Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with³H-arginine,³H-lysine, and³H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by³H-thymidine autoradiography and was measured to be 13.0 hr (G₁ 1.3 hr, S 6.5 hr, G₂ 3.8 hr and M 1.4 hr).³H-arginine labelled proteins which were synthesized at S and G₂ were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G₁, at the transition phase from late S to early G₂, or at the mitotic phase. Such varied incorporation was also found in³H-lysine labelled proteins, but not in³H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G₂. Some of the³H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.     

The secretory activity of rat nasal glands and the effect of cholinergic drugs

Summary The secretory behaviour of rat nasal glands, under normal conditions and after the application of cholinergic drugs, has been studied using morphological and radiobiochemical techniques.Autoradiography and electrophoresis provide evidence for the selective incorporation of ³H-arginine into the glycoprotein containing fraction of the nasal glandular secretion. Radiobiochemical experiments show that labelled arginine is rapidly incorporated into the acinar cells of unstimulated glands, although it takes approximately 4 h before the labelled secretory proteins leave the cells. The secretion of proteins is stimulated by the parasympathetic agonist pilocarpine, whose main action is to promote discharge. Histological sections show a depletion of secretory granules after pilocarpine treatment. The cholinergic antagonist atropine inhibits the secretion; the acinar cells are completely filled with secretory granules following this treatment. The time course of the events following atropine administration suggests that there is no feed-back system controlling glycoprotein synthesis.The techniques employed here therefore appear to be useful for studying the effects of drugs that interfere with the secretory activity of the nasal glands.     

Occlusion of basic proteins by fibrin: Studies with 14C-arginine labelled proteins

With the use of ¹⁴C-arginine labelled basic proteins isolated from cytoplasma of Ehrlich ascites tumor cells it has been shown that these proteins are occluded by fibrin clots under the influence of thrombin. The occlusion of these proteins depends on their concentration. The experiments indicated that 2.5 μg of these proteins were occluded per one mg of fibrinogen in the presence of thrombin. The basic proteins occluded by fibrin make the clots resistant to the fibrinolityc action of plasmin. The clinical role of arginine-rich basic proteins appearing in circulation in malignancy have been discussed briefly.     

Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent.

Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

On coupling bovine fibrinogen to the surface of malignant murine plasma cells by means of transglutaminase.

It was found that native, as well as¹²⁵I labelled fibrinogen, may be coupled by means of transglutaminase to surface proteins of malignant plasma cells of the mouse. Binding of fibrinogen, although greatly affecting the [¹⁴C] putrescine labelling of several surface proteins, leaves the cells viable and malignant.     

Biosynthesis of major platelet proteins in human blood platelets

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]carbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, IIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

Effect of endotoxin on plasma albumin and fibrinogen synthesis rates in rabbits as measured by the [14C]carbonate method

1. Rates of synthesis of plasma albumin and fibrinogen were measured by the [(14)C]carbonate method in normal rabbits and in animals that received a single intravenous injection of Shigella endotoxin 14-48hr. earlier. 2. The accuracy of the method was improved by introducing refinements into procedures for measuring (14)C radioactivities associated with both urea and proteins that are lost from the plasma during the synthesis interval. 3. The synthesis interval (time between injecting carbonate and measuring specific radioactivities of protein guanidine carbon in plasma) can be shortened with advantage to 3-4hr. 4. Injection of endotoxin markedly decreased the fractional rate of loss in the first few hours of injected radioiodine-labelled fibrinogen and to a smaller extent of similarly labelled albumin from the plasma. The absolute rate of synthesis of fibrinogen was increased in endotoxin-treated rabbits by more than 400% compared with normal animals, and the rate of synthesis of albumin was increased by about 60%.     

Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin

Zona-free hamster eggs were fertilized in vitro with human spermatozoa in a culture medium enriched with either 3H-arginine or 3H-tryptophan. Autoradiography was used to investigate decondensing sperm heads and all pronuclei for the presence of newly synthesized, 3H-labelled proteins. In the case of 3H-arginine-labelled proteins, an intense accumulation of radioactivity was detected in all autoradiograms of chromatin structures. On the other hand, no comparable accumulation was seen for 3H-tryptophan-labelled proteins up to the progressed-pronucleus stage. It is concluded that, as a part of changes of the nucleoproteins in decondensing sperm chromatin, there is an accumulation in the male (as well as in the female) pronucleus of basic nuclear proteins synthesized by the egg during fertilization. Since non-histone, 3H-tryptophan-labelled proteins were not incorporated in the same way, these 3H-arginine-labelled proteins accumulating in pronuclear chromatin during the earliest phase of pronucleus formation are probably histones.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

Plasmid-associated bacteriocin production in a JK-type coryneform bacterium

Abstract The outer membrane of Escherichia coli K-12 has a variety of proteolytic activities. We were able to label several outer membrane proteins with [³H]diisopropylfluorophosphate (DFP). This suggests that they are serine proteases. The number of labelled proteins detected varied with the E. coli K-12 strain used. Strains bearing a tolC mutation, in addition, gave better labelling and/or had more labelled proteins. A previously described [³H]DFP-labelled outer membrane protein was shown not to be the TolC protein since it has a slightly lower M _r, it is not labelled more intensely in a TolC-overproducing strain, and it is still labelled in tolC mutant strains.     

Structural analysis of fibrinogen synthesized by cultured chicken hepatocytes in the presence or absence of dexamethasone

Hepatocyte monolayers, derived from chick embryos and cultured in chemically defined medium without hormones, synthesize and secrete fibrinogen that resembles chicken plasma fibrinogen immunochemically and structurally. Addition of a synthetic glucocorticoid, dexamethasone, to the cultured cells resulted in an appreciable and relatively selective increase in fibrinogen synthesis. Autoradiography of fibrinogen that had been metabolically labelled with [35S]methionine and then subjected to SDS-polyacrylamide gel electrophoresis, unreduced or under disulfide-reducing conditions, revealed that only dimeric forms of fibrinogen, containing undegraded A alpha, B beta, and gamma chains, were secreted under stimulated and unstimulated culture conditions.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.     

Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin

Summary Zona-free hamster eggs were fertilized in vitro with human spermatozoa in a culture medium enriched with either ³H-arginine or ³H-tryptophan. Autoradiography was used to investigate decondensing sperm heads and all pronuclei for the presence of newly synthesized, ³H-labelled proteins.In the case of ³H-arginine-labelled proteins, an intensen accumulation of radioactivity was detected in all autoradiograms of chromatin structures. On the other hand, no comparable accumulation was seen for ³H-tryptophan-labelled proteins up to the progressed-pronucleus stage. It is concluded that, as a part of changes of the nucleoproteins in decondensing sperm chromatin, there is an accumulation in the male (as well as in the female) pronucleus of basic nuclear proteins synthesized by the egg during fertilization. Since non-histone, ³H-tryptophan-labelled proteins were not incorporated in the same way, these ³H-arginine-labelled proteins accumulating in pronuclear chromatin during the earliest phase of pronucleus formation are probably histones.