Technical note on the demonstration of rheumatoid factor by immunofluorescence

The authors present a new technic to point out the rheumatoid factor by immunofluorescence and compare this technic and the others to classical technics of agglutination. This technic allows to avoid the absorption of rheumatoid factor in the serological reactions using the immunofluorescence.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Study of quercetin and fisetin synergistic effect on breast cancer and potentially involved signaling pathways

Naturally-derived drugs have drawn much attention in recent decades. Efficiency, lower toxicity, and economic reasons are some of their advantages that justify this broad range of administration for different diseases, including cancer. If we can find a specific combination that boosts the effects of their single therapy, leading to synergism effect, increased efficiency, and decreased toxicity, they can act even better. Quercetin and fisetin, two well-known flavonoids, have been used to fight against various cancers. In this study, we investigated their possible synergism quercetin and fisetin on MCF7, MDA-MB-231, BT549, T47D, and 4T1 breast cancer cell lines. Then the optimum combined dose was used to study their impacts on wound healing abilities and clonogenic properties. The real-time qPCR was used to study the expression of their validated downstream effectors in predicted pathways. A significant synergism effect (p < .01, combination index: <1) was observed for all cell lines. Combination therapy was significantly more effective in colony formation (p < .0001) and wound healing assays (p < .001) compared to single therapies. The expression level of potential effectors was also showed a greater change. In vivo study confirmed the in vitro results and showed how significantly (p < .001) their synergism promotes their singular function in inhibiting cancer progression. The breast cancer mouse models receiving combined therapy lived longer with higher average body weight and smaller tumor sizes. These results exhibit that quercetin and fisetin inhibit cancer cell proliferation, migration and colony formation synergistically, and matrix metalloproteinase signaling and apoptotic pathways are relatively responsible for inhibitory activities.     

Synthesis,Biological Evaluation,and Molecular Modeling Studies of New 1,3,4-Thiadiazole Derivatives as Potent Antimicrobial Agents

In this work, the synthesis, characterization, and biological activities of a new series of 1,3,4-thiadiazole derivatives were investigated. The structures of final compounds were identified using ¹H-NMR, ¹³C-NMR, elemental analysis, and HRMS. All the new synthesized compounds were then screened for their antimicrobial activity against four types of pathogenic bacteria and one fungal strain, by application of the MIC assays, using Ampicilin, Gentamycin, Vancomycin, and Fluconazole as standards. Among the compounds, the MIC values of 4 and 8 μg/mL of the compounds 3f and 3g , respectively, are remarkable and indicate that these compounds are good candidates for antifungal activity. The docking experiments were used to identify the binding forms of produced ligands with sterol 14-demethylase to acquire insight into relevant proteins. The MD performed about 100 ns simulations to validate selected compounds’ theoretical studies. Finally, using density functional theory (DFT) to predict reactivity, the chemical characteristics and quantum factors of synthesized compounds were computed. These results were then correlated with the experimental data. Furthermore, computational estimation was performed to predict the ADME properties of the most active compound 3f .     

Two New C19-Diterpenoid Alkaloids from Delphinium shawurense

Shawurenine C ( 1a ) and D ( 1b ), a new pair of regioisomeric C₁₉-diterpenoid alkaloids, and five known C₁₉-diterpenoid alkaloids ( 2 – 6 ) were isolated from the aerial part of Delphinium shawurense W. T. Wang. The chemical structures of new compounds were established based on spectroscopic analyses: HR-ESI-MS, and 1D, 2D NMR spectroscopic data. The anti-inflammatory and cytotoxic activities of these diterpenoid alkaloids were also evaluated.     

Design,Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC₅₀ values) for the synthesized compounds ranged from 0.12 to 11.92 μM and 0.04 to 24.36 μM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC₅₀ value of 0.12 μM, whereas the highest BChE inhibition was achieved by structure 7 b (IC₅₀=0.04 μM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b . The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.