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PubMedDate not supplied

Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts.

The 67-kD elastin-laminin receptor (ELR) subunit which carries the recognition site for elastin peptides (EP) is a lectin. Its binding with galactosides can modulate the kinetics of its interaction with its ligand, EP. In this study the biosynthesis of proteins, collagen and fibronectin were evaluated in the presence of agonists and antagonists of the receptor on human skin fibroblasts. The biosynthesis of total proteins determined by 3H-proline incorporation and of fibronectin (by immunoprecipitation) were shown to increase with passage number. The presence of 1 microg/ml kappa-elastin (EP) in the culture medium increased both total proteins and fibronectin biosynthesis. Melibiose, an agonist of the receptor at 5 microg/ml (140 microM), decreased both proteins and fibronectin biosynthesis in the culture medium of human skin fibroblast at the 10th and 15th passage. These results show that the ELR can control the biosynthetic mechanisms of some of the macromolecular constituents of extracellular matrix such as fibronectin and moderate its age and passage-dependent upregulation.

Cells, Cultured↗

PubMed1996

Regulation of fibrinogen biosynthesis by cytokines, consequences on the vascular risk.

High level of fibrinogen in plasma is recognised as an important vascular risk factor. However, it is not known if the increase in fibrinogen is directly responsible for the vascular risk or is a marker of vascular inflammation. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since a parallel effect of cytokines on fibrinogen biosynthesis and on vascular injury was noted. Among the cytokines which induce the synthesis of fibrinogen, oncostatin M (OSM) is the most potent cytokine synthesised by activated monocytes for inducing fibrinogen synthesis by Hep G2 cells (human hepatoma cell line). Interestingly at the same concentrations needed for fibrinogen biosynthesis, OSM induces smooth muscle cell proliferation. In contrast, the cytokines IL-4, IL-10 and IL-13 which have a protective effect against vascular injury leading to atherosclerosis, dose dependently down regulate the biosynthesis of fibrinogen. This was due to both a decrease of IL-6 induced fibrinogen synthesis by hepatocytes, evidenced by a decrease in fibrinogen secretion in the medium and beta chain mRNA expression and to an inhibition of production of the hepatocyte-stimulating activity for fibrinogen biosynthesis (HSF) by LPS-activated monocytes. Noteworthingly, IL-10 induces a significant decrease of the production of OSM by LPS-activated monocytes. In situ activation of monocytes by cytokines in the vessel wall could also contribute to the deposition of fibrin(ogen) derivatives, identified as pathogenic factor.

Arteriosclerosis↗

PubMed1986

Biosynthesis of nuclear proteins after stimulation of quiescent Swiss mouse 3T3 cells.

Stimulation of quiescent Swiss mouse 3T3 fibroblasts either by serum or by the low molecular weight hormones, prostaglandin F2 alpha and insulin, induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or these pure hormones there is an overall increase of two- to fourfold in the rate of biosynthesis of nuclear proteins. In addition, there is a relative decrease in some proteins (Mr = 200 X 10(3), pI 6.0-6.5), while others increase (e.g. actin). Two polypeptides show specific correlations with the exit from G0. The synthesis of p30 (Mr = 30 X 10(3), pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (Mr = 36 X 10(3), pI = 4.25) is first seen at 10-20 h after restimulation. Synthesis of p36 correlates well with the initiation of DNA synthesis. The synthesis of both proteins is stimulated by serum and by the hormones. Thus there are common biosynthetic responses to different stimuli indicating convergent pathways leading to DNA biosynthesis. Addition of hydrocortisone with the growth-stimulatory hormones inhibits both entry into the S phase and biosynthesis of p36. In contrast, hydrocortisone does not alter the biosynthesis of p30. This 'early' protein, p30, is different from the products of both c-fos and c-myc. Therefore, we have identified two specific components that might participate in the regulation of cell proliferation.

Animals↗

PubMed1998

Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (proTRH).

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

Animals↗

PubMedDate not supplied

[Effect of chlorpromazine on K+-dependent regulation of aldosterone biosynthesis in guinea pig adrenal cortex].

The effects of chlorpromazine, a calmodulin inhibitor, on K(+)-dependent regulation of aldosterone biosynthesis were studied in sections and dispersed adrenocortical cells of guinea pigs. K ions in concentrations 5-11 mM significantly (p < 0.01) stimulated aldosterone and protein biosynthesis and protein phosphorylation in sections and cells of the guinea pig adrenal cortex. Using the radioautography of P-labeled polypeptides separated in polyacrylamide gel, the authors have demonstrated K+ enhancement of phosphate incorporation in the proteins with molecular masses of 40 and 75 kD in the cytosols and of 75 kD proteins in the mitochondria. Chlorpromazine in concentration 50 microM reduced K(+)-stimulated steroidogenesis and protein phosphorylation, simultaneously increasing labeled phosphate incorporation in the proteins in low K+ concentrations, not stimulating steroidogenesis. K(+)-stimulated protein biosynthesis was virtually unchanged in the presence of chlorpromazine. Increased K+ concentration in the medium resulted in reduction of cGMP concentration in adrenal cortex sections. The nucleotide level grew in the presence of chlorpromazine in elevated concentrations of K+ and reduced if the ion concentrations were low (1-3 mM). The mechanisms of chlorpromazine effect on K(+)-dependent steroidogenesis changes, protein phosphorylation and cGMP levels in the adrenocortical tissue are discussed, as is the role of calmodulin in K(+)-dependent regulation of aldosterone biosynthesis.

Adrenal Cortex↗

PubMed1991

Cholesterol esterase biosynthesis in rat pancreatic AR42J cells. Post-transcriptional activation by gastric hormones.

This study used the rat pancreatoma AR42J as a model system to investigate the possible regulation of cholesterol esterase biosynthesis in pancreas. Initial experiments were performed to verify the synthesis of pancreatic cholesterol esterase by the AR42J cells. Results indicated that this pancreatoma cell line synthesized two forms of cholesterol esterase (Mr = 71,000 and 74,000). The 74-kDa protein is most likely the precursor protein, and the 71-kDa protein is the matured enzyme secreted by the AR42J cells. The synthesis and secretion of cholesterol esterase were stimulated 2-5-fold by incubating the cells with 0.5-4 nM of cholecystokinin or 0.5-2 nM of secretin. Analysis of the RNA isolated from the hormone-stimulated cells revealed that stimulation of cholesterol esterase biosynthesis was not due to an increase in cholesterol esterase mRNA. Furthermore, inhibition of transcription with actinomycin D has no effect on the hormone-induced cholesterol esterase biosynthesis. Therefore, the mechanism of hormone stimulation was not dependent on de novo RNA synthesis. In vitro translation studies showed that the cholesterol esterase mRNA isolated from stimulated AR42J cells were translated more efficiently than those from control cells. Thus, the increased cholesterol esterase biosynthesis induced by gastric hormones was most likely mediated by posttranscriptional modification of the cholesterol esterase mRNA.

Animals↗

PubMed1988

Localization of sites of lipid biosynthesis in mammalian epidermis.

The end-product of epidermal differentiation is a stratified layer of corneocytes whose extracellular lipid bilayers provide a permeability barrier. It is generally accepted that the epidermis synthesizes most if not all of the lipids found in this tissue and that extra-epidermal tissues contribute very little to this lipid content. Moreover, the individual epidermal strata in which epidermal lipid biosynthesis occurs are not known. To address this question, we examined [3H]H2O incorporation into nonsaponifiable and saponifiable lipids in individual epidermal cell layers 3 hr after intraperitoneal injection into neonatal mice, and compared this to protein and DNA synthesis using intraperitoneal [3H]leucine and [3H]thymidine incorporation, respectively. Lipid biosynthesis was also assessed by [14C]acetate incorporation into lipid fractions in organ cultured skin and in epidermal subpopulations. The in vivo studies demonstrated that the biosynthetic activity of both saponifiable and nonsaponifiable lipids was comparable to, if not greater, in the stratum granulosum (SG) than in basal/spinous (SB + SS) layer, despite significantly lower levels of both protein and DNA synthesis in the SG. On a mass basis, the SG accounts for about four times the biosynthetic activity of the combined SB + SS layers. The lipid biosynthetic activity in vitro also was two- to fivefold higher in the SG, regardless of whether the epidermis was separated into individual cell layers before or after incubations with radiolabel. Moreover, this difference could not be ascribed to increased acetate pools or to elevated metabolism in the SG versus the SB + SS since the rates of CO2 production were much lower in the SG fraction. The increase in lipid biosynthesis in SG over SB + SS was greatest for phospholipids, followed by glycosphingolipids, and free sterols but was observed in almost all lipid classes. These studies show not only that mammalian epidermis is an active site of de novo lipid biosynthesis, but also that this activity remains high in the stratum granulosum, while other forms of metabolic activity are diminishing. These observations are consistent with the knowledge that lipids extruded from the stratum granulosum layer provide the hydrophobic permeability barrier, and further suggest that elevated synthetic activity in the stratum granulosum would allow rapid replenishment in the event that the barrier is damaged.

Animals↗

PubMedDate not supplied

A decreased influence of cannabinoids on macromolecular biosynthesis and cell proliferation in human cells which metabolize polycyclic hydrocarbon carcinogens.

The influence of psychoactive and nonpsychoactive cannabinoids on macromolecular biosynthesis and cell proliferation was compared in normal and transformed human cells which differ with respect to their abilities to metabolize polycyclic hydrocarbon-containing carcinogens. delta 9-THC, delta 8-THC, 11-OH-delta 9-THC, cannabinol and cannabidiol cause a dose-dependent inhibition of [3H]thymidine, [3H]uridine and [3H]leucine incorporation and a dose-dependent inhibition of cell growth in both normal and transformed human cells. The effectiveness of cannabinoids in modifying nucleic acid biosynthesis, protein biosynthesis, and cell growth does not appear to be related to expression of the transformed phenotype; however, cells with enhanced drug metabolizing capacities, including abilities to metabolize polycyclic hydrocarbon-containing carcinogens, are significantly less sensitive to cannabinoid-induced effects on macromolecular biosynthesis and cell growth.

Cannabinoids↗

PubMed1997

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis.

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE2 was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE2, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGF beta and tumor necrosis factor-alpha(TNF). The production of 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) and of PGF2 alpha were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGF beta and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE2 biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6-12 h, and levels had appreciably diminished by 24 h after treatment. Both TGF beta and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE2 resulting from TNF stimulation was blocked by the addition of an interleukin-1 beta (IL-1 beta) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1 beta production. TGF beta regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines.

Adult↗

PubMed2002

Prostaglandin biosynthesis by fat body from larvae of the beetle Zophobas atratus.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from larvae of the tenebrionid beetle, Zophobas atratus. PG biosynthesis was sensitive to incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions of those we examined included 2 mg of microsomal-enriched protein, incubated at 22 degrees C for 2 min at pH 6. These preparations yielded four major PGs: PGA(2), PGE(2), PGD(2) and PGF(2 alpha). PGA(2) and PGF(2 alpha) were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis in low concentrations. In vitro PG biosynthetic reaction conditions, using vertebrate or invertebrate enzyme sources, usually include a cocktail of reaction co-factors. The Z. atratus preparation similarly performs better in the presence of co-factors. Arch.

Animals↗

PubMed2003

Caenorhabditis elegans ubiquinone biosynthesis genes.

Ubiquinone (coenzyme Q, Q) is an essential lipid electron carrier in the mitochondria respiratory chain, and also functions as antioxidant and participates as a cofactor of mitochondrial uncoupling proteins. Caernorhabditis elegans synthesize Q9, but both dietary Q8 intake and endogenous Q9 biosynthesis determine Q balance. Thus, it is of current interest to know the regulatory mechanisms of Q9 biosynthesis in this nematode. Here we review results that leaded to identification of genes involved in Q9 biosynthesis in this nematode using the RNA interference technology. C. elegans coq genes were silenced and depletion of Q content was observed, indicating that the genes related here participate in Q9 biosynthesis. Silenced populations showed an extension of adult life span, probably by the decrease of endogenous oxidative stress produced in mitochondria. We also report the heterologous complementation of C. elegans coq-5 and coq-7 genes in their homologue yeast coq null mutants, leading to restore its ability to growth in non-fermentable sugars. These complemented yeast strains accumulated Q6 but also the intermediate demethoxy-Q6. These findings support the conservative functional homology of these genes.

Animals↗

PubMed1975

Collagen biosynthesis in normal and hypertrophic scars and keloid as a function of the duration of the scar.

The rates of collagen biosynthesis and the tissue concentrations of collagen in normal scar, hypertrophic scar and keloid were determined as a function of the duration of the lesions. The rate of collagen synthesis in normal scar was approximately constant between 6 months and 20 years after the initial wounding, but in both hypertrophic scar and keloid the rate was initially approximately twice that in normal scar, and 2-3 years after wounding it fell to approximately the same level as in normal scar. The tissue concentration of collagen appeared to be relatively constant in normal scar with respect to the time elapsed after wounding, but in both types of abnormal scar it was initially somewhat lower than normal scar and then rose to values higher than in normal scar after about 2-3 years. Since similar results were obtained for both types of abnormal scar in terms of the rates of collagen biosynthesis with respect to the time elapsed after wounding and also in terms of the tissue concentrations of collagen, this may indicate that the events in the pathogenesis of both abnormal scar types are similar. The results also raise the possibility that the formation of both types of abnormal scar may occur in two phases: an initial phase characterized by abnormally high rates of collagen biosynthesis and a later phase characterized by essentially normal rates of collagen biosynthesis.

Cicatrix↗

PubMed1995

Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages.

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381-388, 1992) using THP-1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP-1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reduction in cholesterol biosynthesis.

Acetates↗

PubMed2004

Breakdown of the regulatory control of pyrimidine biosynthesis in human breast cancer cells.

The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.

Allosteric Regulation↗

PubMed1986

Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro.

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.

Animals↗

PubMed1989

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport.

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.

Animals↗

PubMed2002

Ethanol-induced increase of sphingosine recycling for ganglioside biosynthesis: a study performed on cerebellar granule cells in culture.

The effect of ethanol on ganglioside metabolism was assessed in cultured rat cerebellar granule cells. Cells were incubated in the presence of tritiated serine or galactose, and the synthesis of radioactive gangliosides was followed. The rate of de novo biosynthesis of gangliosides labeled in the oligosaccharide moiety (deriving from tritiated galactose) was not affected by the presence of ethanol. On the contrary, the biosynthesis of gangliosides labeled in the ceramide long chain base moiety (deriving from tritiated serine), dramatically decreased in the presence of alcohol. These results suggest that the gap between the extent of the biosynthesis of lipid and polar portions observed in the presence of ethanol, is filled by an increased recycling of sphingosine produced from ganglioside degradation. This hypothesis was confirmed by pulse-chase experiments with GM1 ganglioside, tritiated in the sphingosine moiety, and following radiolabeled gangliosides deriving from its metabolic processing. In fact, the radioactivity carried by gangliosides whose labeling could derive exclusively (GD1b + GT1b) or partially (GD1a) from the recycling of catabolic radiolabeled sphingosine, dramatically increased in ethanol-treated cells during the chase period. Taken together, these results suggest that ethanol increases ceramide sphingosine recycling for ganglioside biosynthesis.

Animals↗

PubMed2006

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis.

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.

Amino Acid Sequence↗