scholarly journals Slob, a Slowpoke channel–binding protein, modulates synaptic transmission

2011 ◽  
Vol 137 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Huifang Ma ◽  
Jiaming Zhang ◽  
Irwin B. Levitan
Keyword(s):  
Action Potential ◽  
Synaptic Transmission ◽  
Muscle Cell ◽  
Binding Protein ◽  
P Element ◽  
Macromolecular Complex ◽  
Functional Consequences ◽  
Targeted Expression

Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel–binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission.

scholarly journals Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1465-1475 ◽  
Author(s):  
T Kozlova ◽  
G V Pokholkova ◽  
G Tzertzinis ◽  
J D Sutherland ◽  
I F Zhimulev ◽  
...  
Keyword(s):  
Pupal Stage ◽  
Transcription Unit ◽  
P Element ◽  
Specific Response ◽  
Orphan Receptors ◽  
Mrna Isoforms ◽  
A Cell ◽  
In Vivo Analysis

Abstract DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

scholarly journals C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Kosuke Sasaki ◽  
Shigetsugu Takano ◽  
Satoshi Tomizawa ◽  
Yoji Miyahara ◽  
Katsunori Furukawa ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Binding Protein ◽  
Combined Treatment ◽  
Tumor Infiltrating Lymphocytes ◽  
Pdac Cell ◽  
Α Chain ◽  
Infiltrating Lymphocytes

Abstract Background Recent studies indicate that complement plays pivotal roles in promoting or suppressing cancer progression. We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). However, its mechanism of action remains unclear. Here, we elucidated the functional roles of C4BPA in PDAC cells and the tumor microenvironment. Methods We assessed stromal C4BPA, the C4BPA binding partner CD40, and the number of CD8+ tumor-infiltrating lymphocytes in resected human PDAC tissues via immunohistochemical staining. The biological functions of C4BPA were investigated in peripheral blood mononuclear cells (PBMCs) and human PDAC cell lines. Mouse C4BPA (mC4BPA) peptide, which is composed of 30 amino acids from the C-terminus and binds to CD40, was designed for further in vitro and in vivo experiments. In a preclinical experiment, we assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in a mouse orthotopic transplantation model. Results Immunohistochemical analysis revealed that high stromal C4BPA and CD40 was associated with favorable PDAC prognosis (P=0.0005). Stromal C4BPA strongly correlated with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001). In in vitro experiments, flow cytometry revealed that recombinant human C4BPA (rhC4BPA) stimulation increased CD4+ and CD8+ T cell numbers in PBMCs. rhC4BPA also promoted the proliferation of CD40-expressing PDAC cells. By contrast, combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis rate. mC4BPA peptide increased the number of murine T lymphocytes in vitro and the number of CD8+ tumor-infiltrating lymphocytes surrounding PDAC tumors in vivo. In a preclinical study, GnP/ICBs/mC4BPA peptide treatment, but not GnP treatment, led to the accumulation of a greater number of CD8+ T cells in the periphery of PDAC tumors and to greater tumor regression than did control treatment. Conclusions These findings demonstrate that the combination of GnP therapy with C4BPA inhibits PDAC progression by promoting antitumor T cell accumulation in the tumor microenvironment.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

scholarly journals In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

1998 ◽  
Vol 13 (3) ◽  
pp. 291-301 ◽  
Author(s):  
John H. Butler ◽  
Shiquan Hu ◽  
Shari R. Brady ◽  
Michael W. Dixon ◽  
Gloria K. Muday
Keyword(s):  
Binding Protein ◽  
Acid Binding Protein ◽  
Naphthylphthalamic Acid

scholarly journals Aloe-Emodin Relieves High-Fat Diet Induced QT Prolongation via MiR-1 Inhibition and IK1 Up-Regulation in Rats

2017 ◽  
Vol 43 (5) ◽  
pp. 1961-1973 ◽  
Author(s):  
Yan Bai ◽  
Zhenli Su ◽  
Hanqi Sun ◽  
Wei Zhao ◽  
Xue Chen ◽  
...  
Keyword(s):  
Action Potential ◽  
Protein Expression ◽  
High Fat Diet ◽  
Qt Prolongation ◽  
Electrical Remodeling ◽  
High Fat ◽  
Treatment Groups ◽  
Aloe Emodin

Background/Aims: High-fat diet (HFD) causes cardiac electrical remodeling and increases the risk of ventricular arrhythmias. Aloe-emodin (AE) is an anthraquinone component isolated from rhubarb and has a similar chemical structure with emodin. The protective effect of emodin against cardiac diseases has been reported in the literature. However, the cardioprotective property of AE is still unknown. The present study investigated the effect of AE on HFD-induced QT prolongation in rats. Methods: Adult male Wistar rats were randomly divided into three groups: control, HFD, and AE-treatment groups. Normal diet was given to rats in the control group, high-fat diet was given to rats in HFD and AE-treatment groups for a total of 10 weeks. First, HFD rats and AE-treatment rats were fed with high-fat diet for 4 weeks to establish the HFD model. Serum total cholesterol and triglyceride levels were measured to validate the HFD model. Afterward, AE-treatment rats were intragastrically administered with 100 mg/kg AE each day for 6 weeks. Electrocardiogram monitoring and whole-cell patch-clamp technique were applied to examine cardiac electrical activity, action potential and inward rectifier K+ current (IK1), respectively. Neonatal rat ventricular myocytes (NRVMs) were subjected to cholesterol and/or AE. Protein expression of Kir2.1 was detected by Western blot and miR-1 level was examined by real-time PCR in vivo and in vitro, respectively. Results: In vivo, AE significantly shortened the QT interval, action potential duration at 90% repolarization (APD90) and resting membrane potential (RMP), which were markedly elongated by HFD. AE increased IK1 current and Kir2.1 protein expression which were reduced in HFD rats. Furthermore, AE significantly inhibited pro-arrhythmic miR-1 in the hearts of HFD rats. In vitro, AE decreased miR-1 expression levels resulting in an increase of Kir2.1 protein levels in cholesterol-enriched NRVMs. Conclusions: AE prevents HFD-induced QT prolongation by repressing miR-1 and upregulating its target Kir2.1. These findings suggest a novel pharmacological role of AE in HFD-induced cardiac electrical remodeling.

scholarly journals The thalamic low-threshold Ca 2+ potential: a key determinant of the local and global dynamics of the slow (<1 Hz) sleep oscillation in thalamocortical networks

2011 ◽  
Vol 369 (1952) ◽  
pp. 3820-3839 ◽  
Author(s):  
Vincenzo Crunelli ◽  
Adam C. Errington ◽  
Stuart W. Hughes ◽  
Tibor I. Tóth
Keyword(s):  
Action Potential ◽  
Slow Oscillation ◽  
Global Dynamics ◽  
Action Potential Firing ◽  
Local And Global Dynamics ◽  
Up States ◽  
Potential Firing ◽  
Low Threshold

During non-rapid eye movement sleep and certain types of anaesthesia, neurons in the neocortex and thalamus exhibit a distinctive slow (<1 Hz) oscillation that consists of alternating UP and DOWN membrane potential states and which correlates with a pronounced slow (<1 Hz) rhythm in the electroencephalogram. While several studies have claimed that the slow oscillation is generated exclusively in neocortical networks and then transmitted to other brain areas, substantial evidence exists to suggest that the full expression of the slow oscillation in an intact thalamocortical (TC) network requires the balanced interaction of oscillator systems in both the neocortex and thalamus. Within such a scenario, we have previously argued that the powerful low-threshold Ca 2+ potential (LTCP)-mediated burst of action potentials that initiates the UP states in individual TC neurons may be a vital signal for instigating UP states in related cortical areas. To investigate these issues we constructed a computational model of the TC network which encompasses the important known aspects of the slow oscillation that have been garnered from earlier in vivo and in vitro experiments. Using this model we confirm that the overall expression of the slow oscillation is intricately reliant on intact connections between the thalamus and the cortex. In particular, we demonstrate that UP state-related LTCP-mediated bursts in TC neurons are proficient in triggering synchronous UP states in cortical networks, thereby bringing about a synchronous slow oscillation in the whole network. The importance of LTCP-mediated action potential bursts in the slow oscillation is also underlined by the observation that their associated dendritic Ca 2+ signals are the only ones that inform corticothalamic synapses of the TC neuron output, since they, but not those elicited by tonic action potential firing, reach the distal dendritic sites where these synapses are located.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

2012 ◽  
Vol 442 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Gráinne Barkess ◽  
Yuri Postnikov ◽  
Chrisanne D. Campos ◽  
Shivam Mishra ◽  
Gokula Mohan ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Splice Variants ◽  
Binding Protein ◽  
Histone H3 ◽  
Camp Response Element Binding ◽  
Element Binding Protein ◽  
H3k14 Acetylation ◽  
H3 Acetylation

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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