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1.
Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.  相似文献   

2.
The surface of single cells of Dictyostelium discoideum during growth and aggregation has been observed by scanning electron microscopy (SEM). Our results show a series of morphological changes associated with the transition from growth to development which appear to be cellcontact dependent. In addition, from results using Concanavalin A as a biochemical probe, we conclude that the mannose-containing surface glycoproteins are accessible during growth, but become inaccessible during aggregation.  相似文献   

3.
A study of the effects of steroid hormones on the development of Dictyostelium discoideum has shown that 4 × 10?5M progesterone, and to a lesser extent dehydro-epiandrosterone, oestradiol and testosterone, inhibit both the growth and aggregation of these amoebae. Pregnenolone is active at lower concentrations (3 × 10?6M), but at the level of growth only. Progesterone exerts its inhibitory action throughout the aggregation phase. The steroid prevents starved cells from becoming aggregation competent, and inhibits the aggregation of amoebae previously allowed to achieve the competent stage. However, unlike other agents such as dinitrophenol or EDTA, the steroid does not considerably affect cell morphology. Upon addition of progesterone cells become rounded, but after a brief lag they regain their ability to adhere to solid supports and form pseudopods. The fact that the steroids active on Dictyostelium are among the most liposoluble might indicate that their inhibitory action is dependent upon their incorporation into membrane lipids.  相似文献   

4.
Influence of proteins from the Agkistrodon blomhoffii ussuriensis snake venom on platelet activation and aggregation was developed on different model systems in vitro. It was shown that novel disintegrin (Blomus-B) and phospholipase A2 (Blopholipase) from the venom, activated platelets and inhibited their aggregation. Fibrino(geno)lityc enzyme (Blomulyse) does not activate platelets and has no effect on their aggregation stimulated by collagen, but inhibit ADP and adrenalin-stimulated platelet aggregation. Thrombin-like enzyme (Ancistron-Bu) activates platelets but has no effect on their aggregation. Obtained proteins can be used under development of new antiplatelet agents and as instruments for detailed elaboration and deep investigation of processes which proceed with participation of platelets.  相似文献   

5.
Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.  相似文献   

6.
7.
Phorbol myristate acetate (PMA) at nanomole concentrations induces a rapid aggregation of guinea pig macrophages. This aggregation is dependent on extracellular Mg2+ (but not Ca2+) for its development. Additionally, it is inhibited by some agents which also inhibit aggregation induced by lymphokines and the ionophore A23187. Although the mechanism of aggregation has not been determined, it does not appear to be related to either arachidonic acid metabolites or products of the respiratory burst. In this respect, it resembles the PMA-induced aggregation of neutrophils.  相似文献   

8.
The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.  相似文献   

9.
Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.  相似文献   

10.
Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.  相似文献   

11.
The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.  相似文献   

12.
Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets   总被引:3,自引:0,他引:3  
We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.  相似文献   

13.
We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.  相似文献   

14.
Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1–7, 8–12, 13–17 and 28–32 of amylin would inhibit the aggregation of the amyloidogenic cores 18–22, 23–27, 33–37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1–7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18–22, 23–27, 33–37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1–7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.  相似文献   

15.
The fusion of cells of complementary mating types to produce giant cells has been shown to be the critical event to induce macrocyst formation in Dictyostelium discoideum. We have examined the way in which giant cells use diffusible factors to influence the developmental mode of nearby cells using an experimental design in which NC4 cells are allowed to develop on a dialysis membrane above a suspension of giant cells. We have observed that giant cells are able to inhibit independent aggregation and stream formation in the upper cells and become the dominant aggregation centers. In addition giant cells are able to redirect local amoeba away from the fruiting-body and toward the macrocyst mode of development. We show that these effects are mediated by diffusible factors of under 2,000 MW. and discuss possible mechanisms of action.  相似文献   

16.
Concanavalin Å is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin Å requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl β-d-galactopyranoside did not inhibit the aggregation of concanavalin Å by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin Å may have an additional, ligand-binding site which is netal-dependent and which can be modified by the addition of a saccharide ligand.  相似文献   

17.
新生肽链折叠过程中容易出现错误折叠与聚沉,从而导致折叠病等病理现象. 分子伴侣具有辅助其他蛋白质正确折叠,保护蛋白质分子结构的功能.本文选用人肌肌酸激酶为靶蛋白,研究了肽基脯氨酰顺反异构酶人亲环素18(human cyclophilin 18,hCyp18)对人肌肌酸激酶去折叠的作用,发现hCyp18能够抑制人肌肌酸激酶在热变性与化学变性过程中的失活与构象变化,并抑制人肌肌酸激酶在化学变性过程中的聚沉,因此推断hCyp18具有针对人肌肌酸激酶的分子伴侣功能.本文同时研究了hCyp18与人肌肌酸激酶的结合作用,对hCyp18的作用机制进行了初步探讨.  相似文献   

18.
N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.  相似文献   

19.
In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.  相似文献   

20.
C Y Lin  S Smith 《Life sciences》1976,18(6):563-568
The hypolipidemic drugs halofenate and clofibrate inhibit the aggregation of human platelets induced by ADP, collagen and epinephrine. The effect of the drugs is primarily on the “second wave” of aggregation which is associated with the platelet release reaction. The drugs also inhibit the release of serotonin from platelets suggesting that the effect on aggregation is attributable to an inhibition of the release reaction. Halofenate is much more potent than clofibrate in inhibiting both the release of serotonin and the second wave of aggregation.  相似文献   

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