Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆. The subcellular localization of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ was determined by transient expression in Nicotiana benthamiana. The function of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT₆₂₇₈₈ was also able to transport mannose. The expression of IbSUT₆₂₇₈₈ was higher in leaves, lateral branches and main stems, and the expression of IbSUT₈₁₆₁₆ was higher in lateral branches, stems and storage roots. After IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT₆₂₇₈₈ increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT₈₁₆₁₆ increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.
Ipomoea batatas/metabolism* ; Arabidopsis/metabolism* ; Sucrose/metabolism* ; Saccharomyces cerevisiae/metabolism* ; DNA, Complementary ; Phylogeny ; Plants, Genetically Modified/genetics* ; Membrane Transport Proteins/metabolism* ; Starch/metabolism* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

OBJECTIVE: To explore the clinical features and genetic variant in a child with Cerebral creatine deficiency syndrome (CCDS). METHODS: A child who had presented at the Affiliated Children's Hospital of Fudan University on March 5, 2021 was selected as the study subject. Whole exome sequencing (WES) was carried out for the child, and candidate variant was verified by Sanger sequencing. The level of creatine in the brain was determined by magnetic resonance spectroscopy. RESULTS: The patient, a 1-year-and-10-month male, had presented with developmental delay and epilepsy. Both his mother and grandmother had a history of convulsions. MRS showed reduced cerebral creatine in bilateral basal ganglia and thalamus. The child was found to harbor a hemizygous splicing variant of the SLC6A8 gene, namely c.1767+1_1767+2insA, which may lead to protein truncation. The variant was not found in the public databases. Both his mother and grandmother were heterozygous carriers for the same variant. CONCLUSION The hemizygous c.1767+1_1767+2insA variant of the SLC6A8 gene probably underlay the CCDS in this child. Discovery of the novel variant has also expanded the mutational spectrum of the SLC6A8 gene.
Humans ; Male ; Amino Acid Metabolism, Inborn Errors ; Brain ; Creatine/genetics* ; Heterozygote ; Mothers ; Nerve Tissue Proteins ; Plasma Membrane Neurotransmitter Transport Proteins/genetics* ; Infant

Heme/metabolism* ; Cryoelectron Microscopy ; Membrane Transport Proteins ; Escherichia coli Proteins/metabolism*

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.
3,4-Dihydroxyphenylacetic Acid/pharmacology* ; Animals ; Antidepressive Agents/therapeutic use* ; Chromatography, Liquid ; Depression/drug therapy* ; Disease Models, Animal ; Dopamine ; Eosine Yellowish-(YS)/pharmacology* ; Hematoxylin/pharmacology* ; Hippocampus/metabolism* ; Homovanillic Acid/pharmacology* ; Hydroxyindoleacetic Acid/metabolism* ; Methoxyhydroxyphenylglycol/pharmacology* ; Monoamine Oxidase/metabolism* ; Neurotransmitter Agents/metabolism* ; Norepinephrine/pharmacology* ; Plant Extracts ; Rats ; Rehmannia/chemistry* ; Serotonin/metabolism* ; Serotonin Plasma Membrane Transport Proteins/pharmacology* ; Stress, Psychological/metabolism* ; Tandem Mass Spectrometry ; Tryptophan Hydroxylase/metabolism*

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

NRT1 family proteins play an important roles for absorbing and transporting of nitrate in different plants. In order to identify the NRT1 family genes of Rehmannia glutinosa, this study used 11 NRT1 homologous proteins of Arabidopsis as probe sequences and aligned with the transcriptome data of R. glutinosa by using NCBI BLASTN software. Resulting there were 18 NRT1 proteins were identified in R. glutinosa. On basis of this, a series of the molecular characteristics of R. glutinosa NRT1 proteins including the conserved domains, the transmembrane structure, the subcellular location and phylogenetic features were in detail analyzed. At same time, it were systematically analyzed that the temporal and spatial expression patterns and characteristics of R. glutinosa NRT1 family genes in response to different stress factors. The results indicated that 18 R. glutinosa NRT1 family genes with the length of coding region from 1 260 bp to 1 806 bp, encoded proteins ranging from 419 to 601 amino acids, and all of they owned the domains of typical peptide transporter with 7 to 12 transmembrane domains. These R. glutinosa NRT1 family proteins mostly were found to locate on cellular plasma membrane, and belonged to the hydrophobic proteins. Furthermore, the evolutionary analysis found that the 18 R. glutinosa NRT1 protein family could be divided into two subfamilies, of which 14 NRT1 family genes might occur the positive selection, and 4 genes occur the passivation selection during the evolution process of R. glutinosa. In addition the expression analysis showed that 18 R. glutinosa NRT1 family genes have the distinct expression patterns in different tissues of R. glutinosa, and their expression levels were also obvious difference in response to various stress. These findings infield that 18 R. glutinosa NRT1 family proteins might have obviously different functional roles in nitrate transport of R. glutinosa. In conclusion, this study lays a solid theoretical foundation for clarifying the absorption and transport molecular mechanism of N element during R. glutinosa growth and development, and at same time for deeply studying the molecular function of R. glutinosa NRT1 proteins in absorption and transport of nitrate.
Anion Transport Proteins ; Membrane Transport Proteins ; Nitrates ; Phylogeny ; Plant Proteins/metabolism* ; Rehmannia/genetics* ; Transcriptome

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

Proteins are the physical basis of life and perform all kinds of life activities. Proteins have different orientations and function in different tissues. The same protein, located in different subcellular regions, can perform different and even opposite functions. Both functional and structural proteins are capable of undergoing re-localization which can directly or indirectly participate in signal transduction. Due to abnormal transduction of signals during carcinogenesis, the proteins originally expressed in the cytoplasm are translocated into the nucleus and lead to functional changes in the tumor tissue. The changes of protein localization are affected by many factors, including the interaction between proteins, expression level of proteins and the cleaved intracellular domain of transmembrane protein.
Carcinogenesis ; pathology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Membrane Proteins ; metabolism ; Protein Domains ; Protein Transport ; physiology ; Signal Transduction

Polyamines (putrescine, spermidine, and spermine) are essential polycations that play important roles in various physiological and pathophysiological processes in mammalian cells. The study was to investigate their role in cardioprotection against ischemia/reperfusion (I/R) injury and the underlying mechanism. Isolated hearts from male Sprague-Dawley rats were Langendorff-perfused and cardiac I/R was achieved by 30 min of global ischemia followed by 120 min of reperfusion. Different concentrations of polyamines (0.1, 1, 10, and 15 μmol/L of putrescine, spermidine, and spermine), cyclosporin A (0.2 μmol/L), or atractyloside (20 μmol/L) were given 10 min before the onset of reperfusion. The hemodynamics were monitored; the lactate dehydrogenase (LDH) levels in the coronary effluent were measured spectrophotometrically; infarct size was determined by the 2,3,5-triphenyltetrazolium chloride staining method; and mitochondrial permeability transition pore (MPTP) opening was determined spectrophotometrically by the Ca-induced swelling of isolated cardiac mitochondria. The results showed that compared to I/R alone, 0.1 and 1 μmol/L polyamines treatment improved heart function, reduced LDH release, decreased infarct size, and these effects were inhibited by atractyloside (MPTP activator). In isolated mitochondria from normal rats, 0.1 and 1 μmol/L polyamines treatment inhibited MPTP opening. However, 10 and 15 μmol/L polyamines treatment had the opposite effects, and these effects were inhibited by cyclosporin A (MPTP inhibitor). Our findings showed that polyamines may have either protective or damaging effects on hearts suffering from I/R by inhibiting or activating MPTP opening.
Animals ; Cyclosporine ; pharmacology ; Male ; Mitochondria, Heart ; physiology ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocardial Reperfusion Injury ; physiopathology ; Polyamines ; metabolism ; Rats ; Rats, Sprague-Dawley