scholarly journals Phospholipase C in mouse oocytes: characterization of β and γ isoforms and their possible involvement in sperm-induced Ca2+ spiking

1996 ◽  
Vol 316 (2) ◽  
pp. 583-591 ◽  
Author(s):  
Genevieve DUPONT ◽  
Orla M. McGUINNESS ◽  
Martin H. JOHNSON ◽  
Michael J. BERRIDGE ◽  
Franck BORGESE
Keyword(s):  
Monoclonal Antibodies ◽  
Phospholipase C ◽  
Kinase Inhibitors ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Functional Studies ◽  
Mouse Oocytes ◽  
Mature Mouse

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLCβ1, PLCβ3 and PLCγ isoenzymes. Furthermore, immunodetection of PLCγ1 using monoclonal antibodies reveals that PLCγ1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLCβ protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 μM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 μM), herbimycin (10 μM) and geldanamycin (0.6 μM) which could affect signalling through PLCγ hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.

scholarly journals The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs

2021 ◽  
Author(s):  
Anna Storey ◽  
Khalil Elgmati ◽  
Yisu Wang ◽  
Paul Knaggs ◽  
Karl Swann
Keyword(s):  
Phospholipase C ◽  
Egg Activation ◽  
Apparent Difference ◽  
Free Medium ◽  
Inositol 1 ◽  
Atp Levels ◽  
Mature Mouse ◽  
Differential Ability ◽  
Mouse Eggs

Abstract At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1-4-5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI. Mouse eggs incubated in Ca2+-free, Sr2+-containing medium immediately underwent Ca2+ oscillations but human eggs consistently failed to undergo Ca2+ oscillations in the same Sr2+ medium. We tested the InsP3-receptor (IP3R) sensitivity directly by photo-release of caged InsP3 and found that mouse eggs were about 10 times more sensitive to InsP3 than human eggs. There were no major differences in the Ca2+ store content between mouse and human eggs. However, we found that the ATP concentration was consistently higher in mouse compared to human eggs. When ATP levels were lowered in mouse eggs by incubation in pyruvate-free medium, Sr2+ failed to cause Ca2+ oscillations. When pyruvate was added back to these eggs, the ATP levels increased and Ca2+ oscillations were induced. This suggests that ATP modulates the ability of Sr2+ to stimulate IP3R-induced Ca2+ release in eggs. We suggest that human eggs may be unresponsive to Sr2+ medium because they have a lower level of cytosolic ATP.

scholarly journals The Effect of Octapeptide Repeats on Prion Folding and Misfolding

2021 ◽  
Vol 22 (4) ◽  
pp. 1800
Author(s):  
Kun-Hua Yu ◽  
Mei-Yu Huang ◽  
Yi-Ru Lee ◽  
Yu-Kie Lin ◽  
Hau-Ren Chen ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Age Of Onset ◽  
Prion Diseases ◽  
Critical Role ◽  
Threshold Effect ◽  
Central Feature ◽  
Terminal Domain ◽  
Amyloid Aggregates

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.

scholarly journals Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-6
Author(s):  
Yanli Zhang ◽  
Linley R. Schofield ◽  
Carrie Sang ◽  
Debjit Dey ◽  
Ron S. Ronimus
Keyword(s):  
Biosynthetic Pathway ◽  
Critical Role ◽  
Reduction Reaction ◽  
Coenzyme M ◽  
Purification And Characterization ◽  
The Third ◽  
Optimal Activity ◽  
Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

scholarly journals Characterization of COMMD protein–protein interactions in NF-κB signalling

2006 ◽  
Vol 398 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Prim de Bie ◽  
Bart van de Sluis ◽  
Ezra Burstein ◽  
Karen J. Duran ◽  
Ruud Berger ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Copper Metabolism ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Protein Family ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Necrosis Factor

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-κB (nuclear factor κB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-κB signalling through characterization of protein–protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-κB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-κB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-κB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IκBα (inhibitory κBα), indicating that both proteins inhibit NF-κB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein–protein interactions and provide new mechanistic insight into the function of this protein family in NF-κB signalling.

PDGF-AA and its receptor influence early lung branching via an epithelial-mesenchymal interaction

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2559-2567 ◽  
Author(s):  
P. Souza ◽  
M. Kuliszewski ◽  
J. Wang ◽  
I. Tseu ◽  
A.K. Tanswell ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Critical Role ◽  
Polymerase Chain Reaction Analysis ◽  
Branching Morphogenesis ◽  
Inhibitory Effect ◽  
Embryonic Lung ◽  
Morphometric Analyses ◽  
Terminal Buds ◽  
Lung Branching

The biological role of platelet-derived growth factor (PDGF)-AA in lung morphogenesis was investigated by incubating embryonic lung explants with phosphorothioate antisense PDGF-A oligonucleotides, which decreased PDGF-AA but not PDGF-BB protein content. Antisense PDGF-A oligonucleotides inhibited DNA synthesis. This inhibitory effect of antisense PDGF-A was reversed by the addition of exogenous PDGF-AA but not PDGF-BB. Morphometric analyses of antisense-treated cultures showed a significant reduction in lung size. The number of terminal buds of the lung explants was significantly decreased by antisense PDGF-A oligonucleotides. PDGF-AA but not PDGF-BB attenuated the inhibitory effect of antisense PDGF-A on early lung branching. Sense PDGF-A had no effect on DNA synthesis and early lung branching. Reverse transcriptase-polymerase chain reaction analysis revealed PDGF-A mRNA expression in the epithelial component of the embryonic lung, while message for PDGF alpha-receptor was expressed in the mesenchyme. Incubation of explants with neutralizing PDGF-AA antibodies also reduced DNA synthesis and early branching morphogenesis. We conclude that PDGF-AA and its receptor represent an important epithelial-mesenchymal interaction which plays a critical role in early lung branching morphogenesis.

scholarly journals A VH1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite

2020 ◽  
Vol 6 (38) ◽  
pp. eabb1328 ◽  
Author(s):  
Sonu Kumar ◽  
Bin Ju ◽  
Benjamin Shapero ◽  
Xiaohe Lin ◽  
Li Ren ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Neutralizing Antibodies ◽  
Critical Role ◽  
Viral Escape ◽  
Germline Gene ◽  
Broadly Neutralizing Antibodies ◽  
Functional Studies ◽  
Single Genome ◽  
Hiv 1

An oligomannose patch around the V3 base of HIV-1 envelope glycoprotein (Env) is recognized by multiple classes of broadly neutralizing antibodies (bNAbs). Here, we investigated the bNAb response to the V3 glycan supersite in an HIV-1–infected Chinese donor by Env-specific single B cell sorting, structural and functional studies, and longitudinal analysis of antibody and virus repertoires. Monoclonal antibodies 438-B11 and 438-D5 were isolated that potently neutralize HIV-1 with moderate breadth, are encoded by the VH1-69 germline gene, and have a disulfide-linked long HCDR3 loop. Crystal structures of Env-bound and unbound antibodies revealed heavy chain–mediated recognition of the glycan supersite with a unique angle of approach and a critical role of the intra-HCDR3 disulfide. The mechanism of viral escape was examined via single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Akt Activation Regulates Macrophage Survival and Differentiation: Role of M-CSF and Endogenous ROS.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2208-2208
Author(s):  
Yijie Wang ◽  
Mandy M. Zeigler ◽  
Greg K. Lam ◽  
Melissa G. Hunter ◽  
Tim D. Eubank ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Kinase Inhibitors ◽  
Critical Role ◽  
Annexin V ◽  
Mononuclear Phagocytes ◽  
Ros Generation ◽  
Blood Monocytes ◽  
Human Alveolar Macrophage

Abstract Previous reports from our laboratory showed M-CSF promotes PI 3-kinase activation resulting in the production of reactive oxygen species (ROS) and PI 3-kinase inhibitors, and the antioxidants diphenyleneiodonium (DPI) and n-acetyl cysteine (NAC) suppressed M-CSF-stimulated Erk activation. In this study, we hypothesized that M-CSF-induced generation of ROS affected Akt1 activation and sought to define the role of Akt1 in monocyte/macrophage survival and differentiation. We found that the production of ROS following M-CSF-treatment was inhibited by the antioxidant DPI. The addition of either DPI or NAC to the monocytes in the presence of M-CSF resulted in decrease cell survival as measured by Annexin V/PI and DNA fragmentation. In the cells treated with the antioxidants, there was a reduction in pAKT protein levels compared to M-CSF alone treatment suggesting that ROS contributed to Akt activity and cell survival. Macrophages from p47phox −/− mice, lacking a key component of the NADPH oxidase complex required for ROS generation were examined for M-CSF-induced survival and Akt1 activation. Bone marrow macrophages from p47phox −/ − mice and wild type (WT) littermates were isolated and differentiated in RPMI-1640 medium in the presence of 20 ng/ml of M-CSF for 5 days. We observed a reduction in Akt1 phosphorylation, cellular survival and increase in apoptosis measured by Annexin V/PI staining in p47phox −/ − macrophages compared to WT controls. Since macrophages from the p47phox−/− mice had reduced Akt1 activity and cell survival to M-CSF stimulation, we next wanted to independently evaluate the role of Akt in macrophage survival and differentiation. We therefore examined macrophages from mice that had targeted expression of activated Akt1 (Myr-Akt1) in mononuclear phagocytes. Bone marrow from Myr-Akt1 mice and (WT) littermates was isolated and cultured in the presence of 20 ng/ml of M-CSF for 5 days. We found that Myr-Akt1 cells had enhanced survival and reduced apoptosis versus WT cells. Interestingly, Myr-Akt1 mice had normal circulating numbers of monocytes, but had splenomegaly and increased numbers of mature macrophages in their spleens by CD68 staining. Since the expression of Myr-Akt1 in BMM enhanced cell survival, we were interested in the effect in human monocytes. Peripheral blood monocytes (PBM) were transiently transfected with Myr-Akt1 constructs. We observed a decrease in Annexin V/PI staining indicating promotion of cell survival in PBM expressing Myr-Akt compared to untransfected PBM. Since Akt1 appeared to be important for macrophage survival/differentiation, we examined its contribution in human alveolar macrophage homeostasis and found that freshly isolated macrophages had constitutive Akt1 activity and had increased levels of Akt1 protein. This data support a critical role for Akt1 in macrophage differentiation in mice. Together, our findings may provide insight in the pathogenesis of monocyte/macrophage homeostasis.

Personalized Medicine—Some Things To Consider

2013 ◽  
Vol 09 (01) ◽  
pp. 75
Author(s):  
J Marc Pipas ◽  
Lionel D Lewis ◽  
Marc S Ernstoff ◽  
Gregory J Tsongalis ◽  
◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Genome Project ◽  
Kinase Inhibitors ◽  
Healthcare Providers ◽  
Genome Project ◽  
Designer Drugs ◽  
Genomic Testing ◽  
Personalized Cancer ◽  
The Human Genome Project

The promise of individualized therapy has resonated with scientists, healthcare providers, and patients since the inception of the Human Genome Project. The ability to use designer drugs, such as novel monoclonal antibodies and molecularly targeted tyrosine kinase inhibitors, has created a new hope in the management of the cancer patient by individualizing therapy based on the unique molecular mutations in the cancer and the patient’s germline DNA. The nuances of implementing germline and somatic genomic testing in a clinical setting are discussed and the future role of pharmacogenomics testing in personalized cancer treatment is examined.

scholarly journals The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract

2016 ◽  
Vol 84 (9) ◽  
pp. 2697-2702 ◽  
Author(s):  
Zhangsheng Yang ◽  
Lingli Tang ◽  
Lili Shao ◽  
Yuyang Zhang ◽  
Tianyuan Zhang ◽  
...  
Keyword(s):  
Genital Tract ◽  
Chlamydial Infection ◽  
Critical Role ◽  
Content Type ◽  
Lower Genital Tract ◽  
Successful Infection ◽  
Multiple Strains

Despite the extensivein vitrocharacterization of CPAF (chlamydialprotease/proteasome-likeactivityfactor), its role in chlamydial infection and pathogenesis remains unclear. We now report that aChlamydia trachomatisstrain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficientC. trachomatisstrain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promotingC. trachomatissurvival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.

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