1,25-dihydroxyvitamin D stimulation of specific membrane proteins in chick intestine

Vitamin D-deficient chicks were injected intracardially with physiological doses of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) and the formation of intestinal brush-border proteins was followed in vitro. Within 4 h of receiving the hormone the incorporation of radioactive leucine into at least two proteins in the brush-borders was increased. The apparent molecular weights of these proteins were 45 000 and 84 000. The change in the synthesis of these proteins was followed with time and compared with the concomitant changes in intestinal calcium transport. The relationship of these changes is such that there is a strong possibility that the proteins are involved in calcium absorption.     

Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line

HUT-14 cells, tumorigenic human fibroblasts, express a mutant beta-actin which has a single amino acid substitution at position 244 (glycine to aspartic acid), in addition to normal beta- and gamma-actin. In order to characterize the biochemical function of the mutant beta-actin, actins were extracted and purified from HUT-14 cells. The partially purified actin fraction contained beta-, gamma-, and mutant beta-actins in the ratio of 1:1:1, the same ratio as in the cells. When the actin of this fraction was purified through a polymerization step, mutant beta-actin was always less incorporated into actin filaments than beta- and gamma-actin. When the polymerization ability of purified HUT-14 actins was examined by sedimentation technique, it was lower than those of muscle and of cytoplasmic actins from another human cell line (HUT-11) which expresses only normal beta- and gamma-actin, in the ratio of 2:1. The deficient polymerization of mutant beta-actin was also observed by examining the ratio of beta-, gamma-, and mutant beta-actins incorporated into actin filaments. The ratio of mutant beta-actin in polymerized actins under all conditions examined was always less than that before polymerization. These results indicate that the single amino acid substitution at position 244 caused the reduction of incorporation of the mutant beta-actin into actin filaments in vitro.     

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

Developmental change of protein constituents in chicken gizzards

Developmental change of protein constituents of chick gizzard smooth muscle was described by the fluorescent antibody technique and two-dimensional polyacrylamide gel electrophoresis. Myosin heavy chain, tropomyosin, and desmin were immunohistologically detected in 5-day-old gizzard primordia, but myoglobin was detected after 19 days of incubation. Two-dimensional polyacrylamide gel electrophoresis revealed that most structural proteins including beta- and gamma-actin are synthesized almost simultaneously in the primordium, and accumulate in three patterns by which the proteins examined are classified: (1) gradually increasing protein (gamma-actin, tropomyosin, desmin), (2) abruptly increasing protein at a certain stage (myosin, myoglobin), (3) decreasing or constantly kept protein (tubulin, beta-actin). Based on the quantitative analysis of protein constituents, the nature of regulatory system of protein synthesis in smooth muscle and the possible functional difference between beta- and gamma-actin are discussed.     

High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human beta-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The beta-actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the beta-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human beta-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant beta-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the beta-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

Noncoding regions of the gamma-actin gene influence the impact of the gene on myoblast morphology

We have addressed the question of whether two highly conserved noncoding regions of the gamma-actin gene are of functional importance. Human gamma-actin gene constructs deleted for either the entire 3' untranslated region (UTR) and 3' flank or intron III sequences were transfected into mouse myoblasts and the resulting clones were analyzed for cell morphology and actin protein expression. Transfectants carrying the gamma-actin gene deleted for the 3' end (gamma 22) exhibited numerous long pseudopods and increased surface area. In contrast, transfectants expressing the gamma-actin gene deleted for intron III (gamma 156) were rounded with blebs over the cell surface and showed decreased surface area. The relative expression of beta- to gamma-actin protein decreased for both transfectant types. The total actin protein levels remained constant for the gamma 22 cells but decreased for the gamma 156 cells. The results indicate that alterations to transfectant cell morphology can be influenced by the presence or absence of different noncoding regions in the transfected gamma-actin gene. The mechanisms by which noncoding regions of the gamma-actin gene influence the impact of the gene are unknown. Nevertheless, these noncoding regions are isoform specific and highly conserved in evolution. We propose that the functional significance of the different actin isoforms may involve the properties of these noncoding regions in addition to the differences in protein sequence.     

Possible Role of Non-Muscle Alpha-Actinins in Muscle Cell Mechanosensitivity

The main hypothesis suggested that changes in the external mechanical load would lead to different deformations of the submembranous cytoskeleton and, as a result, dissociation of different proteins from its structure (induced by increased/decreased mechanical stress). The study subjects were fibers of the soleus muscle and cardiomyocytes of Wistar rats. Changes in external mechanical conditions were reconstructed by means of antiorthostatic suspension of the animals by their tails for 6, 12, 18, 24 and 72 hours. Transversal stiffness was measured by atomic force microscopy imaging; beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 levels in membranous and cytoplasmic fractions were quantified by Western blot analysis; expression rates of the corresponding genes were studied using RT-PCR. Results: In 6 hours, alpha-actinin 1 and alpha-actinin 4 levels decreased in the membranous fraction of proteins of cardiomyocytes and soleus muscle fibers, respectively, but increased in the cytoplasmic fraction of the abovementioned cells. After 6–12 hours of suspension, the expression rates of beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 were elevated in the soleus muscle fibers, but the alpha-actinin 1 expression rate returned to the reference level in 72 hours. After 18–24 hours, the expression rates of beta-actin and alpha-actinin 4 increased in cardiomyocytes, while the alpha-actinin 1 expression rate decreased in soleus muscle fibers. After 12 hours, the beta- and gamma-actin content dropped in the membranous fraction and increased in the cytoplasmic protein fractions from both cardiomyocytes and soleus muscle fibers. The stiffness of both cell types decreased after the same period of time. Further, during the unloading period the concentration of nonmuscle actin and different isoforms of alpha-actinins increased in the membranous fraction from cardiomyocytes. At the same time, the concentration of the abovementioned proteins decreased in the soleus muscle fibers.     

Incorporation of [h]leucine and [h]valine into protein of freshwater bacteria: uptake kinetics and intracellular isotope dilution

Incorporation of [H]leucine and [H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of H and C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 x 10 and 6.6 x 10 cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.     

Modulation of microfilament protein composition by transfected cytoskeletal actin genes.

HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.     

Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.     

Stimulation of rat intestinal protein synthesis by 1,25-dihydroxyvitamin D3

To study general stimulatory effects of 1,25-dihydroxyvitamin D3 on intestinal protein synthesis, slices of duodenal villi from 1,25-dihydroxyvitamin D3-treated and vitamin D-deficient rats were incubated in vitro for 90 min at the surface of medium containing [3H]leucine. Incorporation of the [3H]leucine into TCA-precipitated protein, which was shown to be linear for 12 h and 90% inhibited by cycloheximide, was increased by 50-60% at 26 h after a single injection of 125 ng of 1,25-dihydroxyvitamin D3 (three experiments, P less than 0.001). The increase, which was not due to circadian rhythm fluctuations of the intestine, was in synchrony with the second Ca2+ transport response observed by Halloran and DeLuca (Arch. Biochem. Biophys. 208, 477-486, 1981). However, no significant difference in [3H]leucine incorporation was observed before or during the initial Ca2+ transport response observed by Halloran and DeLuca, i.e., at 1.0, 3.0, and 6.5 h following an injection of 1,25-dihydroxyvitamin D3. The late onset of the 1,25-dihydroxyvitamin D3-induced increase in total protein synthesis implies that it is an indirect rather than a direct effect of the hormone.     

Protein synthesis during hyperthermia in ground squirrels]

The intensity of [14C]leucine incorporation into heart, liver, brain, muscle, and blood plasma protein in gophers under deep artificial hypothermia has been studied. It was shown that the intensity of protein synthesis decreased sharply under these conditions. Insignificant incorporation of [14C]leucine into proteins was observed during the first two hours after the onset of hypothermia and then ceased completely.     

Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.

Two different mutant human beta-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant beta-actin containing a single mutation (Gly-244----Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant beta-actin in excess of normal beta-actin were morphologically altered. In contrast, a second mutant beta-actin gene containing two additional mutations (Gly-36----Glu-36 and Glu-83----Asp-83, as well as Gly-244----Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous beta- and gamma-actin decreased in response to high-level expression of the stable mutant beta-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous beta- and gamma-actins were equal, the ratio of net beta-actin (mutant plus normal) to gamma-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant beta-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on beta-actin/gamma-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the beta-actin mutations.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Uptake of (14C) leucine and protein synthesis in potato tuber buds and effect of abscisic acid on these processes]

The uptake of [14C] leucine and its incorporation into proteins of dormant and growing potato tuber buds were studied. It was found that the label uptake was increased at the beginning of the growth period, whereas the dynamics of this process were not changed in comparison with the dormant buds samples. The rate of [14C] leucine incorporation into proteins was increased in the growing buds; this increase was not, however, due to the increase in the uptake of the labelled precursor and was probably caused by activation of the protein synthesis. In contrast, the activation of protein synthesis was accompanied by changses is the dynamic incorporation of [14C] leucine into the protein at the end of dormancy. The effect of abscisic acid (10(-7) M) on the protein synthesis was not connected with its action of the uptake of labelled precursor and depended on the physiological state of buds and incubation time. A possible mechanism of regulatory effect of abscisic acid on protein synthesis in potato tuber buds is discussed.     

Glutamine,arginine, and leucine signaling in the intestine

Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.