scholarly journals Distribution of proteins between nucleus and cytoplasm of amoeba proteus

1981 ◽  
Vol 88 (3) ◽  
pp. 516-525 ◽  
Author(s):  
L Goldstein ◽  
C Ko
Keyword(s):  
Amoeba Proteus ◽  
Cellular Protein ◽  
The Other ◽  
Nuclear Transplantation ◽  
Main Function ◽  
Functional Sites ◽  
Protein Mass ◽  
High Affinity ◽  
Nuclear Binding

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but - because the cytoplasm is 50 times larger than the nucleus - about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic "contamination" of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm's large volume ensures that CP will be abundant there. Extending Bonner's idea of "quasi-functional nuclear binding sites" for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.

Platelets Lacking Both Pleckstrin and Pleckstrin-2 Have a Marked Deficient Cell Spreading and Actin Assembly

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 253-253
Author(s):  
Yanfeng Wang ◽  
Lurong Lian ◽  
John H. Hartwig ◽  
Charles S. Abrams
Keyword(s):  
Actin Cytoskeleton ◽  
Signaling Pathways ◽  
Cellular Protein ◽  
Genetically Engineered ◽  
The Other ◽  
Actin Organization ◽  
Genetically Engineered Mice ◽  
Membrane Bound ◽  
Coated Surfaces

Abstract Pleckstrin makes up approximately one percent of total cellular protein within platelets and leukocytes, a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin-2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To understand the true in vivo role of these two proteins, we genetically engineered mice to lack individual or both pleckstrin isoforms. Pleckstrinnull platelets exhibit mildly impaired aggregation in response to thrombin, but fail to aggregate in response to thrombin in the presence of PI3K inhibitors. This suggests that a PI3K-dependent signaling pathway compensates for the loss of pleckstrin. Platelets lacking pleckstrin exhibit a marked defect in the secretion of delta and alpha granules following exposure to the PKC stimulant, PMA. Although pleckstrin-null platelets centralized and merged their granules in response to stimulation of PKC, they failed to empty their contents into the open canalicular system. These results differ from that seen with platelets lacking the other pleckstrin isoform, pleckstrin-2. Platelets derived from pleckstrin-2 null mice secrete and aggregate normally in response to thrombin and PMA. In addition, unlike the effect seen on pleckstrin knockout platelets, inhibitors of PI3K had no effect on the aggregation or secretion of pleckstrin-2 knockout platelets. Also in contrast to pleckstrin knockout platelets, pleckstrin-2 null platelets fail to secrete in response to thrombin when they were exposed to inhibitors of either PLC or PKC. These data demonstrate that pleckstrin-2 knockout platelets compensate for their secretion defect by a pathway dependent on PLC and PKC. It is notable that PI3K or PKC inhibitors only minimally affected the thrombin-induced secretion of wild-type platelets unless both inhibitors were used together. Together, these results suggest that platelets utilize parallel signaling pathways, one dependent on PKC and pleckstrin, and the other on PI3K and pleckstrin-2. Studies in platelets and neuronal cells suggest that disassembly of the actin cytoskeleton is required for secretion. Since overexpression studies have suggested that both pleckstrin and pleckstrin-2 can modulate the actin cytoskeleton, we hypothesized that both pleckstrin isoforms affect secretion through an actin-dependent pathway. To test this hypothesis, we analyzed the effect of the pleckstrin and pleckstrin-2 null mutations on actin organization within platelets. When pleckstrin null platelets were allowed to adhere to immobilized fibrinogen, or when they were flowed over collagen-coated surfaces, they exhibited impaired adherence and spreading. Phalloidin staining indicated that they also assembled less F-actin than normal platelets. Similarly, platelets lacking pleckstrin-2 also adhered and spread poorly. Since we have shown that pleckstrin and pleckstrin-2 perform analogous roles in complementary signaling pathways, we bred mice to generate a murine lacking both pleckstrin isoforms. Platelets lacking both pleckstrin and pleckstrin-2 exhibited a marked spreading defect in response to PMA (0% of control) or thrombin (18% of control). Following stimulation with PMA, platelets containing the double null mutation also failed to increase in their F-actin content during the spreading process (8% of control). Electron micrographs of platelets lacking both pleckstrin and pleckstrin-2 revealed that the double null platelets fail to extend any broad lamellipodia, and instead, only extended small membrane blebs. These data show that pleckstrin and pleckstrin-2 are absolutely essential for the cytoskeletal organization that occurs during platelet adhesion. These data also demonstrate that adhesion-induced cytoskeletal changes within platelets can be mediated by one of two parallel pathways, the first involving PKC and pleckstrin, and the second involving PI3K and pleckstrin-2.

scholarly journals Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response

1976 ◽  
Vol 156 (2) ◽  
pp. 391-398 ◽  
Author(s):  
T C Spelsberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Polymerase Activity ◽  
Rna Polymerase I ◽  
Target Tissue ◽  
Functional Sites ◽  
Nuclear Binding ◽  
Polymerase I

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.

Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms

2000 ◽  
Vol 113 (21) ◽  
pp. 3795-3803 ◽  
Author(s):  
A. Di Nardo ◽  
R. Gareus ◽  
D. Kwiatkowski ◽  
W. Witke
Keyword(s):  
Alternative Splicing ◽  
Cell Motility ◽  
Hela Cells ◽  
The Other ◽  
Actin Dynamics ◽  
High Affinity ◽  
Other Hand ◽  
The Brain

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.

Inhibition of 2,3-oxidosqualene: β-amyrin-cyclase, S-adenosyl-l-methionine: cycloartenol C-24-methyltransferase and cycloeucalenol: obtusifoliol isomerase by rationally designed molecules containing a tertiary amine function

1983 ◽  
Vol 11 (5) ◽  
pp. 537-543 ◽  
Author(s):  
ALAIN RAHIER ◽  
PIERRETTE BOUVTER ◽  
LUIGI CATTEL ◽  
ACHARAN NARULA ◽  
PIERRE BENVENISTE
Keyword(s):  
Tertiary Amine ◽  
Inhibitory Action ◽  
Plant Sterol ◽  
The Other ◽  
Maize Seedlings ◽  
Higher Plant ◽  
High Affinity ◽  
Amine Function

25-Azacycloartanol (I), 2-aza-2-dihydrosqualene (II) and Tridemorph (2,6-dimethyl-N-tridecylmorpholine)(III) are potent inhibitors of higher plant sterol biosynthesis. The first two molecules have been designed using rational enzymological concepts. I, II and III were shown to inhibit the S-adenosyl-l-methionine: cycloartenol C-24-methyltransferase, the 2,3-oxidosqualene: β- amyrin-cyclase and the cycloeucalenol: obtusifoliol isomerase, respectively. Inhibition was demonstrated either in vivo on bramble cell suspensions or in vitro on microsomes from maize seedlings. Each inhibitor has been shown to have a high affinity for its presumed enzymic target and only negligible inhibitory action on the other two enzymes. The applications of these results to further physiological studies are discussed.

Synthesis and biological activity of histogranin and related peptides

1995 ◽  
Vol 73 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Jyoti A. Prasad ◽  
Vijay K. Shukla ◽  
Simon Lemaire
Keyword(s):  
Nmda Receptor Antagonist ◽  
The Other ◽  
Peptide Fragments ◽  
Antagonist Activity ◽  
Brain Membrane ◽  
High Affinity ◽  
Aspartate Receptor ◽  
Vitro Binding

Histogranin (HN) was first isolated from bovine adrenal medulla and shown to be a pentadecapeptide displaying N-methyl-D-aspartate (NMDA) receptor antagonist activity. To determine the active pharmacophore of HN, fragments of the peptide were synthesized and their structure–activity relationships studied by measuring their ability to displace the binding of [125I][Ser1]HN to rat brain membrane preparations and to block NMDA-induced convulsions in mice. In the binding assay, only the full length peptide HN and HN(1–10) displayed a high affinity (Ki of 72 and 162 nM, respectively). All other tested fragments with deletions at the N- and (or) C-terminals of the molecule showed large (16- to 2500-fold) decreases in potency. The least active peptide fragment tested was HN(6–10) (Ki of 164 μM). In vivo, HN and HN(2–15) (100 nmol; i.c.v.) produced 94 and 40% protection against NMDA-induced convulsions in mice, respectively. None of the other peptide fragments displayed significant anticonvulsant activity. The protective activity of HN (60 and 100 nmol) was markedly antagonized by coadministration of HN(1–10) (100 nmol). The results indicate that the in vivo anti-NMDA and in vitro binding activities of HN and related peptides, with the exception of HN(1–10), depend upon the integrity of the molecule. On the other hand, the high affinity of HN(1–10) for HN binding sites correlates well with its antagonist effects towards the activity of the parent peptide.Key words: histogranin, peptide receptor, N-methyl-D-aspartate receptor, anticonvulsant.

scholarly journals Acquisition of hyaluronate-binding affinity in vivo by newly synthesized cartilage proteoglycans

1989 ◽  
Vol 258 (3) ◽  
pp. 875-880 ◽  
Author(s):  
J D Sandy ◽  
J R O'Neill ◽  
L C Ratzlaff
Keyword(s):  
Binding Affinity ◽  
Gradient Centrifugation ◽  
The Other ◽  
Binding Properties ◽  
High Affinity ◽  
Cscl Gradient ◽  
Cartilage Proteoglycans ◽  
Relationship Of ◽  
Total Tissue

We have studied the hyaluronate-binding properties of aggregating cartilage proteoglycans synthesized in vivo by immature (6-week), mature (25-week) and aged (75-week) rabbits. Precursor isotope (35SO4) was given by intra-articular injection and articular cartilage was removed from rabbits after periods ranging from 1.5 h to 168 h. Proteoglycans were extracted with 4 M-guanidinium/HCl and monomers were isolated by CsCl gradient centrifugation under dissociative conditions. The percentages of both radiolabelled and total tissue monomers with a high affinity for hyaluronate [that is, capable of forming aggregates on Sepharose CL-2B in the presence of 0.8% (w/w) hyaluronate] were then determined. For all samples about 30% of the tissue monomers were high-affinity; however, less than 5% of the radiolabelled monomers were high-affinity at 1.5 h after injection, and this figure increased gradually with time in vivo. The increase was rapid in immature rabbits, such that after 24 h, about 30% of the radiolabelled monomers were high-affinity; on the other hand for mature and aged rabbits the increase was markedly slower such that 30% high-affinity was attained only after about 72 h. The results show that aggregating cartilage proteoglycans are secreted in vivo in a ‘precursor’ form with a low affinity for hyaluronate, and suggest that conversion of these monomers to a form with a higher binding affinity occurs with a half-time of about 12 h in immature cartilages but greater than 24 h in mature cartilages. The possible relationship of these findings to the process of proteoglycan aggregation in vivo is discussed.

scholarly journals 117 CYTOPLASMIC FACTORS INFLUENCE DEVELOPMENTAL POTENTIAL OF SAMP1/Yit MOUSE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 209
Author(s):  
J. Otsuka ◽  
H. Funabashi ◽  
T. Kono
Keyword(s):  
Blastocyst Stage ◽  
Development Rate ◽  
Mouse Embryos ◽  
The Other ◽  
Nuclear Transplantation ◽  
Chi Square ◽  
Developmental Potential ◽  
Reconstructed Embryos

Nuclear transplantation is an efficient means to investigate nucleo-cytoplasmic interactions of mammalian embryos during early development. A recent study has shown that the developmental potential of embryos is affected by the type of cytoplasm. The SAMP1/Yit mouse, an inbred strain that develops spontaneous chronic ileitis resembling Crohn's disease (Matsumoto S 1999 Bioscience Microflora 18, 1–9), has poor reproductive performance, and the developmental ability of embryos is low (unpublished data). Therefore we need to enhance productivity of the SAMP1/Yit mouse. Recently it was reported that cytoplasm of F1 mouse egg supported the development of embryos which have low developmental ability (Muggleton-Harris A et al. 1982 Nature 299, 460–462). In the present study, we examined the influences of the nucleus and cytoplasm on the development of reconstructed embryos in vitro and in vivo, using reciprocal nuclear transplantation between SAMP1/Yit and B6P1F1 (C57BL/6J × SAMP1/Yit) mouse embryos. We evaluated the developmental ability of reconstructed embryos by the development rate into blastocysts in vitro and by the rate of offspring after transfer of blastocysts to recipient mice. Pronuclear transplantation was carried out as reported previously (McGrath J and Solter D 1983 Science 220, 1300–1302). Briefly, karyoplasts from one-cell SAMP1/Yit embryos were introduced into enucleated B6P1F1 zygotes (SAMP1/B6P1F1) and fused by addition of inactivated HVJ (2700 U L−1). The other group of reconstructed embryos (B6P1F1/SAMP1) was manipulated similarly. After fusion, reconstructed embryos were cultured in drops of KSOM medium for 120 h at 37°C in 5% CO2 in humidified air. Some reconstructed and control (unmanipulated) embryos that developed to the blastocyst stage were transferred to the uteri of recipient mice. Data were compared using chi-square test; differences were considered significant at P < 0.01. The development rate of [SAMP1/B6P1F1] embryos to the blastocyst stage was significantly (P < 0.01) higher (75.0%) than that of SAMP1/Yit controls (39.1%). The rate of offspring in [SAMP1/B6P1F1] was also significantly (P < 0.01) higher (47.5%) than that of SAMP1/Yit controls (22.1%). On the other hand, [B6P1F1/SAMP1] embryos showed low developmental potential compared to B6P1F1 control embryos. These results indicate that the source of the cytoplasm strongly influences the development of reconstructed embryos containing SAMP1/Yit karyoplasts. Table 1.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Reparação óssea no implante dental

2020 ◽  
Vol 12 (45) ◽  
pp. 63-66
Author(s):  
Halim Nagem Filho ◽  
Reinaldo Francisco Maia ◽  
Reinaldo Missaka ◽  
Nasser Hussein Fares
Keyword(s):  
Gene Expression ◽  
Titanium Surface ◽  
Close Contact ◽  
The Other ◽  
Main Function ◽  
Indirect Interaction ◽  
M2 Macrophages ◽  
Activated Macrophages ◽  
M1 Macrophages ◽  
High Production

The osseointegration is the stable and functional union between the bone and a titanium surface. A new bone can be found on the surface of the implant about 1 week after its installation; the bone remodeling begins between 6 and 12 weeks and continues throughout life. After the implant insertion, depending on the energy of the surface, the plasma fluid immediately adheres, in close contact with the surface, promoting the adsorption of proteins and inducing the indirect interaction of the cells with the material. Macrophages are cells found in the tissues and originated from bone marrow monocytes. The M1 macrophages orchestrate the phagocytic phase in the inflammatory region and also produce inflammatory cytokines involved with the chronic inflammation and the cleaning of the wound and damaged tissues from bacteria. On the other hand, alternative-activated macrophages (M2) are activated by IL-10, the immune complex. Its main function consists on regulating negatively the inflammation through the secretion of the immunosuppressant IL-10. The M2 macrophages present involvement with the immunosuppression, besides having a low capacity for presenting antigens and high production of cytokines; these can be further divided into M2a, M2b, and M2c, based on the gene expression profile.

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