Huaju Xiaoji Formula Regulates ERS-lncMGC/miRNA to Enhance the Renal Function of Hypertensive Diabetic Mice with Nephropathy.

Background: Better therapeutic drugs are required for treating hypertensive diabetic nephropathy. In our previous study, the Huaju Xiaoji (HJXJ) formula promoted the renal function of patients with diabetes and hypertensive nephropathy. In this study, we investigated the therapeutic effect and regulation mechanism of HJXJ in hypertensive diabetic mice with nephropathy. Methods: We constructed a mouse hypertensive diabetic nephropathy (HDN) model by treating mice with streptozotocin (STZ) and nomega-nitro-L-arginine methyl ester (LNAME). We also constructed a human glomerular mesangial cell (HGMC) model that was induced by high doses of sugar (30 mmol/mL) and TGFß1 (5 ng/mL). Pathological changes were evaluated by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and Masson staining. The fibrosis-related molecules (TGFß1, fibronectin, laminin, COL I, COL IV, α-SMA, and p-smad2/3) were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels and protein expression of endoplasmic reticulum stress, fibrosis molecules, and their downstream molecules were assessed using qPCR and Western blotting assays. Results: Administering HJXJ promoted the renal function of HDN mice. HJXJ reduced the expression of ER stress makers (CHOP and GRP78) and lncMGC, miR379, miR494, miR495, miR377, CUGBP2, CPEB4, EDEM3, and ATF3 in HDN mice and model HGMCs. The positive control drugs (dapagliflozin and valsartan) also showed similar effects after treatment with HJXJ. Additionally, in model HGMCs, the overexpression of CHOP or lncMGC decreased the effects of HJXJ-M on the level of fibrosis molecules and downstream target molecules. Conclusion: In this study, we showed that the HJXJ formula may regulate ERS-lncMGC/miRNA to enhance renal function in hypertensive diabetic mice with nephropathy. This study may act as a reference for further investigating whether combining HJXJ with other drugs can enhance its therapeutic effect. The findings of this study might provide new insights into the clinical treatment of hypertensive diabetic nephropathy with HJXJ.

The effect of thyme and tea tree oils on morphology and metabolism of Candida albicans.

Members of Candida species cause significant problems in medicine and in many industrial branches also. In order to prevent from Candida sp. development, essential oils are more and more frequently applied as natural, non-toxic, non-pollutive and biodegradable agents with a broad spectrum of antimicrobial activity. The aim of the research was to determine changes in morphology and metabolic properties of Candida albicans in the presence of thyme and tea tree oils. Changes of enzymatic activity of isolates were observed in the presence of both tested essential oils, and they were primarily associated with loss or decrease of activity of all enzymes detected for control. Furthermore, only for 3 out of 11 isolates additional activity of N-acetyl-ß-glucosaminidase, α-mannosidase, α-fucosidase and trypsin was detected. Vivid changes in biochemical profiles were found after treatment with tea tree oil and they were related to loss of ability to assimilate D-xylose, D-sorbitol and D-trehalose. The main differences in morphology of isolates compared to the control strain concerned formation of pseudohyphae structures. Both examined essential oils caused changes in cell and colony morphology, as well as in the metabolism of Candida albicans. However, the extent of differences depends on the type and concentration of an essential oil. The most important finding is the broad spectrum of changes in yeast enzymatic profiles induced by thyme and tea tree oils. It can be supposed that these changes, together with loss of ability to assimilate saccharides could significantly impact Candida albicans pathogenicity.

Virtual screening and QSAR study of some pyrrolidine derivatives as α-mannosidase inhibitors for binding feature analysis.

Virtual screening and QSAR analysis were carried out to investigate the binding features of (2R, 3R, 4S)-2-aminomethylpyrrolidine 3,4-diol and the functionalized pyrrolidine derivatives to the α-mannosidase I and II enzymes. The QSAR models (possessed considerable R(2), Q(2) values, etc.) suggested that the presence of polar property on the vdW surface (vsurf_W, vsurf_Wp, etc.) of the molecules is important along with the presence of aromatic rings (opr_violation) in the molecules (which also provide hydrophobicity to the molecules). The docking study performed on α-mannosidase I and II enzymes pointed that the main interactions occur by hydrogen bonds, hydrophobic π-π stacking contacts and salt bridges with the cation calcium (for α-mannosidase I) and close interaction with zinc ion (α-mannosidase II), respectively. The bond flexibility orientates the aromatic ring in the molecules toward the hydrophobic cavity for π-π stacking contacts with the aromatic amino acids (Phe528, Phe329 and Phe659 for α-mannosidase I and Trp95, Tyr269, Phe312, Tyr102 for α-mannosidase II). The pharmacophore analysis also supports the results derived from the docking and QSAR studies. Our results suggest that the best compound to inhibit both classes of α-mannosidase is the compound 30, which may be used to design similar and better inhibitors to next generation drugs.

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa.

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P<0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co2+, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn2+, and not activated by Co2+ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.

Human lysosomal alpha-mannosidases exhibit different inhibition and metal binding properties.

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal alpha-mannosidase (hLM, MAN2B1) and the human core alpha-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II alpha-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi alpha-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal alpha-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

Effect of Bu-Zhong-Yi-Qi-Tang on deficiency of N-glycan/nitric oxide and islet damage induced by streptozotocin in diabetic rats.

AIM: To investigate the effect of Bu-Zhong-Yi-Qi-Tang (Decoction for Reinforcing Middle Jiao and Replenishing Qi) on deficiency of N-glycan/nitric oxide (NO) and islet damage induced by injecting two medium doses of streptozotocin (STZ). METHODS: Diabetes was induced by intraperitoneal injection of STZ at 55 mg/kg on day 1 and day 8. Islet damage was evaluated using a scoring system. Nitrite, nitrate, alpha-mannosidase and amylase activities were measured by colorimetry. N-glycan patterns of amylase were determined with lectin [ConA, pisum sativum agglutinin (PSA), peanut agglutinin (PNA), and lens culinaris agglutinin (LCA)] affinity precipitation method. RESULTS: Severe islet necrosis and mild islet atrophy were observed in diabetic rats. The number and size of islets, the activities of alpha-mannosidase, amylase and nitrite were decreased, while the binding of PNA and LCA to amylase was increased. All of which were improved after treatment with Bu-Zhong-Yi-Qi-Tang. Islet damage was significantly correlated with nitrite, nitrate, alpha-mannosidase, amylase and the binding of LCA, PNA, and PSA to amylase. CONCLUSION: STZ-induced islet damage is related to N-glycan deficiency in proteins by blocking alpha-mannosidase activity and no deficiency, accumulation of unfolded proteins, and endoplasmic reticulum stress and activation of cellular signals, all of which are improved after treatment with Bu-Zhong-Yi-Qi-Tang.

Effects of vitamin C on fibroblasts from sporadic Alzheimer's disease patients.

Several therapies for Alzheimer's Disease (AD) are currently under investigation. Some studies have reported that concentration of vitamins in biological fluids are lower in AD patients compared to control subjects and clinical evidence has shown the therapeutic potential of vitamin C and E in delaying AD progression. However, the molecular mechanism(s) that are engaged upon their administration in the APP metabolism in vitro or in vivo still need clarifying. Here, we investigate the effects of vitamin C supplementation, at physiological concentration, in skin fibroblasts obtained from SAD and FAD patients. This study shows that SAD patients' fibroblasts exhibited the exclusive appearance of C-terminal fragments, derived from APP processing, without giving rise to the beta-amyloid peptide, other than corresponding decreased levels of lysosomal enzymes, such as beta-hexosaminidase, alpha-mannosidase and cathepsins B, L, and D.

[Changes in glycosidases and cellulase activities, and cell wall composition in strawberry fruits during development and ripening].

Experiments were carried out with two strawberry (Fragaria x nanassa Duch.) cultivars Fengxiang and Hongfeng, with different softening characteristics during growth, ripening and postharvest storage. The fruits were harvested at different stages of growth and ripening, as assessed by size and the coloration of the surface of the fruits. We selected the following stages: small and green (S1), large and green (S2), white (S3), reddish (S4), and fully red (S5). The main results were as follows. Both alpha- and beta-galactosidase activities were changed with ripening of strawberry fruits (Fig.1A, B). Of the strawberry cultivars tested, no correlation was found between glucosidase activity and fruit ripening (Fig.1C, D). Alpha-mannosidase is an enzyme being ionically bound with cell wall and its activities is correlated with the softening of strawberry fruits (Fig.1E, F). No beta-mannosidase has been detected in strawberry. The activities of cellulase increased as the strawberry fruits developed from stage of small and green to stage of overripe (Fig.2A). The activities of PME increased during the development of strawberry fruits (Fig.2B). Endo-PG was not detected in strawberry, and exo-PG was not related to fruit ripening (Fig.2C). Changes in cell wall component contents were clearly related to the changes in the firmness of strawberry fruits. The increase in soluble pectin, together with reduction of ionically bound pectin content, covalently bound pectin content (Fig.3A, B) and cellulose (Fig.3A, B) resulted in softening of strawberry fruits.