scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

2004 ◽  
Vol 286 (5) ◽  
pp. G814-G821 ◽  
Author(s):  
Bi-Guang Tuo ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett ◽  
Jon I. Isenberg
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bicarbonate Secretion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers ◽  
Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

scholarly journals Effect of Palmitic Acid on Exosome-Mediated Secretion and Invasive Motility in Prostate Cancer Cells

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2722
Author(s):  
Ivan V. Maly ◽  
Wilma A. Hofmann
Keyword(s):  
Prostate Cancer ◽  
Palmitic Acid ◽  
Cancer Cells ◽  
Dependent Manner ◽  
Prostate Cancer Cells ◽  
Concentration Dependent Manner ◽  
Fat Consumption ◽  
Progression Markers

High fat consumption can enhance metastasis and decrease survival in prostate cancer, but the picture remains incomplete on the epidemiological and cell-biological level, impeding progress toward individualized recommendations in the clinic. Recent work has highlighted the role of exosomes secreted by prostate cancer cells in the progression of the disease, particularly in metastatic invasion, and also the utility of targeting these extracellular vesicles for diagnostics, as carriers of disease progression markers. Here, we investigated the question of a potential impact of the chief nutritional saturated fatty acid on the exosome secretion. Palmitic acid decreased the secretion of exosomes in human prostate cancer cells in vitro in a concentration-dependent manner. At the same time, the content of some prospective metastatic markers in the secreted exosomal fraction was also reduced, as was the ability of the cells to invade across extracellular matrix barriers. While by themselves our in vitro results imply that on the cell level, palmitic acid may be beneficial vis-à-vis the course of the disease, they also suggest that, by virtue of the decreased biomarker secretion, palmitic acid has the potential to cause unjustified deprioritization of treatment in obese and lipidemic men.

scholarly journals Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- and filament-dependent manner

2019 ◽  
Vol 30 (23) ◽  
pp. 2913-2928 ◽  
Author(s):  
Konstantinos Nakos ◽  
Megan R. Radler ◽  
Elias T. Spiliotis
Keyword(s):  
Membrane Trafficking ◽  
Binding Proteins ◽  
Live Cells ◽  
Dependent Manner ◽  
Guanosine Diphosphate ◽  
In Vitro Reconstitution ◽  
In Trans ◽  
In Cis ◽  
Precise Role

Septins (SEPTs) are filamentous guanosine-5′-triphosphate (GTP)-binding proteins, which affect microtubule (MT)-dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus-end growth and elongation, while higher and intermediate concentrations inhibit and pause plus-end growth, respectively. We show that SEPT2/6/7 has a modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5′- O-[γ-thio]triphosphate (GTPγS)-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus-end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus-end growth and the tracking of plus end–binding proteins and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment. [Media: see text]

The Role of β2-Glycoprotein I in the Clearance of Platelet Microvesicles.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 145-145
Author(s):  
Hanan Abdel-Monem ◽  
Swapan Kumar Dasgupta ◽  
Anhquyen Le ◽  
Anthony Prakasam ◽  
Perumal Thiagarajan
Keyword(s):  
Plasma Proteins ◽  
Plasma Concentrations ◽  
Major Protein ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Concentration Dependent Manner ◽  
Glycoprotein I

Abstract Abstract 145 The physiological function of β2-glycoprotein I is unclear and several studies suggest a role in the clearance of anionic phospholipid containing membranes. Anionic phospholipid containing liposomes are cleared rapidly from the circulation by the reticuloendothelial cells. In rats, uptake of liposomes by Kupffer cells requires that the liposomes bind to plasma proteins. In mice, the clearance of liposomes from the circulation is related to their ability to interact with plasma proteins. β2-glycoprotein I was identified as a major protein associated with rapid clearance of liposomes and pretreating the mice with antiβ2- glycoprotein I antibodies was found to significantly increase the half-life of the liposome. In vitro, β2-glycoprotein I was also shown to promote the phagocytosis of phosphatidylserine containing liposomes and apoptotic tumor cells. In conditions associated with increased microvesicles generation such as disseminated intravascular coagulation, plasma levels of β;2-glycoprotein I was reduced presumably due to its consumption. Antibodies to β2 glycoprotein I are frequently seen in patients with systemic lupus erythematosus and at times, in otherwise normal individuals. A subset of these antibodies prevents the assembly of the prothrombinase and the tenase complexes on phospholipid membrane, leading to the lupus anticoagulant effect. The presence of these antibodies is clinically very significant, as individuals harboring these antibodies are at risk for thromboembolic manifestations. We studied the role of β-glycoprotein I in the clearance of procoagulant platelet microvesicles and the effect of the auto antibodies in the phagocytosis of platelet microvesicles. We labeled β2-glycoprotein I with BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-hydrazide and β2-glycoprotein I incorporated 1.8 mole of BODIPY /mole. Labeling of β2-glycoprotein I with BODIPY did not change the binding efficacy of β2-glycoprotein I to cardiolipin as determined by Elisa assay. Binding of BODIPY-β2-glycoprotein I to platelet microvesicles was analyzed by flow cytometry. BODIPY- β2-glycoprotein I bound to phosphatidylserine-expressing platelet microvesicles in a concentration-dependent manner. Binding was inhibited by 50 fold molar excess of unlabeled β2-glycoprotein I, annexin A5 and the phosphatidylserine-binding C1C2 fragment of lactadherin. β2-glycoprotein I also promoted the phagocytosis of platelet microvesicles by THP-1 derived macrophages in vitro at physiological plasma concentrations with a half maximal effect at ∼10 ug/ml. β2-glycoprotein I-mediated phagocytosis was inhibited by annexin V and the C1C2 fragment of lactadherin. Furthermore, immunoaffinity purified β2-glycoprotein I-dependent antiphospholipid antibodies from 5 patients inhibited the phagocytosis in a concentration dependent manner. These studies suggest β2-glycoprotein I binding to phosphatidylserine-expressing procoagulant platelet microvesicles promotes their clearance by macrophages and autoantibodies to β2-glycoprotein I inhibit the process. The predictive value of antiβ-2 glycoprotein I for thrombosis is highly variable but the correlation is stronger in patients with lupus. In lupus, there is impaired clearance of procoagulant apoptotic cells. β2-glycoprotein I may have a significant role in their clearance and antibodies to β2-glycoprotein I may causally related to the thrombosis in these patients by inhibiting the clearance. Disclosures: No relevant conflicts of interest to declare.

The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

1988 ◽  
Vol 116 (2) ◽  
pp. 231-239 ◽  
Author(s):  
M. S. Johnson ◽  
R. Mitchell ◽  
G. Fink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

scholarly journals Bni5p, a Septin-Interacting Protein, Is Required for Normal Septin Function and Cytokinesis in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (19) ◽  
pp. 6906-6920 ◽  
Author(s):  
Philip R. Lee ◽  
Sukgil Song ◽  
Hyeon-Su Ro ◽  
Chong J. Park ◽  
John Lippincott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Localization ◽  
Growth Defect ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Growth Defects ◽  
Bud Emergence ◽  
Bud Neck ◽  
And Function

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Δ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Δ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

Inhibition of Apoptosis in Early Tooth Development Alters Tooth Shape and Size

2006 ◽  
Vol 85 (6) ◽  
pp. 530-535 ◽  
Author(s):  
J.-Y. Kim ◽  
Y.-G. Cha ◽  
S.-W. Cho ◽  
E.-J. Kim ◽  
M.-J. Lee ◽  
...  
Keyword(s):  
Tooth Development ◽  
Tooth Germ ◽  
Dependent Manner ◽  
Crown Height ◽  
Concentration Dependent Manner ◽  
Molar Teeth ◽  
In Vitro Organ Culture ◽  
Shape And Size

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

scholarly journals Direct synergistic effects of interleukin-7 on in vitro myelopoiesis of human CD34+ bone marrow progenitors

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 775-779 ◽  
Author(s):  
FW Jacobsen ◽  
LS Rusten ◽  
SE Jacobsen
Keyword(s):  
Colony Formation ◽  
Synergistic Effects ◽  
Dependent Manner ◽  
Morphological Examination ◽  
Concentration Dependent Manner ◽  
Interleukin 7 ◽  
Human Cd34 ◽  
Cd34 Cells

Interleukin-7 (IL-7) is an important growth factor in B and T lymphopoiesis in mouse and human, whereas IL-7 has been regarded to lack proliferative effects on cells within the myeloid lineage. However, we have recently reported that IL-7 potently can enhance colony stimulating factor (CSF)-induced myelopoiesis from primitive murine hematopoietic progenitors, showing a novel role of IL-7 in early murine myelopoiesis. Using CD34+ human hematopoietic progenitor cells, we show here a similar role of IL-7 in human myelopoiesis, although interesting differences between the two species were found as well. Although purified recombinant human (rh)IL-7 alone did not induce any proliferation of CD34+ cells, IL-7 in a concentration-dependent manner enhanced the colony formation induced by all four CSFs up to threefold. Furthermore, stem cell factor (SCF)-induced granulocyte-macrophage (GM) colony formation was increased fourfold in the presence of IL-7. Single- cell cloning assays showed that these synergistic effects of IL-7 were directly mediated on the targeted progenitors, and that IL-7 increased the number, as well as the size of the colonies formed. Morphological examination showed that IL-7 affected the progeny developed from CD34+ cells stimulated by G-CSF or IL-3, increasing the number of CFU-M (colony forming unit-macrophage) and CFU-granulocyte-macrophage, whereas the number of CFU-granulocyte were unaltered.

scholarly journals BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation

2016 ◽  
Vol 113 (27) ◽  
pp. 7655-7660 ◽  
Author(s):  
Dongqing Xu ◽  
Yan Jiang ◽  
Jigang Li ◽  
Fang Lin ◽  
Magnus Holm ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
26S Proteasome ◽  
Light Signaling ◽  
Dependent Manner ◽  
Positive Regulator ◽  
Molecular Base ◽  
Precise Role

BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub.

scholarly journals Direct synergistic effects of interleukin-7 on in vitro myelopoiesis of human CD34+ bone marrow progenitors

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 775-779 ◽  
Author(s):  
FW Jacobsen ◽  
LS Rusten ◽  
SE Jacobsen
Keyword(s):  
Colony Formation ◽  
Synergistic Effects ◽  
Dependent Manner ◽  
Morphological Examination ◽  
Concentration Dependent Manner ◽  
Interleukin 7 ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Interleukin-7 (IL-7) is an important growth factor in B and T lymphopoiesis in mouse and human, whereas IL-7 has been regarded to lack proliferative effects on cells within the myeloid lineage. However, we have recently reported that IL-7 potently can enhance colony stimulating factor (CSF)-induced myelopoiesis from primitive murine hematopoietic progenitors, showing a novel role of IL-7 in early murine myelopoiesis. Using CD34+ human hematopoietic progenitor cells, we show here a similar role of IL-7 in human myelopoiesis, although interesting differences between the two species were found as well. Although purified recombinant human (rh)IL-7 alone did not induce any proliferation of CD34+ cells, IL-7 in a concentration-dependent manner enhanced the colony formation induced by all four CSFs up to threefold. Furthermore, stem cell factor (SCF)-induced granulocyte-macrophage (GM) colony formation was increased fourfold in the presence of IL-7. Single- cell cloning assays showed that these synergistic effects of IL-7 were directly mediated on the targeted progenitors, and that IL-7 increased the number, as well as the size of the colonies formed. Morphological examination showed that IL-7 affected the progeny developed from CD34+ cells stimulated by G-CSF or IL-3, increasing the number of CFU-M (colony forming unit-macrophage) and CFU-granulocyte-macrophage, whereas the number of CFU-granulocyte were unaltered.

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