Amounts Of Fetal Bovine Serum Research Articles

Elucidating the unexpected cell adhesive properties of agarose substrates. The effect of mechanics, fetal bovine serum and specific peptide sequences

2D agarose substrates have recently been surprisingly shown to be permissive for cell adhesion, depending on their mechanics and the use of the adhesive proteins of fetal bovine serum (FBS) in the cell culture medium. Here, we elucidate how the cells exhibit two anchoring mechanisms depending on the amount of FBS. Under low FBS conditions, the cells recognize the surface-coupled adhesive sequences of fibronectin via the binding of the heterodimer α5β1 integrin. Functionality of the actomyosin axis and mechanoactivation of focal adhesion kinase (FAK) are essential for the stretching of the protein, thereby accessing the “synergy” PPSRN site and enhancing cell adhesion in combination with the downstream RGD motif. Under high FBS conditions, the specific peptide sequences are much less relevant as the adsorbed serum proteins conceal the coupled fibronectin and the cells recognize the adhesive protein vitronectin, which is constitutively present in FBS, via the binding of the heterodimer αvβ3 integrin. Similarly, the intracellular tension and FAK activity are decisive, which collectively indicate that the cells stretch the partially cryptic RGD site of vitronectin and thus make it more accessible for integrin binding. Both anchoring mechanisms only work properly if the agarose substrate is mechanically compliant in terms of linear stress-strain response, unraveling a critical balance between the mechanics of the agarose substrate and the presentation of the adhesive peptides. Statement of significanceIn the context of biomaterial design, agarose hydrogels are known to lack intrinsic cell-adhesive peptide motifs and are therefore commonly used for the development of non-permissive 2D substrates. However, we unexpectedly found that agarose hydrogels can become permissive substrates for cell adhesion, depending on a compliant mechanical response of the substrate and the use of fetal bovine serum (FBS) as protein reservoir in the cell culture medium. We describe here two anchoring mechanisms that cells harness to adhere to agarose substrates, depending on the amount of FBS. Our results will have a major impact on the field of mechanobiology and shed light on the central role of FBS as a natural source of adhesive proteins that could promote cell anchoring.

Insulin-transferrin-selenium promote formation of tissue-engineered vascular grafts in early stage of culture

To create tissue-engineered vascular grafts (TEVGs) in vitro, vascular smooth muscle cells (VSMCs) must function effectively and produce sufficient extracellular matrix (ECM) in a three-dimensional space. In this study, we investigated whether the addition of insulin-transferrin-selenium (ITS), a medium supplement, could enhance TEVG formation. PGA fabric was used as the scaffold, and 1% ITS was added to the medium. After two weeks, the tissues were examined using electron microscopy and staining. The ITS group exhibited a denser structure and increased collagen production. VSMCs were cultured in two dimensions with ITS and assessed for collagen production, cell growth, and glucose metabolism. The results showed that ITS supplementation increased collagen production, cell growth, glucose utilization, lactate production, and ATP levels. Furthermore, reducing the amount of fetal bovine serum (FBS) in the medium did not affect the TEVGs or VSMCs when ITS was present. In conclusion, ITS improves TEVG construction by promoting VSMCs growth and reducing the need for FBS.

An effective cytokine combination for ex vivo expansion of porcine muscle stem cells

Cultured meat technology is a novel and promising strategy for sustainable and effective meat production. Muscle stem cells are widely used as seed cell populations because of their ease of access and myogenic differentiation potential. However, muscle stem cells are difficult to continuously propagate ex vivo and are heavily dependent upon serum for survival and growth, which impedes their commercial use as a cultured meat source. Herein, we identified an effective four-cytokine combination to promote long-term proliferation of porcine muscle stem cells using a serial elimination screening approach, which was consisted of long chain human insulin growth factor-1, platelet derived growth factor BB, basic fibroblast growth factor, and epidermal growth factor. The expansion of muscle stem cells with the four-cytokine combination achieved a 6.31 × 107-fold increase in cell number after 28 days of culture with retained cell myogenic differentiation potential, and most importantly, reduced the amount of fetal bovine serum required for cell culture by at least 5%. Furthermore, the four-cytokine combination exerted the pro-proliferative function by activating PI3K/Akt/mTOR and MEK/ERK signaling pathways. This approach provides for a new means by which to industrialize the production of cultured meat.

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon.

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.

Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment.

Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment.

Adsorption of plasma proteins and fibronectin on poly(hydroxylethyl methacrylate) brushes of different thickness and their relationship with adhesion and migration of vascular smooth muscle cells.

The surface-grafted poly(hydroxylethyl methacrylate) (PHEMA) molecules were demonstrated to show a brush state regardless of their molecular length (molecular weight). Adsorption of proteins from 10% fetal bovine serum (FBS), fibronectin (Fn) and bovine serum albumin (BSA) was quantified by ellipsometry, revealing that the amounts of FBS and Fn decreased monotonously along with the increase of PHEMA thickness, whereas not detectable for BSA when the PHEMA thickness was larger than 6 nm. Radio immunoassay found that the adsorption of Fn from 10% FBS had no significant difference regardless of the PHEMA thickness. However, ELISA results showed that the Arg-Gly-Asp (RGD) activity of adsorbed Fn decreased with the increase of PHEMA thickness. By comparison of cellular behaviors of vascular smooth muscle cells (VSMCs) being cultured in vitro in the normal serum-containing medium and the Fn-depleted serum-containing medium, the significant role of Fn on modulating the adhesion and migration of VSMCs was verified. Taking account all the results, the Fn adsorption model and its role on linking the biomaterials surface to the VSMCs behaviors are proposed.

51 ARTIFICIAL DORMANCY OF BOVINE EMBRYOS FOR A MAXIMUM OF 7 DAYS USING A SIMPLE MEDIUM

The worldwide pregnancy rate using cryopreserved mammalian embryos has not improved over the past 2 decades, probably because the freeze-thawing processes cause significant damage. Therefore, it is now relevant to examine the feasibility of short-term non-freezing preservation, and whether this could be applied to embryos that have high vitality and are to be transferred into recipients within several days. We introduce here an artificial dormancy fluid that can extend the hypothermic storage period of bovine embryos for a maximum of 7 days. First, to examine the effect of different basal media and the optimal concentration of fetal bovine serum (FBS) for hypothermic preservation, bovine blastocysts produced in vitro were stored at 4°C in a plastic ministraw in 1 of the following 3 media: PBS, medium 199, or Leibovitz L15 with various amount of FBS (0, 5, 20, 50, or 100%) for 3 days. Second, to examine the effect of Good's buffers, bovine embryos produced in vivo (morula to blastocyst stages) were stored at 4°C in a plastic ministraw in medium 199 plus 50% FBS supplemented with various Good's buffers [HEPES, TES, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), MOPS, and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS)] for 7 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability rate of the embryos was assessed at the end of the culture period. Finally, to observe the pregnancy rate of chilled embryos, 32 embryos produced in vivo were stored at 4°C for 7 days in medium 199 plus 50% FBS supplemented with HEPES. Following hypothermic preservation, the chilled embryos were transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda electronics Co. Ltd, Toyohashi, Japan) on Day 60 of gestation. Data were analysed using the chi-squared test. The viability rate of the embryos after hypothermic storage for 3 days was significantly increased for medium 199 plus 50% FBS [27/30 (90%)] compared with PBS [18/30 (60%)] or Leibovitz L15 [15/30 (50%)] plus 50% FBS (P < 0.05). Chilled embryos stored for 7 days in medium 199 plus 50% FBS supplemented with HEPES had much higher survival than embryos stored in the same medium with other Good's buffers. The pregnancy rate of the chilled embryos stored for 7 days was extremely high [24/32 (75%)] and normal live calves were delivered at term. In conclusion, maintaining artificial dormancy of bovine embryos for 7 days using a simple medium appears to be feasible. This is the first documented success of storing chilled mammalian embryos in a viable state for 7 days. To be of practical value, bovine embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

Nuclear Receptor CAR Requires Early Growth Response 1 to Activate the Human Cytochrome P450 2B6 Gene

The nuclear receptor CAR (constitutive active/androstane receptor) is a drug-sensing transcription factor, regulating the hepatic genes that encode various drug-metabolizing enzymes. We have now characterized the novel regulatory mechanism by which the signal molecule EGR1 (early growth response 1) determines CAR-mediated activation of the human CYP2B6 (cytochrome P450 2B6) gene. The CYP2B6 enzyme metabolizes commonly used therapeutics and also activates pro-drugs. The CAR directly binds to the distal enhancer element of the CYP2B6 promoter, which is essential in converging to its drug-sensing function onto promoter activity. However, this binding alone is not sufficient to activate the CYP2B6 promoter; the promoter requires EGR1 to enable CAR to activate the CYP2B6 promoter. Upon stimulation by protein kinase C, EGR1 directly binds to the proximal promoter and coordinates the nearby HNF4alpha (hepatocyte-enriched nuclear factor 4alpha) with CAR at the distal enhancer element to activate the promoter. Thus, synergy of drug activation and the stimulation of cellular signal are necessary for CAR to activate the CYP2B6 gene.

두 가지 곤충 세포주에 대한 배양 및 바이러스 증식을 위한 최적 FBS 농도 결정

곤충 세포주 Sf21과 Bm5 세포주에 대해 세포 배양과 바이러스의 증식을 위한 최적 FBS 의 농도를 결정하기 위하여 다양한 FBS 농도에서 세포 및 바이러스의 증식 곡선을 비교하였다. 세포의 생존율, 증식속도, 증식량 그리고 FBS 함량을 모두 고려할 때 Sf21 에 대해서는 7%가, Bm5에 대해서는 5% FBS 가 최적 농도로 결정되었다. 바이러스의 증식은 감염 후 5일째에 두 세포주 모두 모든 FBS 농도에서 유사한 증식량을 보였으나, 감염 후 2 일과 3일에 있어서는 Sf21 은 각각 10%와 3%가 Bm5 에 대해서는 양일 모두 5% FBS 농도에서 가장 증식량이 높았다. 이러한 결과는 목적에 따라 세포 및 바이러스 증식을 위한 적정 FBS 농도의 결정이 필요함을 제시하는 것이다. To determine the optimal concentration of fetal bovine serum (FBS) on the growth of insect cells and the multiplicity of viruses, the growth of cells (Sf21 and Bm5) and viruses were examined on the various concentrations of FBS. In view of the viability, growth speed, proliferation of cells and the amount of FBS, the most proper concentration for the cell culture were 7% and 5% for Sf21 and Bm5, respectively. The multiplicity of viruses at the various concentrations of FBS was similar in both cell lines at 5 days post-infection (p.i.). However, it differed significantly at 2 and 3 days p.i. The proper concentration of FBS were 10% and 3% for Sf21 at 2 and 3 days p.i., respectively, and 5% for Bm5 at both 2 and 3 days p.i. These results suggested that the optimal concentration of FBS should be determined according to the used cell lines and viruses for their optimum production.

Stimulation of cell growth by erythropoietin in RAW264.7 cells: Association with AP-1 activation

Erythropoietin (EPO), a hematopoietic factor, is required for normal erythrocyte developments, but it has been demonstrated to have many other functions, and its receptor is localized in other tissues. In the present study, we investigated whether EPO can promote other cell proliferation and possible molecular mechanisms. EPO restored the inhibition of the RAW264.7 and PC12 cell growth by fetal bovine serum (FBS) withdrawal in a dose dependent manner, but not that of other cell types tested. The restoring effect of EPO was completed when the RAW264.7 cells were cultured in the medium containing as low as 3% of FBS, and 10 U/mL EPO could replace FBS. The restoring effect of EPO in the RAW264.7 cells was associated with the increased of c-Fos and c-Jun expression as well as AP-1 activation. These data demonstrate that EPO can stimulate RAW264. 7 cell as well as PC12 cell growth even when the cells were cultured without FBS or in the presence of small amounts of FBS in the medium, and this stimulating effect is associated with the activation of AP-1 transcription factor.

The influence of cell growth media on the stability and antitumour activity of methionine enkephalin

Studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. The objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. The degradation kinetics of the model peptide methionine enkephalin (Met-E, Tyr-Gly-Gly-Phe-Met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of fetal bovine serum (FBS). The influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the Met-E degradation was also investigated. The results obtained in the Dulbecco's modified Eagle medium containing 10% FBS indicated a rapid degradation of Met-E (t(1/2) = 2.8 h). Preincubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of Met-E, as shown by the increased half-life to 10 h. The in vitro activity of Met-E against poorly differentiated cells from lymph node metastasis of colon carcinoma (SW620) and human larynx carcinoma (HEp-2) cells was determined. Tumour cells were grown for 3 weeks prior to the experiment in a medium supplemented with 10%, 5% or 2% FBS. Statistically significant to mild or no suppression of cell proliferation was observed in all cultures. In both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and Met-E, compared with cells exposed to the peptide alone and cells grown in the absence of Met-E, was observed. This study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays.

Tissue Engineering of Fibroblast Constructs and Anisotropic Collagen Gels

AbstractThe creation of anin vitrofunctional tendon construct will enable testing of the influence of mechanics and nutrients on the development and remodeling of tendon under known controlled stimuli which is difficult to achievein vivo. Tendon constructs were engineeredin vitrovia stress-mediated self organization of fibroblasts and ECM on a laminin coated elastomer substrate. Varying the laminin density and the amount of fetal bovine serum on the substrate affected the ability of tendon fibroblasts to form a confluent cell layer and the time to layer delamination. Understanding the factors that promote self-assembly of tendon constructs will enable their combination with already developedin vitromuscle constructs.

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.

Chemical decomposition of glutamine in cell culture media: effect of media type, pH, and serum concentration.

The chemical decomposition of glutamine to ammonia and pyrrolidonecarboxylic acid was studied at 37 degrees C in a pH range of 6.8-7.8 in different media preparations containing various amounts of fetal bovine serum. The media type influenced the decomposition rate, and the first-order rate constants increased with increasing pH values. The serum concentration had little or no effect on the decomposition rate. The importance of chemical decomposition of glutamine on the analysis of glutamine and ammonia metabolism was illustrated by an example of batch cultivation of a hybridoma cell line. The difference between the apparent uptake rate of glutamine and the actual uptake rate (which is corrected for the chemical decomposition) is shown to be as high as 200%. Similar discrepancy between the apparent and actual ammonia production rate is observed. Mathematical analysis was carried out to develop the relationship between the apparent and actual glutamine uptake and ammonia production rates. The analysis reveals that there are three important dimensionless parameter ratios that govern the difference between the apparent and actual glutamine uptake and ammonia production rates.

Physiological variables affecting collagen lattice contraction by human dermal fibroblasts

Normal human dermal fibroblasts cultured in collagen lattices can compact that matrix by the process known as lattice contraction. That process is a model of the pathological one of scar contracture or wound contraction and is affected by several factors. Lattice contraction is promoted by the addition of adequate amounts of fetal bovine serum to the medium (maximum contraction with 10% serum). The process requires energy, of which glucose and pyruvate have been shown to be adequate sources. When glucose is used as the substrate, the major pathway of energy generation appears to be anaerobic metabolism. When pyruvate is the only substrate, aerobic metabolism may be crucial. The synthesis of DNA is not required for lattice contraction, while protein synthesis is, although the identities of the specific proteins are unknown. Impairment of calcium ion transport inhibits lattice contraction, and the specific inhibition of calmodulin-calcium interactions by W-7 blocks contraction. W-7 at a concentration of 6 × 10 −6 M blocks lattice contraction completely, while it has no effect at any lower concentration. Impairing dynamic microtubule activity impairs contraction. Disrupting microfilaments by cytochalasin B completely blocks lattice contraction. Microfilament function and calcium-calmodulin may be linked by a mechanism involving myosin-ATPase. The process of cell-mediated lattice contraction requires the production of energy, protein synthesis, and a functional cytoskeleton.

Kinetics of monoclonal antibody production in low serum growth medium

Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies. The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media. The radial immunodiffusion assay, employing rabbit anti-mouse IgG antibodies in the immobile phase and the monoclonal antibody (an IgG2a to Rhizobium japonicum cells) as the antigen in the mobile phase, was more reproducible and reliable for determining antibody levels in culture broth than was an indirect enzyme-linked immunosorbent assay. Addition of 0.25% Primatone RL and 0.01%Pluronic F-68 to Dulbecco's modified Eagle medium allowed cells to adapt to growth in medium containing as little as 1% fetal bovine serum; without these additives, 5% serum was the lowest level attained. For the kinetic studies, cells were grown in the low-protein medium in 3 liter spinner flasks. Antibody production occured during the growth phase, however, significant amounts were also produced during later phases when the cells had stopped growing. Final titers were 100–200 μg/ml. It was concluded that maintenance of cell viability is more important than growth rate in production of antibody. This conclusion, confirmed in other studies, has developed into the major underlying strategy employed in subsequent investigations to maximize antibody production in stirred reactors.

The effects of division rate-limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells.

Chick embryo fibroblasts serially propagated in media containing division rate-limiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating 3H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated "senescent" cultures returned to 10% serum containing medium had proliferative rates, incorporated 3H-thymidine, and attained saturation densities at confluency similar to younger cells. "Senescent" cells serially propagated in low serum and returned to 10% serum, achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis because of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative life-span per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.

Beneficial effects of embryo extract on cultured rabbit cartilage cells.

AbstractGrowth and the expression of cartilage‐like differentiation by adult rabbit chondrocytes is stimulated greatly by the Sephadex G‐25 macromolecular fraction of chicken embryo extract (CEEM). A small amount of fetal bovine serum or the macromolecular fraction of fetal bovine serum (FBSM) is also needed for growth. Medium F12 supplemented with CEEM plus FBSM supports moderate clonal growth of the rabbit chondrocytes with excellent expression of differentiated properties. The effects of CEEM on growth and the expression of differentiation have been quantitated by measurement of radioactive sulfate incorporation and total protein synthesis.