Plasma Membrane-Localized Transporter MdSWEET12 Is Involved in Sucrose Unloading in Apple Fruit.

Sugar content is an important factor determining the flavor in apple fruit. Sugar unloading is a prerequisite step for sugar accumulation. However, little is known about sugar unloading mechanisms in apple. Transcriptomic sequencing of two apple varieties, "Envy" and "Pacific Rose," with significantly different sugar content was performed. MdSWEET12a from the SWEET transporter family was differentially expressed. Further study of the MdSWEET12a showed that this plasma membrane-localized transporter protein-encoding gene was mainly expressed in sieve element-companion cells (SE-CC) in the fruit, which was positively correlated with the sucrose accumulation during the development of "Envy" apple. Consistently manipulating the gene expression through either transient overexpression or silencing significantly increased or decreased the sugar content in apple fruit, respectively. Complementary growth experiments in mutant yeast cells indicated that MdSWEET12a transported sucrose. Heterologous expression of MdSWEET12a in tomato increased the expression of genes related to sugar metabolism and transport, leading to increased sugar content. These findings underpin the involvement of MdSWEET12a in sugar unloading in apple fruit.

HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1­pAFP-TK and targeted killing effect.

Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non­toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1­pAFP­TK, carrying the suicide gene driven by the alpha­fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription­polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1­pAFP­TK plasmid, whereas HSVtk gene expression was not detected in normal HL­7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1­pAFP­TK decreased in a dose­dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1­pAFP­TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase­3 staining demonstrated that activated caspase­3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase­3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

AKAP95 promotes cell cycle progression via interactions with cyclin E and low molecular weight cyclin E.

AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle.

[Relationship between AKAP95, cyclin E1, cyclin D1, and clinicopathological parameters in lung cancer tissue].

OBJECTIVE: To investigate the correlation between expression of A-kinase anchoring protein 95 (AKAP95) and protein expression of cyclin E1 and cyclin D1 in lung cancer tissue. METHODS: Fifty-one cases of lung cancer were included in the study. The protein expression of AKAP95, cyclin E1, and cyclin D1 were measured by immunohistochemistry. RESULTS: The protein expression of cyclin E1 in lung cancer tissues was significantly higher than that in para-cancerous tissues (positive rate: 75.56%vs 20%, P < 0.01); its expression showed no relationship with histopathological type, lymph node metastasis, and cellular differentiation (P > 0.05). The protein expression of cyclin D1 in lung cancer tissues was higher than that in para-cancerous tissues (positive rate: 69.39% vs 14.29%); its expression showed a significant relationship with histopathological type (P < 0.05). The expression of AKAP95 was correlated with the protein expression of cyclin E1 and cyclin D1 in lung cancer tissues (P < 0.01). CONCLUSION: Cyclin E1 and cyclin D1 are highly expressed in lung cancer tissue, suggesting that they play an important role in the development and progression of lung cancer. The protein expression of cyclin E1 has no relationship with cellular differentiation, lymph node metastasis, and histopathological type of lung cancer, and the protein expression of cyclin D1 has a significant relationship with histopathological type. The expression of AKAP95 is correlated with the protein expression of cyclin E1 and cyclin D1 in lung cancer tissue.

[Comparison of the methods of separating high-quality sperm for intrauterine insemination].

OBJECTIVE: To analyze and compare the pregnancy rates of intrauterine insemination (IUI) achieved by 3 optimized methods of separating high-quality sperm. METHODS: The data from 452 infertile couples who underwent 671 IUI cycles in our reproductive medicine center were retrospectively analyzed. The patients were divided into three groups: 5% HSA Earle's swim-up, SpermRinse swim-up and SupraSperm density gradient centrifugation according to different methods for separating high-quality sperm, and the clinical pregnancy rates were compared. RESULTS: In the 5% HSA Earle's swim-up group, 21 pregnancies were achieved in 221 cycles (9.5%) and in the SpermRinse swim-up group, 34 in 215 cycles (15.8%), with a significantly higher rate in the latter than in the former (P < 0.05). In the SupraSperm density gradient centrifugation group, there were 34 pregnancies in 235 cycles (14.5%), with no statistically significant difference from the other two groups (P > 0.05). CONCLUSION: The SpermRinse swim-up method can improve the clinical pregnancy rate and is suitable for various types of sterile patients. SupraSperm density gradient centrifugation, as an effective method available for separating high-quality sperm, is particularly suitable for those with lots of inflammatory cells and dead and abnormal sperm in the semen.