Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

Effect of local anesthetics on plasma protein secretion by rat hepatocytes

The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with l-[¹⁴C]leucine for 9 min at 37°C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpomazine, (3 · 10^?5 M), dibucaine (10^?5 M), lidocaine (10^?3 M) and procaine (5 · 10^?5 M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of l-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phopholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, howover, procaine did not cause the retained plasma proteins to accumulate in Goli-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.     

Dissecting the human plasma proteome and inflammatory response biomarkers

A central focus of clinical proteomics is to search for biomarkers in plasma for diagnostic and therapeutic use. We studied a set of plasma proteins accessed from the Healthy Human Individual's Integrated Plasma Proteome (HIP²) database, a larger set of curated human proteins, and a subset of inflammatory proteins, for overlap with sets of known protein biomarkers, drug targets, and secreted proteins. Most inflammatory proteins were found to occur in plasma, and over three times the level of biomarkers were found in inflammatory plasma proteins and their interacting protein neighbors compared to the sets of plasma and curated human proteins. Percentage overlaps with Gene Ontology terms were similar between the curated human set and plasma protein set, yet the set of inflammatory plasma proteins had a distinct ontology‐based profile. Most of the major hub proteins within protein‐protein interaction networks of tissue‐specific sets of inflammatory proteins were found to occur in disease pathways. The present study presents a systematic approach for profiling a plasma subproteome's relationship to both its potential range of clinical application and its overlap with complex disease.     

Protein metabolism. 3. Relationship between albumin metabolism and elevated liver protein synthesis in the guinea pig infected with Trichostrongylus colubriformis

Studies of the incorporation of ¹⁴C-l-leucine into polypeptides by isolated liver ribosomes from guinea pigs confirmed previous in vivo studies that showed that Trichostrongylus colubriformis infection results in an elevated hepatic protein synthesis.The increased rate of protein synthesis was associated with the membrane-bound ribosomes that synthesize the circulating plasma proteins. Inappetance of infected animals was not resposible for the increased rate of synthesis by the membrane-bound ribosomes, but it was found that undernourishment may stimulate synthesis by free ribosomes.Plasma albumin turnover rate and loss into the intestine were both significantly increased in infected guinea pigs. It was concluded that these changes stimulated protein synthesis by membrane-bound ribosomes.The importance of elevated liver protein synthesis and loss of plasma protein in gastrointestinal nematode infections is discussed.     

The measurement of synthesis rates of albumin and fibrinogen in rabbits

1. A formula is proposed for calculating fractional synthesis rates of liver-produced plasma proteins that dispenses with urinary information or information about the size of the urea pool in the body or the fraction of urea that is endogenously catabolized. 2. Synthesis rates obtained for albumin and fibrinogen agreed well with corresponding catabolic rates for the ¹³¹I-labelled proteins except in two of the fibrinogen measurements. 3. Significant reutilization of ¹⁴C occurs in some animals after [¹⁴C]carbonate injections, giving rise to errors in the calculation of protein synthesis rates. These can best be avoided by using results obtained by injecting [¹³C]urea simultaneously. [¹⁵N]urea is shown not to be satisfactory for this purpose.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS AND MIGRATION IN LIVER

The sites of synthesis of proteins and their subsequent migration in rat liver have been studied during a 75 min period after labeling of liver-slice proteins by exposure to leucine-H³ for 2 min. Incorporation of the label into protein began after 1 min and was maximal by 4 min. Electron microscopic radioautography showed that synthesis of proteins in hepatocytes occurs mainly on ribosomes, particularly those in rough endoplasmic reticulum and, to some extent, in nuclei and mitochondria. Most of the newly formed proteins leave the endoplasmic reticulum in the course of 40 min, and concurrently labeled proteins appear in Golgi bodies, smooth membranes, microbodies, and lysosomes. A likely pathway for the secretion of some or all plasma proteins is from typical rough endoplasmic reticulum to a zone of reticulum which is partially coated with ribosomes, to the Golgi apparatus, and thence to the cell periphery. The formation of protein by reticuloendothelial cells was measured and found to be about 5% of the total protein formed by the liver.     

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K⁺ conditions. In the present study, we addressed whether hERG internalization occurs under normal K⁺ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K⁺-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K⁺ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K⁺ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

1H, 13C and 15N resonance assignments of the pyrin domain from human PYNOD

PYNOD is a novel protein belonging to a large family of proteins containing the nucleotide-binding and oligomerization domain (NOD) involved in inflammation and apoptosis. Human PYNOD inhibits inflammatory response mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). Here we report the ¹H, ¹³C and ¹⁵N resonance assignments and secondary structure identification of the pyrin domain (PYD) of human PYNOD as the first step towards elucidating the structural basis of the anti-inflammatory activity of PYNOD.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

Subacute exposure to residual oil fly ash (ROFA) increases eHSP70 content and extracellular-to-intracellular HSP70 ratio: a relation with oxidative stress markers

The purpose of this study was to evaluate whether exposure to particles induces an imbalance in 70-kDa heat shock proteins (HSP70). Since intracellularly (iHSP70) it has anti-inflammatory roles whereas extracellularly (eHSP70) it has pro-inflammatory roles, we evaluate the effect of residual oil fly ash (ROFA) exposure on eHSP70-to-iHSP70 ratio (H index), a biomarker of inflammatory status that is related to oxidative stress in plasma and lymphoid tissue. Wistar rats that received ROFA suspension for three consecutive days (750 μg) showed an increase in plasma eHSP70 levels (mainly the 72-kDa inducible form). Also, ROFA promoted alterations on plasma oxidative stress (increased protein carbonyl groups and superoxide dismutase activity, and decrease sulfhydryl groups). There was an increase in H index of the plasma/thymus with no changes in circulating leukocyte level, iHSP70, or oxidative stress markers in lymphoid tissues. Our results support the hypothesis that eHSP70 content and H index represent inflammatory and oxidative biomarkers.     

Cell death: Are new proteins synthesized during hormone-induced tadpole tail regression?

The possibility that tadpole tail regression might be initiated by thyroid hormone-induced synthesis of new proteins was investigated. Changes in the newly-synthesized proteins of cultured Xenopus laevis tadpole tails treated with 1.5 × 10^?7 M tri-iodothyronine (T₃) were studied, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate for protein separation. After initial studies of unfractionated tail proteins, fractionated mixtures of [³H]methionine and [³⁵S]methionine labelled proteins derived from control and hormone-treated tails respectively were examined for hormone-induced changes. Using a new procedure developed to allow effective analysis of small differences in distribution of two isotopes within gel slices, it was shown that no significant changes in synthesis of fractionated tail proteins are induced by the hormone during the first 3–4 days in culture. The average detection limit was approx. 0.02% of total tail protein synthesis. Although no changes in the synthesis of the tissue structural or enzymic proteins are induced by the hormone this study still leaves open the possibility of changes in the synthesis of regulatory proteins. Based on the known method of activation of the tadpole tail collagenase (which is shown here directly for the first time to be involved in T₃-induced tail regression), it is suggested that many of the initial hormone-induced changes might result from T₃-induced activation of proteolytic “cascades”.     

Changes in protein synthesis in rats in response to endurance training

Experiments were conducted to investigate the influence of endurance exercise training on protein synthesis in skeletal muscle, heart, and liver. Training decreased incorporation of [¹⁴C]-leucine into proteins of the stromal fraction of muscle but there was no change in amino acid incorporation into proteins of the sarcoplasmic and myofibrillar fractions. Incorporation of [¹⁴C]-leucine into the protein of heart, liver, and plasma was depressed in trained rats compared to untrained rats. The specific radioactivity of [¹⁴C]-leucine was similar in tissues of trained and untrained rats and thus the depressed amino acid incorporation represents a decrease in the rate of protein synthesis. These observations demonstrate that the adaptation of muscle protein metabolism to endurance training is quite different than the alterations during work-induced hypertrophy of muscle. The difference in adaptation probably relates to the functional differences between the types of exercise. However depression of protein synthesis in trained rats is a general effect in several tissues and not an effect localized in muscle tissue.     

Binding of lithium and boron to human plasma proteins

The binding of lithium and boron, at normal physiological levels, to plasma proteins has been investigated by the techniques of precipitation with ethyl alcohol and gel chromatography. Assays of lithium and boron were made by thermal neutron activation and mass spectrometric assay of³He and⁴He. Results of alcohol precipitation experiments for plasma from two apparently healthy donors showed that 13 ± 4% and 16 ± 3% of the lithium in plasma is protein bound, but essentially no boron is bound under the conditions used. We believe that because of denaturation of proteins which occurs during alcohol precipitation, these percentages represent lithium and boron tightly bound to protein molecules. The results of the gelchromatography experiment, on the other hand, showed that lithium and boron are bound to a wide range of plasma proteins, from low (∼ 60,000 amu) to high (∼ 1,000,000 amu) molecular weights, and to very low- (∼ 6000 amu) molecular-weight ligands. Although a clear identification of the specific proteins which bind lithium and boron cannot be made at present, some possibilities can be suggested. Certain commercial equipment, instruments, or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.     

Inhibition of protein synthesis elicits early grey crescent formation in the axolotl oocyte

Summary In the axolotl (Ambystoma mexicanum Shaw), it was recently shown that cycloheximide (CH) could induce early grey crescent formation (EGC) in non-activated oocytes, maturing in vitro (Grinfeld and Beetschen 1982). Since it was not proved that EGC was a consequence of protein synthesis inhibition rather than a side-effect of the drug, experiments were performed using microinjections of a quite different inhibitor, diphtheria toxin (DT). This toxin also appeared to elicit EGC. Incorporation of (³H) leucine into oocyte proteins in the presence of increasing DT concentrations (10^–11 to 10^–6 M) was then investigated. The frequency of EGC closely parallels the level of protein synthesis inhibition, which is higher in symmetrized oocytes. The lowest CH concentration which can still elicit EGC also exerts a fairly strong inhibition of (³H) leucine incorporation into proteins. It is concluded that protein synthesis inhibition in the late maturing oocyte actually creates specific conditions which allow cytoplasmic rearrangements to occur, leading to grey crescent formation. These results support the interpretation that (a) proteinic inhibitor (s) of symmetrization could be synthesized in the normal maturing oocyte.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.