Characterization of the beta-adrenergic receptors of cultured human epidermal keratinocytes

The presence of beta-adrenergic receptors has been demonstrated in membrane preparations from passaged human epidermal keratinocytes. The receptors were characterized in terms of density and binding properties. Using the titrated beta-adrenergic antagonists dihydroalprenolol and propranolol, the equilibrium dissociation constant (Kd) was found to be about 1.4 nM for the two antagonists with a receptor density of approximately 280 fmol/mg membrane protein. Stereospecificity of the binding sites was shown by the much lowered affinity to D-isoproterenol as compared to that of L-isoproterenol. By the use of subtype specific antagonists, the receptors were classified as beta 2 adrenoceptors. This finding is supported by the relative order of affinities of the agonists isoproterenol greater than epinephrine greater than norepinephrine. The Kd value for dihydroalprenolol was approximately the same when determined from equilibrium binding studies or from association and dissociation kinetics, suggesting that the ligand binding is a single step bi-molecular reaction.     

Purification of three arginine esterases from the venom of the Western Diamondback rattlesnake (Crotalus atrox)

—Three acidic arginine esterases have been isolated from the venom of the Western Diamondback rattlesnake (Crotalus atrox). These components demonstrated marked differences in ionic characteristics, as noted by KCl gradient elution from a DEAE-Sephadex A-50 column. Molecular weights, as determined from gel permeation chromatography, were estimated at 25,100 (fraction D), 24,000 (B); and 22,900 (F) for the three separate enzymes. The two larger enzymes (B and D) exhibited similar activities toward the synthetic substrates α-N-benzoyl-l-arginine ethyl ester and α-N-benzoyl-dl-arginine-p-nitroanilid Hydrolysis rates were similar to commercial trypsin preparations. Fraction F exhibited a markedly lower activity as an arginine esterase and negligible activity as an arginine amidase. Arginine esterase activity was evident for all three enzymes in the presence of ethylenediamine tetra-acetic acid.     

Interaction of bromosulfophthalein with mitochondrial membranes-inhibition of respiration

Bromosulfophthalein reversibly inhibits mitochondrial respiration. Most sensitive is state 3 respiration (K_i about 3 nmole/mg protein independent of substrate). At higher concentrations (20–100 nmole/mg protein) state 4 respiration and uncoupled respiration are also inhibited. This inhibition is substrate dependent. With succinate, inhibition appears to be noncompetitive at low concentrations and competitive above 1 mM succinate (half-maximal inhibition at 9–17 nmole/mg protein, dependent on succinate concentration). Substrate permeation seems to be not the only sensitive step in oxidation, as is deduced from similar results obtained on glycerol phosphate respiration. By use of artificial electron shunts and by difference spectroscopy, individual dehydrogenases have been made probable as the site of bromosulfophthalein action. It is suggested that bromosulfophthalein acts via the electro-static effects of the increased negative surface charge, making dehydrogenases less accessible for their substrates.     

[3H]cocaine binding in brain is inhibited by tris (hydroxymethyl) aminomethane

High-affinity binding of [³H]cocaine to membranes of mouse cerebral cortex is inhibited by Tris (hydroxymethyl) aminomethane, the buffer commonly used in receptor binding assays. This inhibition is not due to an effect of ionic strength in general. Comparison of binding in Tris buffer with that in sodium phosphate buffer indicates a more than 4-fold higher K_d in the former buffer, with no differences in the B_max values.     

Two new phenol derivatives from Stereum hirsutum FP-91666

Two new phenol derivatives, 2-(3-methyl-2-buten-1-yl)-4-methoxyethyl-phenol (1) and 5-hydroxy-4-(hydroxymethyl)-2-(3-methylbut-2-en-1-yl)cyclohex-4-en-1-one (2), together with eight known compounds consisting of phenol derivatives (3 and 4), niacinamide (5), and five ergosta type compounds (6–10), were isolated from solid fermentation products of Stereum hirsutum FP-91666. Two new structures were elucidated by extensive spectroscopic methods, including 1D NMR and 2D NMR, and HR-EI-MS experiments.     

Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential

Glutamate induces reactive oxygen species formation (ROS) in neurons. Free radicals can potentially be synthesized by NADPH oxidase or mitochondria. The primary source of ROS origin has yet to be identified. In addition, pro-oxidant action of glutamate receptors on neuronal presynaptic terminals is still not characterized. We investigated the influence of glutamate and agonists of its ionotropic receptors on ROS formation detected by fluorescent dye DCFDA in rat brain synaptosomes. Glutamate in concentration 10 and 100μM led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100μM of NMDA or 100μM of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10μM), NO synthase (NOS) inhibitor l-NAME (100μM) and NADPH oxidase inhibitors DPI (30μM) and not affected by mitochondrial uncoupler CCCP (10μM) and mitochondrial toxins rotenone (10μM)+oligomycin (5μg/ml). We also showed that 100μM of glutamate leads to a decrease of intrasynaptosomal mitochondrial potential monitored by fluorescent dye Rhodamine-123. Hence, the depolarization of intrasynaptosomal mitochondria is not a primary cause of glutamate-induced ROS formation in neuronal presynaptic terminals. Activation of NMDA receptors might be responsible for a certain part of glutamate pro-oxidant action. Most likely, sources of glutamate-induced ROS formation in neuronal presynaptic terminals are NADPH oxidase and NOS activation.     

Acridine derivatives as anti-BVDV agents

Twenty-six 9-aminoacridine derivatives were evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. While seven compounds (9, 10, 14, 19, 21, 22, 24) did not affect any virus and two (6, 11) were moderately active against CVB-5 or Reo-1, 17 compounds exhibited a marked specific activity against BVDV, prototype of pestiviruses which are responsible for severe diseases of livestock. Most anti-BVDV agents showed EC₅₀ values in the range 0.1-8 μM, thus comparing favorably with the reference drugs ribavirine and NM 108. Some compounds, particularly those bearing a quinolizidinylalkyl side chain, displayed pronounced cytotoxicity. Further studies are warranted in order to achieve still better anti-BVDV agents, and to explore the potential antiproliferative activity of this kind of compounds.     

合子草化学成分的研究(Ⅱ)

目的研究合子草Actinostemma lobatum全草的化学成分。方法综合应用正、反相硅胶,Sephadex LH-20以及HPLC等现代色谱技术进行分离纯化,根据化合物的理化性质和波谱学性质鉴定化合物的结构。结果从合子草全草的75%乙醇提取物中分离得到12个化合物,分别鉴定为lobatoside B(1)、土贝母皂苷乙(2)、土贝母皂苷戊(3)、没食子酸(4)、没食子酸甲酯(5)、原儿茶酸(6)、原儿茶酸甲酯(7)、对羟基苯乙酮(8)、对羟基苯甲酸(9)、对羟基苯甲醛(10)、5-羟甲基-2-呋喃甲醛(11)、5-羟甲基-2-呋喃乙酮(12)。结论化合物2、5~12为首次从该属植物中分离得到。     

A novel sulphur glycoside from the seeds of Descurainia sophia (L.)

A new sulphur glycoside, named descurainoside (1), and the known compound sinapic acid (2) have been isolated from the seeds of Descurainia sophia (L.) Webb ex Prantl. The structure of 1 has been identified as (1R,6S,8R,9S,10S)-9,10-dihydroxy-4-[(4-hydroxy-3,5-dimethoxyphenyl)methylene]-8-(hydroxymethyl)-2,7-dioxa-5-thiabicyclo[4.4.0]decan-3-one by means of physico-chemical properties and spectroscopic methods (1D and 2D NMR, HRMS, ESI-MS).     

Release of nucleophosmin from the nucleus: Involvement in aloe-emodin-induced human lung non small carcinoma cell apoptosis

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) is one of the active constituents from the root and rhizome of Rheum palmatum. Our previous study has demonstrated that aloe-emodin induced a significant change in the expression of lung cancer cell apoptosis-related proteins compared to those of control cells. However, the molecular mechanisms underlying the biological effects of aloe-emodin still remain unknown. Based on these reasons, we were interested in the change of aloe-emodin-induced total protein expression by the proteomics technique during aloe-emodin-induced lung cancer cell apoptosis. Our study applied 2D electrophoresis to analyze the proteins involved in aloe-emodin-induced apoptosis in H460 cells. We found that the release of nucleophosmin from the nucleus to the cytosol and the degradation of nucleophosmin were associated with aloe-emodin-induced H460 cell apoptosis. Our study also demonstrated that the gene expression of nucleophosmin remained unchanged after treatment with aloe-emodin. The aloe-emodin-caused increase in the amount of proform and fragment of nucleophosmin in cytoplasm may be one of the important events for aloe-emodin-induced H460 cell apoptosis.