scholarly journals Molecular Cloning and Characterization of Phocein, a Protein Found from the Golgi Complex to Dendritic Spines

2001 ◽  
Vol 12 (3) ◽  
pp. 663-673 ◽  
Author(s):  
Gilbert Baillat ◽  
Abdelaziz Moqrich ◽  
Francis Castets ◽  
Agnès Baude ◽  
Yannick Bailly ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Hela Cells ◽  
Brefeldin A ◽  
Calmodulin Binding ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Clathrin Adaptor

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the ς subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity betweenDrosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.

scholarly journals Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments

2000 ◽  
Vol 11 (1) ◽  
pp. 171-182 ◽  
Author(s):  
William T. Brigance ◽  
Charles Barlowe ◽  
Todd R. Graham
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Proteolytic Processing ◽  
Golgi Compartment ◽  
Specific Antisera ◽  
Sensitive Factor ◽  
Processing Event ◽  
Golgi Network

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.

Serotonin 5HT1B/1D Receptor Agonists Abolish NMDA Receptor-evoked Enhancement of Nitric Oxide Synthase Activity and cGMP Concentration in Brain Cortex Slices

Cephalalgia ◽  
1999 ◽  
Vol 19 (10) ◽  
pp. 859-865 ◽  
Author(s):  
A Stȩpień ◽  
M Chalimoniuk ◽  
J Strosznajder
Keyword(s):  
Nmda Receptor ◽  
Brain Cortex ◽  
Receptor Agonists ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Brain Cortex Slices ◽  
Cortex Slices ◽  
The Brain

Our previous studies indicating that the function of excitatory amino acids, NMDA type receptor, is modulated by serotonin focused on the interaction between serotonin 5HT1B/1D and glutamate, NMDA receptor in brain cortex. The effect of agonists of 5HT1B/1D receptor, sumatriptan, and zolmitriptan on NMDA receptor-evoked activation of nitric oxide (NO) and cGMP synthesis in adult rat brain cortex slices was investigated. Two kinds of experiment were carried out using adult rats. In one of them, sumatriptan or zolmitriptan was administered in vivo subcutaneously (s.c.) in a dose of 0.1 mg per kg body weight. Brain slices were then prepared and used in the experiments or, in the other exclusively in vitro studies, both agonists at 10 μM concentration were added directly to the incubation medium containing adult rat brain cortex slices. The data obtained from these studies indicated that stimulation of NMDA receptor in brain cortex slices. The data obtained from these studies indicated that stimulation of NMDA receptor in brain cortex slices leads to a large increase in calcium, calmodulin-dependent NO synthase (NOS) activity and to significant enhancement of the cGMP level. This NMDA receptor-dependent NO and cGMP release was completely blocked by competitive and noncompetitive NMDA receptor antagonists APV (10 μM) or MK-801 (10 μM.), respectively. The specific inhibitor of Ca2+-dependent isoforms of NOS (N-nitro-1-arginine NNLA and 7-nitroindozole (7-N1)) eliminated the NMDA receptor-mediated enhancement of NO and cGMP release. Moreover, the serotonin 5HT1B/1D receptor agonists sumatriptan and zolmitriptan administrated in vivo (s.c.) or in vitro abolished NMDA receptor-evoked NO signalling in brain cortex. The potency of both agonists investigated directly in vitro was similar to their effect after in vivo administration. These results suggest that both serotonin 5HT1B/1D receptor agonists may play an important role in modulating the NO and cGMP-dependent signal transduction pathway in the brain. This effect of sumatriptan and zolmitriptan on NO signaling in the brain system should be taken into consideration when investigating their mechanism of action in the migraine attack.

scholarly journals Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

1997 ◽  
Vol 137 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Jochen Scheel ◽  
Rainer Pepperkok ◽  
Martin Lowe ◽  
Gareth Griffiths ◽  
Thomas E. Kreis
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Marker Proteins ◽  
Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

scholarly journals Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

2001 ◽  
Vol 69 (9) ◽  
pp. 5626-5634 ◽  
Author(s):  
David A. Lewis ◽  
Marla K. Stevens ◽  
Jo L. Latimer ◽  
Christine K. Ward ◽  
Kaiping Deng ◽  
...  
Keyword(s):  
Hela Cells ◽  
Culture Supernatant ◽  
Cell Envelope ◽  
Rabbit Model ◽  
Haemophilus Ducreyi ◽  
Supernatant Fluid ◽  
E Coli

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtCgenes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtAmutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

scholarly journals The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins

2001 ◽  
Vol 152 (5) ◽  
pp. 923-934 ◽  
Author(s):  
Jason H. Walenta ◽  
Aaron J. Didier ◽  
Xinran Liu ◽  
Helmut Krämer
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Spatial Organization ◽  
Large Fraction ◽  
Coiled Coil ◽  
Brefeldin A ◽  
Associated Proteins ◽  
Terminal Domains

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

Characterization of neural stem cells in the dorsal vagal complex of adult rat by in vivo proliferation labeling and in vitro neurosphere assay

Neuroscience ◽  
2006 ◽  
Vol 138 (1) ◽  
pp. 5-16 ◽  
Author(s):  
C. Charrier ◽  
V. Coronas ◽  
J. Fombonne ◽  
M. Roger ◽  
A. Jean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Dorsal Vagal Complex ◽  
Adult Rat ◽  
Neurosphere Assay

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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