Genome-Wide Analysis, Expression Profile, and Characterization of the Acid Invertase Gene Family in Pepper.

Catalytic decomposition of sucrose by acid invertases (AINVs) under acidic conditions plays an important role in the development of sink organs in plants. To reveal the function of AINVs in the development of pepper fruits, nine AINV genes of pepper were identified. Protein sequencing and phylogenetic analysis revealed that the CaAINV family may be divided into cell wall invertases (CaCWINV1⁻7) and vacuolar invertases (CaVINV1⁻2). CaAINVs contain conserved regions and protein structures typical of the AINVs in other plants. Gene expression profiling indicated that CaCWINV2 and CaVINV1 were highly expressed in reproductive organs but differed in expression pattern. CaCWINV2 was mainly expressed in buds and flowers, while CaVINV1 was expressed in developmental stages, such as the post-breaker stage. Furthermore, invertase activity of CaCWINV2 and CaVINV1 was identified via functional complementation in an invertase-deficient yeast. Optimum pH for CaCWINV2 and CaVINV1 was found to be 4.0 and 4.5, respectively. Gene expression and enzymatic activity of CaCWINV2 and CaVINV1 indicate that these AINV enzymes may be pivotal for sucrose hydrolysis in the reproductive organs of pepper.

Genome-Wide Identification, Expression, and Functional Analysis of the Alkaline/Neutral Invertase Gene Family in Pepper.

Alkaline/neutral invertase (NINV) proteins irreversibly cleave sucrose into fructose and glucose, and play important roles in carbohydrate metabolism and plant development. To investigate the role of NINVs in the development of pepper fruits, seven NINV genes (CaNINV1-7) were identified. Phylogenetic analysis revealed that the CaNINV family could be divided into α and ß groups. CaNINV1-6 had typical conserved regions and similar protein structures to the NINVs of other plants, while CaNINV7 lacked amino acid sequences at the C-terminus and N-terminus ends. An expression analysis of the CaNINV genes in different tissues demonstrated that CaNINV5 is the dominant NINV in all the examined tissues (root, stem, leaf, bud, flower, and developmental pepper fruits stage). Notably, the expression of CaNINV5 was found to gradually increase at the pre-breaker stages, followed by a decrease at the breaker stages, while it maintained a low level at the post-breaker stages. Furthermore, the invertase activity of CaNINV5 was identified by functional complementation of the invertase-deficient yeast strain SEY2102, and the optimum pH of CaNINV5 was found to be ~7.5. The gene expression and enzymatic activity of CaNINV5 suggest that it might be the main NINV enzyme for hydrolysis of sucrose during pepper fruit development.

Fe2O3 nanoparticles suppress Kv1.3 channels via affecting the redox activity of Kvß2 subunit in Jurkat T cells.

Superparamagnetic iron oxide nanoparticles (SPIONs) are promising nanomaterials in medical practice due to their special magnetic characteristics and nanoscale size. However, their potential impacts on immune cells are not well documented. This study aims to investigate the effects of Fe2O3 nanoparticles (Fe2O3-NPs) on the electrophysiology of Kv1.3 channels in Jurkat T cells. Using the whole-cell patch-clamp technique, we demonstrate that incubation of Jurkat cells with Fe2O3-NPs dose- and time-dependently decreased the current density and shifted the steady-state inactivation curve and the recovery curve of Kv1.3 channels to a rightward direction. Fe2O3-NPs increased the NADP level but decreased the NADPH level of Jurkat cells. Direct induction of NADPH into the cytosole of Jurkat cells via the pipette abolished the rightward shift of the inactivation curve. In addition, transmission electron microscopy showed that Fe2O3-NPs could be endocytosed by Jurkat cells with relatively low speed and capacity. Fe2O3-NPs did not significantly affect the viability of Jurkat cells, but suppressed the expressions of certain cytokines (TNFα, IFNγ and IL-2) and interferon responsive genes (IRF-1 and PIM-1), and the time courses of Fe2O3-NPs endocytosis and effects on the expressions of cytokines and interferon responsive genes were compatible. We conclude that Fe2O3-NPs can be endocytosed by Jurkat cells and act intracellularly. Fe2O3-NPs decrease the current density and delay the inactivation and recovery kinetics of Kv1.3 channels in Jurkat cells by oxidizing NADPH and therefore disrupting the redox activity of the Kvß2 auxiliary subunit, and as a result, lead to changes of the Kv1.3 channel function. These results suggest that iron oxide nanoparticles may affect T cell function by disturbing the activity of Kv1.3 channels. Further, the suppressing effects of Fe2O3-NPs on the expressions of certain inflammatory cytokines and interferon responsive genes suggest that iron oxide nanoparticles may exert modulatory effects on T cell immune activities and anti-inflammation effects.

Expression of serum sCD163 in patients with liver diseases and inflammatory disorders.

OBJECTIVE: To investigate the diagnostic values of soluble cluster of differentiation 163 (sCD163) in patients with liver failure or various inflammations. METHODS: Serum samples were collected from patients admitted to the First Affiliated Hospital, Zhejiang University from October 2013 to January 2015 for treatment of with liver diseases, including liver failure (n=38), hepatitis B virus (HBV)-induced liver cancer (HBsAg positive) (n=40), HBV-induced hepatic cirrhosis (HBsAg positive) (n=40), chronic hepatitis B (n=38), HBV carrier (n=40), fatty liver patients without HBV infection (n=40), chronic glomerulonephritis (n=38), community acquired pneumonia (n=38) and acute pancreatitis (n=38). The CD163/sCD163 was determined using commercial ELISA kits according to the manufacturer's instructions. RESULTS: Significant decrease was noticed in the sCD163 in patients with fatty liver and HBV carrier compared with that of patients with chronic hepatitis B (P < 0.05). Compared with the healthy controls, the level of sCD163 was remarkably increased in the other groups (P < 0.05). The serum sCD163 in patients with HBV-induced liver cancer showed statistical difference compared with those of the patients with fatty liver, HBV carrier, as well as those with liver failure (P < 0.05). The expression of sCD163 was remarkably elevated in patients with liver failure compared with the patients with liver cancer, HBV-induced hepatic cirrhosis, chronic hepatitis B, fatty liver, or HBV carrier (P < 0.05). No significant difference was noticed in the sCD163 in patients with chronic hepatitis B, community acquired pneumonia, chronic glomerulonephritis, and acute pancreatitis (P > 0.05). CONCLUSIONS: sCD163 is a sensitive marker protein for liver failure. The elevation of sCD163 was closely related to the progression of the liver failure. No statistical difference was noticed in the sCD163 in patients with inflammatory disorders, indicating sCD163 showed no organ specificity.

[Subacute toxicity and chemical analysis of Tibetan medicine Oxytropis falcata].

Four different extracts of Oxytropis falcata, including the aerial aqueous extract, and the underground aqueous extract, the aerial lipophilic extract, and the underground lipophilic extract were prepared and then administrated orally to mice at the maximum dose (50 g x kg(-1) x d(-1) calculated by raw material) for fifteen days respectively. Compared with the control group, which was administrated of 1.0% tween-80, the treatment groups did not show significant differences in appearance and behavior. However, the organcoefficient, blood biochemical indicator and pathological section results showed that the lipophilic extracts of the aerial and underground parts of O. flacata showed mild injury to the liver of mice, while the aerial and underground aqueous extracts and the underground lipophilic extract showed mild toxicity to the kidney of male mice. Chemical analysis showed that the lipophilic extracts of the aerial and underground parts, especially aerial lipophilic extract, consisted of large amount of flavonoid aglycones with little amount of polysaccharides and proteins, while the aqueous extracts contained much polysaccharides and proteins with almost no flavonoid aglycones detected.

Protective effects of 5-methoxypsoralen against acetaminophen-induced hepatotoxicity in mice.

AIM: To investigate the hepatic protective effects of 5-methoxypsoralen (5-MOP) and to learn if 5-MOP causes hepatotoxicity at protective doses. METHODS: C57BL/6J mice were administrated orally with 5-MOP at doses of 12.5, 25 and 50 mg/kg body weight respectively every morning for 4 d before given acetaminophen (APAP) subcutaneously at a dose of 500 mg/kg. The 5-MOP alone group was treated with 5-MOP orally at a dose of 50 mg/kg body weight for 4 d without APAP. Twenty-four hours after APAP administration, blood samples of mice were analyzed for serum enzyme alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) levels, and malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) of liver tissues were measured and histopathologic changes of the liver were observed. RESULTS: Compared with the vehicle control group, the serum levels (IU/L) of ALT, AST and LDH were all increased significantly in APAP group (8355 ± 3940 vs 30 ± 21, P < 0.05; 6482 ± 4018 vs 146 ± 58, P < 0.05; 24627 ± 10975 vs 1504 ± 410, P < 0.05). Compared with APAP group, the serum ALT levels (IU/L) (1674 ± 1810 vs 8355 ± 3940, P < 0.05; 54 ± 39 vs 8355 ± 3940, P < 0.05; 19 ± 9 vs 8355 ± 3940, P < 0.05), AST levels (IU/L) (729 ± 685 vs 6482 ± 4108, P < 0.05; 187 ± 149 vs 6482 ± 4108, P < 0.05; 141 ± 12 vs 6482 ± 4108, P < 0.05) and LDH levels (IU/L) (7220 ± 6317 vs 24 627 ± 10 975, P < 0.05; 1618 ± 719 vs 24 627 ± 10 975, P < 0.05; 1394 ± 469 vs 24 627 ± 10 975, P < 0.05) were all decreased drastically in the three-dosage 5-MOP pretreatment groups. Pretreatment of 5-MOP could attenuate histopathologic changes induced by APAP, including hepatocellular necrosis and infiltration of inflammatory cells, and the effect was dose-dependent. MDA levels (nmol/mg) were decreased by 5-MOP in a dose-dependent manner (0.98 ± 0.45 vs 2.15 ± 1.07, P > 0.05; 0.59 ± 0.07 vs 2.15 ± 1.07, P < 0.05; 0.47 ± 0.06 vs 2.15 ± 1.07, P < 0.05). The pretreatment of 5-MOP could also increase the GSH/GSSG ratio (3.834 ± 0.340 vs 3.306 ± 0.282, P > 0.05; 5.330 ± 0.421 vs 3.306 ± 0.282, P < 0.05; 6.180 ± 0.212 vs 3.306 ± 0.282, P < 0.05). In the group treated with 5-MOP but without APAP, the serum enzyme levels, the liver histopathologic manifestation, and the values of MDA and GSH/GSSG ratio were all normal. CONCLUSION: 5-MOP can effectively protect C57BL/6J mice from APAP-induced hepatotoxicity and possesses an antioxidative activity, and does not cause liver injury at the protective doses.

[Interaction of microRNA-338 and its potential targeting protein eiF4E3].

OBJECTIVE: To analyze the interaction between the microRNA-338 and its targeting proteins during the cerebral ischemia and reperfusion injury. METHODS: TargetScan was used to predict the targets of microRNA-338. The potential targeting proteins were then selected according to their secondary structures using RNA structure 4.6 software and their involvement in cerebral ischemia and reperfusion injury was studied. Dual-luciferase reporter assay was used to testify whether microRNA-338 can recognize the 3'UTR of target protein. Western blot was applied to analyze the expression of eiF4E3 in both experimental group and control group. RESULT: EiF4E3 was the most likely potential targeting protein of microRNA-338. The secondary structure of local region of eiF4E3 recognizing microRNA-338 was conservative. The ratio of firefly to renilla luciferase activity in the experimental group was much higher than that of control group. However, there was no significant difference in the expression of eiF4E3 between these two groups. CONCLUSION: MicroRNA-338 can recognize the 3'UTR of eiF4E3 while it has no significant effect on the expression of eiF4E3. The post-target-recognizing regulation for miRNA do exist and this mechanism is possibly related to the tertiary structure of target mRNA.

Structure-activity relationships of the dimeric analogues of endomorphin-2 with different lengths of spacers.

In the present study, seven novel dimeric analogues of endomorphin-2 with longer spacers were designed and synthesized. Through dimerization, their affinity for delta-opioid receptor was mostly increased, especially the delta-opioid receptor preferred dimeric analogue, DEM(12). The results were confirmed by the in vitro bioassay. The structure-activity relationships were also discussed.

[Cloning and expression of human calcyclin binding protein (hCacyBP) gene].

Human calcyclin binding protein (hCacyBP) gene was obtained by the screening of a human cDNA library. The full coding region of CacyBP was cloned into E.coli strain pET28, and then was expressed and purified through affinity chromatography. Rabbit anti-human CacyBP polyclonal antibody was obtained by immunizing rabbit with the purified human CacyBP. Western blots showed that it was expressed extensively in many tissues of mouse. The results of immunohistochemistrial staining showed that the location of CacyBP in BT325 cell line before and after differentiation changed from cytoplasm into nucleus and perinucleus cytoplasm.

[Study of Low Dose Irradiation Enhancing the Effect of Chemotherapeutic Agents against Leukemia and Its Mechanism]

For effectively enhancing the anti-leukemia effect of chemotherapeutic agents, and meanwhile decreasing the side effect of these agents, the study has been made to explore the synergistic effect of low dose irradiation (LDI) combined with Ara-C on murine leukemia and its mechanism. Firstly, an optimal scheme of low dose total body irradiation combined with Ara-C was established in L615 leukemia (T lymphocytic leukemia) mouse model. The machanism of the enhancing effect was explored by patho-morphological observation, examination of residual leukemia cells, the expression of GM-CSF on the surface of marrow stromal cells and in the bone marrow cultural supernatants. The results showed that the optimal scheme was 300 cGy irradiation at 4 days after inoculation of leukemic cells followed by Ara-C 30 mg/kg x 3 days in an interval of 1, 2 or 3 days after irradiation. The mean survival time of the L615 leukemia mice in LDI + Ara-C combined treatment groups was longer than that of control groups. The percentage of long-term survival mice (> 30 days) was the highest (58% - 72%), too. 17% of the mice were be cured. The numbers of blood leukocytes and marrow nucleated cells were transiently decreased in combined treatment group, and then recovered rapidly. Slight myelosuppression and marrow sinus dilation and congestion were seen after 300 cGy irradiation. The expression of GM-CSF either on the stromal cells or in marrow cultural supernatant after irradiation increased strikingly (P < 0.05). Therefore, LDI combined with Ara-C possesses synergistic effect. The mechanism is possibly related to three facts: LDI could increase the permeability of bone marrow sinus; LDI could promote marrow stromal cells to produce some cytokines (such as GM-CSF, etc.) which drive leukemia cells into cell cycle to make the cells more sensitive to chemotherapeutic agents; and LDI could augment Ara-C-induced cytotoxicity through the mechanism of apoptosis.