OBJECTIVETo observe the effects of thermal stress on proliferation of human vascular endothelial cells (VECs) and explore its significance.

METHODSChanges of VECs proliferation were investigated with (3)H-TdR incorporation method after ECV304 was treated at 43 degrees for 2 hours, while expressions of intercellular adhesion molecule-1 (ICAM-1), inhibitor of differentiation-1 (ID1), and P16 and P21 proteins were determined by Western Blotting.

RESULTSThe effect of inhibition of VECs growth after thermal stress was detected by (3)H-TdR incorporation experiment. Western blotting showed ICAM-1, a marker of activated endothelial cells, was increased markedly after thermal stress. Expression of ID1 protein declined gradually with increasing expressions of its downstream genes, P16 and P21 following the thermal stress.

CONCLUSIONSThermal stress could strongly activate VECs and inhibit proliferation of VECs through ID1, thus down regulating cyclin-dependent kinase inhibitors, P16 and P21, which might be an essential pathway for recovery of VECs after thermal stress.

Blotting, Western ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Helix-Loop-Helix Motifs ; physiology ; Humans ; Inhibitor of Differentiation Protein 1 ; Intercellular Adhesion Molecule-1 ; metabolism ; Repressor Proteins ; Temperature ; Transcription Factors ; metabolism ; Umbilical Veins ; cytology

This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.
Animals ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; COS Cells ; Cercopithecus aethiops ; Gene Fusion ; Genetic Vectors ; Helix-Loop-Helix Motifs ; genetics ; Leptospira ; genetics ; Lipoproteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.
Blotting, Western ; Cell Differentiation ; Cell Line, Tumor ; Cloning, Molecular ; Cyclic AMP/metabolism ; DNA, Complementary/genetics ; Gene Expression Regulation, Neoplastic ; Helix-Loop-Helix Motifs ; Human ; Nerve Tissue Proteins/chemistry/*genetics/*metabolism ; Neurites/*metabolism ; Neuroblastoma/genetics/*metabolism/*pathology ; Trans-Activation (Genetics) ; Transcription Factors/chemistry/*genetics/*metabolism

Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.
Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; Cells, Cultured ; DNA-Binding Proteins/genetics/*metabolism ; E-Box Elements ; Gene Expression Regulation/physiology ; Helix-Loop-Helix Motifs ; Human ; Islets of Langerhans/cytology/metabolism ; Mice ; Molecular Sequence Data ; Nerve Tissue Proteins/genetics/*metabolism ; Neurons/cytology/metabolism ; Organ Specificity ; Transcription Factors/genetics/*metabolism ; Two-Hybrid System Techniques

Rat hippocampal precursor cells isolated from hippocampi of embryonic day 16.5 (E16.5) rat embryos were found to proliferate in the presence of basic fibroblast growth factor. Addition of soluble neural cell adhesion molecule (NCAM) to these precursor cells reduced cell proliferation in a dose dependent manner and enhanced the induction of precursor cells' differentiation to the neuronal lineage. Given these findings that NCAM induces the differentiation of hippocampal precursor cells, we investigated possible effects of NCAM on the expression of basic helix-loop-helix (bHLH) transcription factors during the differentiation. Soluble NCAM upregulated the transcription of bHLH transcription factors, neurogenin1 and NeuroD, but decreased HES5. Western blot analysis showed that NCAM increased the expression levels of CaMKII, p-MAPK, GluR1 and NR1 but decreased p-STAT3. These results support a role for NCAM in the inhibition of proliferation and the induction of neural differentiation of hippocampal neural precursor cells, and act as developmental regulators of the bHLH families, ultimately leading to the generation of glutamatergic neural cell types in the differentiation of hippocampal precursor cells.
Animals ; Apoptosis/drug effects ; Cell Differentiation/*drug effects ; Cell Division/drug effects ; Cell Lineage/drug effects ; Cells, Cultured ; Helix-Loop-Helix Motifs ; Hippocampus/*cytology/*drug effects ; Neural Cell Adhesion Molecules/*pharmacology ; Neurons/cytology/*drug effects/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Receptors, Glutamate/*metabolism ; Signal Transduction ; Stem Cells/cytology/*drug effects ; Transcription Factors/genetics/metabolism

The EF-hand superfamily is a large group of proteins which contain EF-hand motif formed by helix-loop-helix. These proteins always have the ability of binding metal ions or forming dimmers. Troponin C, known as having ability of binding Ca2+, is one member of the EF-hand superfamily. Troponin C interacts with troponin I and troponin T, forming a troponin complex which takes part in regulating muscle contraction. It is interesting that troponin C was also found in non-muscular tissue, and its function was proved to be different from that of troponin C found in muscular tissue. To date, a lot of researches about troponin C have been carried out widely. However, most of them focused on vertebrate, seldom were done on invertebrate. Our group carried out a research on troponin C from silkworm, a model organism of insects, aiming to clarify the structure and function of silkworm troponin C. Here, we mainly discuss the characters of the EF-hand superfamily and the classification, structure and function of troponin C . We also introduced our work about silkworm troponin C briefly, hoping of making a little contribution to the research of invertebrate troponin C.
Amino Acid Sequence ; Animals ; Binding Sites ; genetics ; Bombyx ; genetics ; metabolism ; Calcium ; metabolism ; EF Hand Motifs ; Molecular Sequence Data ; Phylogeny ; Protein Binding ; Troponin C ; classification ; genetics ; metabolism

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²⁺ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca²⁺ binding. Because the Ca²⁺ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²⁺ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca²⁺ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca²⁺-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca²⁺ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca²⁺. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²⁺ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²⁺ and modulate the activation by Ca²⁺ and voltage.
Amino Acids, Acidic ; Animals ; Calcium ; Calmodulin ; Chloride Channels ; Cryoelectron Microscopy ; Crystallography, X-Ray ; EF Hand Motifs ; Mice ; Models, Molecular ; Mutagenesis ; Nectria ; Protein Isoforms

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.
Antibodies ; Blotting, Western ; Calcium-Binding Proteins ; Cations, Divalent ; Dyneins ; EF Hand Motifs ; Electrophoretic Mobility Shift Assay ; Fasciola hepatica ; Humans ; Molecular Weight ; Opisthorchiasis ; Opisthorchis ; Parasites ; Protein Isoforms

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 micrometer of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.
Transfection ; Sequence Analysis, DNA ; Sequence Alignment ; Microscopy, Electron, Transmission ; Microfilament Proteins/*chemistry/genetics/*metabolism ; EF Hand Motifs ; DNA, Complementary ; Culture Media ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; Actins/*metabolism ; Acanthamoeba/genetics/growth & development/*metabolism

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
Amino Acid Sequence ; Binding Sites ; Calcium ; physiology ; Calcium-Binding Proteins ; chemistry ; Crystallography, X-Ray ; EF Hand Motifs ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Tertiary ; Sequence Alignment ; Sequence Homology, Amino Acid ; Streptomyces coelicolor ; Structural Homology, Protein ; Surface Properties