. Coincident with the masking of the cytoplasmic epitope of erd2P, following lowering of pHi, Hsp47 is not retained but eludes its retention receptor to be expressed on the cell surface. Alkalization of the endosomal compartments by treatment with NH4Cl or chloroquine also results in the loss of Hsp47 to the cell surface, presumably by inhibiting the retrieval of trans-Golgi network proteins from the cell surface. The expression of Hsp47 on the cell surface under conditions of stress and alteration of pHi and pHe posture Hsp47 as a serpin family protein that may modulate cell migration during development and invasion and metastasis in cancer.

Amino Acid Sequence↗

PubMed1996

Epidermal growth factor and epidermal growth factor-receptor expression in the mouse dental follicle during tooth eruption.

When the role of exogenous epidermal growth factor (EGF) during tooth eruption was first demonstrated it was strongly suggested that EGF was a natural regulator of eruption. Recent immunohistochemical studies have shown that EGF and EGF-receptors are localized in the dental follicle, alveolar bone and ameloblasts before and during the prefunctional stage of eruption. Localization of mRNA for EGF has also been successfully attempted in mouse incisors and molars. The purpose now was to study the temporal expression of EGF and EGF-receptor genes in the coronal aspect of the dental follicle. First molars from 2-, 5-, 9- and 11-day-old CD-1 mouse neonates were incubated in 1% trypsin for 1.5 h at 4 degrees C. Follicles were carefully separated from the coronal aspect of the molar and processed for RNA extraction. Reverse transcriptase-polymerase chain reaction was performed on each mRNA sample. EGF expression was detected at day 2, 5 and 9 in the coronal aspect of the follicle whereas EGF-receptor expression was found at day 9 only. These findings strongly suggest that cells of the dental follicle are the target of EGF at a specific stage of their development and therefore may have a very important role during eruption.

Actins↗

PubMed1996

Hsp47 and other ER-resident molecular chaperones form heterocomplexes with each other and with collagen type IV chains.

Collagen type IV is a genetically distinct secretory protein that constitutes a major component of the basement membranes. Despite the differences between collagen types I and IV, it appears that collagen IV alpha-chain translation/translocation through the ER should follow similar pathways to those established for procollagen I. Using a collagen IV-producing mouse teratocarcinoma cell line, we sought to determine if evolving nascent chains of collagen IV are associated with Hsp47 and other ER-resident molecular chaperones. We show that Hsp47, Grp78 and Grp94 appear to form heteromeric complexes associated with each other and with nascent chains of collagen IV. In addition, we investigated whether ATP depletion, a condition known to promote stable association of Grp78 and Grp94 proteins with their chaperone substrates, extended this response to include a chaperone independent of ATP, Hsp47. These studies reveal that ATP depletion increased the levels of newly synthesized collagen IV associated with Hsp47, Grp78, Grp94 and with each other. These results indicate that Hsp47, Grp78 and Grp94 exist as oligomers and form heterocomplexes during the maturation of newly synthesized collagen IV chains.

Adenosine Triphosphate↗

PubMed1995

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

Amino Acid Isomerases↗

PubMed1994

Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design.

The conditions for the high cell density fed-batch culture of a Saccharomyces cerevisiae strain producing recombinant hirudin (rHV2-Lys47) have been established. A Leu+ derivative of S. cerevisiae c13ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding rHV2-Lys47 and driven by the MF alpha 1 promoter. In order to develop the fed-batch culture protocol, the recombinant strains' physiology was first of all investigated in chemostat culture. The maximum respiratory capacity of the recombinant strain was observed to be between dilution rates of 0.2 and 0.26 h-1, which is typical for laboratory strains as compared to values published for baker's yeasts. Furthermore, maximum biomass yield and product secretion were observed at a dilution rate of approx. 0.15 h-1. The plasmid segregational stability of the recombinant strain showed that the expression plasmid was stable, irrespective of the dilution rates used, for more than 80 generations of growth between dilution rates of 0.043 h-1 and 0.3 h-1. The chemostat data was used to define a fed-batch process. The fed-batch results demonstrated a biomass production of 60 g l-1 CDW and a high production level of recombinant hirudin of 500 mg l-1. Stability of the expression of the gene coding for rHV2-Lys47 was maintained during all the studied fed-batch conditions. The plasmid copy number in the fed-batch remained constant at approx. 43 at a specific growth rate of 0.12 h-1, whereas it increased by 60-95% at a lower dilution rate (mu = 0.06 h-1). Although a variation of the plasmid copy number could be expected, it was postulated from the experimental data that the observed amplification could have been influenced by an environmental effect due to an accumulation of medium components in the supernatant. The results presented here illustrate the importance of a well-balanced medium when considering the production of a recombinant protein in a high cell density cultivation process with high production levels.

Base Sequence↗

PubMed1994

Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones.

Hsp47, Grp78, and Grp94 have been implicated with procollagen maturation events. In particular, Hsp47 has been shown to nascent procollagen alpha 1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collagen has previously been suggested to be independent of ATP. Grp78 and Grp94 are known to dissociate from its substrates by an ATP-dependent release mechanism. The early association of Hsp47 with procollagen and its relatively late release suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hsp47. Herein, we examined how these events occur in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the release of Hsp47, Grp78, and Grp94 from maturing procollagen is delayed. Thus, in cells experiencing metabolic stress, newly synthesized procollagen unable to properly fold became stably bound to a complex of molecular chaperones. In that Hsp47, Grp78, and Grp94 could be recovered with nascent procollagen and as oligomers in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.

2,2'-Dipyridyl↗

PubMed1993

[Cardiorespiratory and metabolic changes during prolonged exercise at 50% of the maximal load. Application to rehabilitation of severe cardiac insufficiencies].

The object of physical training in patients with chronic cardiac failure and severe left ventricular dysfunction is to improve skeletal muscle adaptation without impairing myocardial function. It is acknowledged that aerobic exercise limits increased ventricular strain. Therefore, the authors decided to determine whether exercise at 50% maximal power load constituted aerobic training. Eight patients with chronic cardiac failure (age 57 +/- 10 years; resting radionuclide ejection fraction: 25 +/- 5%) performed a rectangular submaximal exercise for 20 minutes at 50% of maximal aerobic load (MAL) as determined by a prior triangular exercise test at 10 W/mn (MAL = 92 +/- 26 Watts-Peak VO2 = 14.8 +/- 2.4 ml/Kg/mn). Gaseous exchanges (VO2, VCO2) minute volume (MV) and serum lactates were measured at rest and every 5 minutes during exercise. MV, VO2 and VCO2 increased significantly (p < 0.005) with a change of less than 5% between the 20th and 10th minute. Respiratory quotient was unchanged until the end of exercise and serum lactate levels stabilised between the 10th (3.4 +/- 0.66 mmol/l) and the 20th minute of exercise (3.5 +/- 0.47 mmol/l). No significant relationship was observed between ventilation and serum lactate or between respiratory quotient and serum lactate. These results confirm that exercise at 50% of maximal load is aerobic training even in patients with severe left ventricular dysfunction. This parameter would appear to be more useful than using the heart rate which is often abnormal in chronic cardiac failure (abnormal chronotropic response, atrial or ventricular arrhythmias).

Aerobiosis↗

PubMed1991

Biotin formation by recombinant strains of Escherichia coli: influence of the host physiology.

Strains of Escherichia coli were transformed with different plasmids bearing the gene clusters bioXWF and bioDAYB isolated from the Gram positive bacterium Bacillus sphaericus. These genes encode for the enzymes involved in the metabolic pathway which synthesizes biotin from the precursor pimelic acid. Transformed E. coli strains were grown in bioreactors to reach a biomass of 18 g l-1 cell dry weight in 1 litre batch culture with substrate feeding and approximately 50 g l-1 in 10 l fed batch culture. Improved yields of total vitamers and biotin formed in these processes were achieved after a comparative analysis of different culture conditions, medium compositions, host strains and expression systems. Production of 27 mg l-1 of biotin and 200 mg l-1 of vitamers was achieved in 1 litre batch culture. Using a 10-1 fed batch process, biotin and vitamer concentrations reached maximum values of 45 mg l-1 and 350 mg l-1, respectively.

Bacillus↗