OBJECTIVETo explore the effect of the total flavonoids of Jiawei Wuzi Yanzong prescription on the Voltage-gated calcium channel of the CA1 pyramidal cell of rat hippocampus.

METHODThe inward calcium current was recorded by the whole-cell patch clamp and the amplitude of it was thought to observe the effect of Abeta25-35 and the total flavonoids. The hippocampus of rat was separated and cut into slices. Active pyramidal cells of slices were chosen for the whole-cell patch clamp recording. After exposure to Abeta25-35, voltage steps (500 ms) were used to depolarize stepwise in a range from 50 to +50 mV (increment: 5 mV). An inward Ca2+ current which was suggested to be survey was evoked. Application of the total flavonoids was to be observed if it had effect on this voltage-depended inward current.

RESULTAbeta25-35 could enhance the calcium current to induce intracellular calcium overload. The amplitude of the control group was--(157.1 +/- 19.9) pA, but after application of Abeta25-35 the current enhanced to--(323.2 +/- 23.4) pA. When the total flavonoids at concentration of 125, 250, 500 g x L(-1) were added, the current declined to--(257.9 +/- 31.6), - (196.4 +/- 29.8) and--(169.3 +/- 34.0) pA, respectively.

CONCLUSIONAbeta-induced intracellular calcium overload may be one of neurotoxic of beta-amyloid peptide. The total flavonoids of Jiawei Wuzi Yanzong prescription can suppress inward calcium current to protect neurons.

Alzheimer Disease ; drug therapy ; metabolism ; physiopathology ; Amyloid beta-Peptides ; metabolism ; Animals ; CA1 Region, Hippocampal ; cytology ; drug effects ; physiology ; Calcium Channels ; genetics ; physiology ; Disease Models, Animal ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Electrophysiology ; Female ; Flavonoids ; chemistry ; pharmacology ; Humans ; Male ; Peptide Fragments ; metabolism ; Prescription Drugs ; chemistry ; pharmacology ; Pyramidal Cells ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the changes in the expressions of p53, DKK (the inhibitor of Wnt pathway) and phosphorylated tau in rat bilateral hippocampus after beta-amyloid peptide (beta-AP) (25-35) injection, and observe the effect of saponin B from Anemarrhena asphodeloides Bunge (SAaB) in this model.

METHODSAfter bilateral injection of beta-AP (25-35) into the hippocampus of rats, RT-PCR was performed for observing the changes in p53 and DKK mRNA expressions and immunochemistry carried out to detect the changes in phosphorylated tau protein.

RESULTSRT-PCR showed increased p53 and DKK mRNA expression and immunochemistry revealed increased phosphorylated tau-positive cells in rat hippocampus after beta-AP (25-35) injection, and administration of SAaB significantly ameliorated these changes.

CONCLUSIONSAaB can significantly ameliorate beta-AP-induced tau hyperphosphorylation by inhibiting increased p53 and DKK mRNA expressions in response to beta-AP injection.

Amyloid beta-Peptides ; pharmacology ; Anemarrhena ; chemistry ; Animals ; Hippocampus ; drug effects ; metabolism ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Male ; Peptide Fragments ; biosynthesis ; genetics ; pharmacology ; Phosphorylation ; drug effects ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins ; pharmacology ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; tau Proteins ; metabolism

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
Animals ; Biosensing Techniques ; Cattle ; Cell Division/drug effects ; Cell Movement/*drug effects ; Cells, Cultured ; Chickens ; Cricetinae ; Endothelial Cells/*cytology/*drug effects ; Humans ; Kinetics ; *Kringles ; Ligands ; Peptide Fragments/*chemistry/genetics/metabolism/*pharmacology ; Protein Binding ; Receptors, Cell Surface/metabolism ; Receptors, Urokinase Plasminogen Activator ; Urokinase-Type Plasminogen Activator/*chemistry/genetics/pharmacology ; Vascular Endothelial Growth Factor A/pharmacology

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.
Amino Acid Sequence ; Animals ; Antithrombin III/metabolism ; Blood Coagulation/drug effects ; Circular Dichroism ; Humans ; Models, Molecular ; Molecular Sequence Data ; Peptide Fragments/*chemistry/pharmacology ; Protein Binding/drug effects ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Rats ; Seminal Vesicle Secretory Proteins/*chemistry/genetics/metabolism ; Thrombin/*chemistry/genetics/metabolism

OBJECTIVETo study the alterations of alpha-7 nicotinic receptor (nAChR) status in human brain tissues with Alzheimer's disease (AD) and mouse brain tissues with Swedish APP670/671 gene mutation, and to study the effect of beta-amyloid peptides (A-beta) on alpha-7 nAChR status in cultured astrocytes and neurons.

METHODSPostmortem brain tissues from patients with AD and mouse brain tissues with Swedish APP mutation were collected. The expression of alpha-7 nAChR on astrocytes and neurons was detected by immunohistochemistry (ABC method). The alpha-7 nAChR protein level was measured by Western blotting. On the other hand, cultured astrocytes and neurons were treated with different concentrations of A-beta 25 - 35. The alpha-7 nAChR protein level was then measured.

RESULTSIncreased number of astrocytes surrounding senile plaques was observed in AD brain tissues. In AD brain tissues, as compared to age-matched controls, alpha-7 nAChR protein level was increased in astrocytes, but decreased in neurons. High level of alpha-7 nAChR protein was also observed in mouse brain tissues with APP mutation. Exposure to A-beta 25 - 35 induced an increase (up to 38%) in alpha-7 nAChR protein level in astrocytes but a decrease (up to 32%) in neurons.

CONCLUSIONSDecrease in alpha-7 nAChR level in neurons may be related to the pathogenesis of AD, whereas an increased level of alpha-7 nAChR in astrocytes, as induced by excessive A-beta, may represent a compensatory neuroprotective response.

Aged ; Aged, 80 and over ; Alzheimer Disease ; genetics ; metabolism ; pathology ; Amyloid beta-Peptides ; chemistry ; genetics ; metabolism ; Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Brain ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Glial Fibrillary Acidic Protein ; analysis ; Humans ; Immunoblotting ; Immunohistochemistry ; Male ; Mice ; Mutation ; Neurons ; cytology ; drug effects ; metabolism ; Peptide Fragments ; pharmacology ; Receptors, Nicotinic ; biosynthesis