The effect of glucocorticoid on the synthesis of biglycan and decorin in human osteoblasts and bone marrow stromal cells

We have previously demonstrated that glucocorticoids induce differentiation of human bone marrow stromal cells (BMSC) into cells expressing mature osteoblast phenotype. As glucocorticoids have marked effects on extracellular matrix protein synthesis in bone, and because proteoglycans are important components of bone matrix and may condition the differentiation and biological activities of osteoblasts, we studied the effects of dexamethasone (Dex) on the synthesis of small proteoglycans [decorin (DCN) and biglycan (BGN)] in adult human BMSC and human osteoblasts (HOB). First passaged HOB and BMSC were treated with either ethanol or 10(-7) M Dex for 7 days. After treatment, the cells were metabolically labeled with either [35S]SO4 alone or [35S]SO4 and [3H]leucine together for 24 h. Conditioned media were collected, and cell layers were extracted with 4 M guanidine HCl. The extracts and conditioned media were subjected to gel filtration and ion exchange chromatography. Fractions containing radiolabeled proteoglycans were analyzed either directly or after immunoprecipitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Dex treatment resulted in a dramatic increase in DCN and an associated decrease in BGN in both the conditioned medium and the cell layer of HOB cultures. In Dex-treated BMSC cultures, BGN was decreased in both the conditioned medium and cell layer, whereas DCN was stimulated in the majority of cultures. Northern blot analysis indicated that steady state messenger RNA (mRNA) concentration of DCN was increased by Dex in all of the HOB cultures and in seven of eight BMSC cultures analyzed. The steady state mRNA level of BGN was decreased by Dex in both HOB and BMSC cultures. The regulation of DCN and BGN mRNA by Dex in both HOB and BMSC (when responsive) was dose dependent. Time-course analysis indicated that as little as 1 day of treatment with Dex was sufficient to decrease BGN mRNA and increase DCN mRNA (when observed) levels in BMSC; the regulation spanned a 4-week interval, during which the extracellular matrix of BMSC was mineralized. The effect of Dex on the steady state mRNA levels of DCN and BGN in HOB was also apparent after 1 day of treatment. These accumulated results suggest that Dex modulates the synthesis of small proteoglycans in both human bone marrow stromal osteoprogenitor cells and mature osteoblasts.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Variation of multifunctional surface binding proteins--a virulence strategy for group A streptococci?

Procollagen and proteoglycan biosynthesis was defined in long-term culture of a human osteogenic sarcoma cell line, SAOS-2. An osteoblast phenotype was maintained by these cells up to 40 days post-confluent in the presence of ascorbic acid. Under these conditions, cells deposited around them an extensive collagenous matrix that was able to mineralize in the presence of an exogenous phosphate donor (beta-glycerophosphate). The collagenous matrix was comprised predominantly of collagen type I with significant amounts of collagen type V, and greater than 80% of the collagen in the matrix was involved in covalent crosslinkages. With increasing time in culture there was a decrease in the collagen synthetic rate, although the collagenous matrix was maintained. The proteoglycans synthesized by nonmineralizing and mineralizing cultures were purified after biosynthetic labeling with [35S]sulfate and [3H]glucosamine. Two major species were apparent: a large chondroitin sulfate proteoglycan (CSPG), and a small chondroitin sulfate proteoglycan, decorin. In nonmineralizing cultures, decorin partitioned equally between the cell layer and culture medium, whereas the large CSPG species partitioned exclusively into the cell layer-associated matrix. In the presence of extensive mineral deposition, greater than 90% of the newly synthesized proteoglycans were secreted into the medium. Northern blot hybridization indicated that SAOS-2 cells express mRNA encoding a range of bone proteins, including decorin, osteonectin, and bone sialoprotein.     

Metabolism of glycine in primary astroglial cells: synthesis of creatine, serine, and glutathione

The metabolism of [2-13C]glycine in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13C]glycine, cell extracts and incubation media were analyzed for 13C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13C]glycine, the amount of de novo synthesized [2-13C]creatine was two-fold increased, and in addition, 13C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-13C] serine, [3-13C]serine, and [2,3-13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.     

The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Ectonucleotidases, purine nucleoside transporter, and function of the bile canalicular plasma membrane of the hepatocyte

Expansion of the glomerular mesangium is a consistent finding of diabetic nephropathy. Negatively charged proteoglycans are an integral part of the mesangium and their synthesis and degradation is disturbed in many forms of glomerulosclerosis. The metabolism of ascorbic acid (AA), which plays an important role in extracellular matrix regulation, is known to be abnormal in diabetes. The action of AA has also been shown to be inhibited by high glucose (HG) concentration. In this study we investigated the effect of AA and HG on proteoglycan (PG) synthesis by examining the incorporation of [35S] sulphate into PG in the cellular, matrix and media components of rat mesangial cell (MC) cultures. MC were grown in 9 or 25 mM glucose for 8 days, with and without the addition of AA. Sulphation of PG was measured by adding 50 microCi of [35S] sulphuric acid to the culture medium and precipitating 35S-labelled PG with cetylpyridinium chloride. In this study AA was shown to have a stimulatory effect on the overall incorporation of [35S] sulphate into cell and matrix PG and this was inhibited by 25 mM glucose. Correcting for protein synthesis and specific activity of [35S] sulphate showed that HG inhibits AA stimulation by decreasing sulphation of the individual PG molecules. These findings may be of particular importance in the pathophysiology of nephropathy in diabetes, a condition where AA concentration is already compromised.     

A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass culture-derived exoantigens

The supernatants of in vitro cultures of Babesia divergens Rouen 1987 in human erythrocytes, obtained by using a semidefined medium based on human high-density lipoproteins, were fractionated by gel filtration chromatography into four fractions, F1 to F4. The crude supernatant as well as each fraction adjuvanted with Quil-A protected gerbils from mortality due to a homologous infectious challenge. Analysis of the humoral response of the 10 protected gerbils with fraction F4, containing major proteins with molecular masses lower than 50 kDa, showed that a few antigens (from 50 to 17 kDa) could be important candidates for an improved vaccine against B. divergens babesiosis. As an immunodominant response was directed against the 37-kDa antigen (Bd37) in two different B. divergens strains tested, a polyclonal antibody directed against Bd37 was produced in a rabbit. In an immunofluorescence assay, the anti-Bd37 antiserum strongly labelled small internal vesicles of the merozoites and the cell surface was diffusely labelled after fixation, whereas on live merozoites, this labelling was not observed. [3H]glucosamine-radiolabelling experiments demonstrate that Bd37 is a glycoprotein. The Bd37 protein can also be labelled with [14C]palmitate but not with [3H]myristic acid. In Triton X-114 temperature phase partitioning of B. divergens-infected erythrocyte extracts, Bd37 was exclusively found into the detergent phase, indicating that the palmitoylated Bd37 protein was in the membrane fraction. In the in vitro supernatant, the glycoprotein Bd37 was found in a nonpalmitoylated form, indicating excretion and/or release of the glycoprotein from the merozoite.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Biosynthesis of growth hormone-releasing factor by fetal rat cerebrocortical and hypothalamic cells

The biosynthesis of growth hormone-releasing factor (GRF) by cerebrocortical tissue is controversial. Although several reports have indicated its presence in certain rat cortical areas and in cultured rat hypothalamic cells, no data exist demonstrating its biosynthesis in these areas. In this study, we have investigated the capacity of fetal rat cerebrocortical and hypothalamic cells in culture for synthesizing GRF. Fetal cerebrocortical and hypothalamic cells were exposed to [3H]Arg for 48 h. Medium and cell extracts were processed and [3H]Arg-IR-rGRF was isolated by affinity chromatography and characterized by HPLC. Intracellular [3H]Arg-IR-rGRF from both hypothalamic and cerebrocortical cells exhibited four major peaks, one of them coeluting with synthetic rGRF. In cerebrocortical cultures, newly synthesized and released [3H]Arg-IR-rGRF showed a similar pattern to the cell content. However, in media from hypothalamic cells, higher hydrophobicity molecular forms were absent. The data demonstrated that fetal cerebrocortical and hypothalamic cells in primary culture synthesize GRF with similar posttranslational processing, but with different molecular patterns of secretion.     

Determinants of tPA antigen and associations with coronary artery disease and acute cerebrovascular disease

A serum-free organ culture model for chondrocyte maturation, using the Avian sternum, was developed. Day-14 chick embryo sterna were placed in organ culture in the presence of defined medium. The optimal medium for chondrocyte terminal differentiation contained specific concentrations of dexamethasone, insulin, thyroid hormone and ascorbic acid. Three parameters, including sternal growth, cell diameter and type X collagen production, were analyzed as indicators of chondrocyte terminal differentiation. These parameters were analyzed in cephalic, middle and caudal regions of the organ-cultured chick sterna and compared to sterna grown in ovo. This study demonstrates that the organ-cultured tissue maintains normal morphological characteristics and terminal differentiation in the cephalic region only, similar to in ovo development, while maintaining normal cell-matrix relationships.     

High-frequency oscillation and centroid frequency of diaphragm EMG during inspiratory loading

Exposure of progenitor cells with chondrogenic potential to recombinant human osteogenic protein-1 [rhOP-1, or bone morphogenetic protein-7 (BMP-7] may be of therapeutic interest in the regeneration of articular cartilage. Therefore, in this study, we examined the influence of rhOP-1 on cartilage formation by human perichondrium tissue containing progenitor cells with chondrogenic potential in vitro. Fragments of outer ear perichondrium tissue were embedded in clotting autologous blood to which rhOP-1 had been added or not (controls), and the resulting explant was cultured for 3 weeks without further addition of rhOP-1. Cartilage formation was monitored biochemically by measuring [³5;S]sulfate incorporation into proteoglycans and histologically by monitoring the presence of metachromatic matrix with cells in nests. The presence of rhOP-1 in the explant at the beginning of culture stimulated [³5;S]sulfate incorporation into proteoglycans in a dose-dependent manner after 3 weeks of culture. Maximal stimulation was reached at 40 microgram/ml. Histology revealed that explants treated with 20-200 microgram/ml rhOP-1, but not untreated control explants, contained areas of metachromatic-staining matrix with chondrocytes in cell nests. These results suggest that rhOP-1 stimulates differentiation of cartilage from perichondrium tissue. The direct actions of rhOP-1 on perichondrium cells to stimulate chondrocytic differentiation and production of cartilage matrix in vitro provide a cellular mechanism for the induction of cartilage formation by rhOP-1 in vivo. Thus, rhOP-1 may promote early steps in the cascade of events leading to cartilage formation. Therefore, rhOP-1 could be an interesting factor for regeneration of cartilage in articular cartilage defects.     

Non-smoking as risk factor for Alzheimer''s disease?

The short-term effect of dexamethasone, a synthetic glucocorticoid, on both fatty acid and cholesterol synthesis has been investigated in rat hepatocyte cultures. Within 4h following hormone addition to the cultures, a noticeable stimulation of labelled acetate incorporation into fatty acids was observed. Similar behaviour was noticed when [3H]H2O was used as an independent index of the lipogenic activity. In the same cultures, however, cholesterol synthesis from both [14C]acetate or [3H]H2O was significantly reduced by dexamethasone addition. In these conditions, no significative variation of cholesterol synthesis starting from labelled mevalonate was observed.     

Analysis of proteins synthesized in mitochondria of cultured mammalian cells. An assessment of current approaches and problems in interpretation

1. The conditions which enable highly efficient utilization of [35S]methionine by cultured mammalian cells and the resolution of selectively labelled mitochondrial products are described. 2. Analysis of mitochondria purified from cells labelled in the presence or absence of inhibitors of cytoplasmic (or mitochondrial) protein synthesis indicated that about 5% of the [35S]methionine incorporated into mitochondrial proteins results from synthesis on mitoribosomes. 3. The electrophoretic profile of the detergent-solubilized proteins of mitochondrial isolated from cells which were labelled in the presence of 50 mug/ml emetine was similar to those obtained with extracts prepared by direct solbuilization of the intact cells after incorporation of label. 4. Pulse-labelling studies suggested that the components resolved by electrophoresis and autoradiography under the conditions described, apparently represent discrete and stable end products radiography under the conditions described, apparently represent discrete and stable end products of mitochondrial protein synthesis. No post-synthetic modification or degradation of these products was detected. 5. Erythromycin was found to suppress the synthesis of additional labelled products which were detected in extracts of one cell line, when analysed by procedures which normally detected only mitochondrially synthesized proteins. These additional bands were attributed to the synthetic activity of Mycoplasma.     

Synthesis of deoxyglucose-1-phosphate, deoxyglucose-1,6-bisphosphate, and other metabolites of 2-deoxy-D-[14C]glucose in rat brain in vivo: influence of time and tissue glucose level

When the kinetics of interconversion of deoxy[14C]glucose ([14C]DG) and [14C]DG-6-phosphate ([14C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [14C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the 14C-labeled compounds in ethanol extracts of brains of rats given [14C]DG. Six 14C-labeled metabolites, in addition to [14C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P2), comprised approximately 5 and approximately 10-15%, respectively, of the total 14C in the brain 45 min after a pulse or square-wave infusion of [14C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P2, and ethanol-insoluble compounds were rapidly labeled after a pulse of [14C]DG, whereas there was a 10-30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [14C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [14C]DG-6-P into other ethanol-soluble and ethanol-insoluble 14C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [14C]DG-1-P can explain the overestimation of the [14C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.     

Cell division and deoxyribonucleic acid (DNA) synthesis in cultures of stimulated lymphocytes

The responses of lymphocytes cultured with various stimulants were analysed with respect to DNA synthesis and cell division. Autoradiographic labelling with [3H]thymidine indicated that similar proportions of cells had incorporated this labelled precursor for DNA synthesis during both short and long periods of exposure to this specific precursor for DNA synthesis. Changes in labelling index (LI) after pulsing these cells with [3H]thymidine showed that exchange of labelled material, which could not be chased out with unlabelled thymidine, was responsible for the increases of LI seen. Failure to prevent this increase with excess unlabelled thymidine indicated that direct incorporation of [3H]thymidine did not account for this exchange. Using hydroxyurea and colcemid arrest to analyse cell cycle events in these cultures, it was shown that approximately 70 per cent of the responding cells in cultures of stimulated lymphocytes, while actively synthesizing DNA, were not in cell cycle for division. It was concluded that DNA turnover, involving synthesis and exchange of newly synthesized material, possibly DNA, was occurring in these cells.     

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.     

Novaco Anger Scale: reliability and validity within an adult criminal sample

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30-100 microM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from approximately 90 microM to less than 1 microM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.     

Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.