Objective: To investigate the clinical presentation and genetic characteristics of malignant infantile osteopetrosis. Methods: This was a retrospective case study. Thirty-seven children with malignant infantile osteopetrosis admitted into Beijing Children's Hospital from January 2013 to September 2022 were enrolled in this study. According to the gene mutations, the patients were divided into the CLCN7 group and the TCIRG1 group. Clinical characteristics, laboratory tests, and prognosis were compared between two groups. Wilcoxon test or Fisher exact test were used in inter-group comparison. The survival rate was estimated with the Kaplan-Meier method and the Log-Rank test was used to compare the difference in survival between groups. Results: Among the 37 cases, there were 22 males and 15 females. The age of diagnosis was 0.5 (0.2, 1.0) year. There were 13 patients (35%) and 24 patients (65%) with mutations in CLCN7 and TCIRGI gene respectively. Patients in the CLCN7 group had an older age of diagnosis than those in the TCIRGI group (1.2 (0.4, 3.6) vs. 0.4 (0.2, 0.6) years, Z=-2.60, P=0.008). The levels of serum phosphorus (1.7 (1.3, 1.8) vs. 1.1 (0.8, 1.6) mmol/L, Z=-2.59, P=0.010), creatine kinase isoenzyme (CK-MB) (457 (143, 610) vs. 56 (37, 82) U/L, Z=-3.38, P=0.001) and the level of neutrophils (14.0 (9.9, 18.1) vs. 9.2 (6.7, 11.1) ×10⁹/L, Z=-2.07, P=0.039) at diagnosis were higher in the CLCN7 group than that in the TCIRG1 group. However, the level of D-dimer in the CLCN7 group was lower than that in the TCIRGI group (2.7 (1.0, 3.1) vs. 6.3 (2.5, 9.7) μg/L, Z=2.83, P=0.005). After hematopoietic stem cell transplantation, there was no significant difference in 5-year overall survival rate between the two groups (92.3%±7.4% vs. 83.3%±7.6%, χ²=0.56, P=0.456). Conclusions: TCIRGI gene mutations are more common in children with osteopetrosis. Children with TCIRGI gene mutations have younger age, lower levels of phosphorus, CK-MB, and neutrophils and higher level of D-dimer at the onset. After hematopoietic stem cell transplantation, patients with CLCN7 or TCIRGI gene mutations have similar prognosis.
Child ; Male ; Female ; Humans ; Osteopetrosis/therapy* ; Retrospective Studies ; Prognosis ; Genes, Recessive ; Phosphorus ; Chloride Channels/genetics* ; Vacuolar Proton-Translocating ATPases/genetics*

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg²⁺), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.
Sphingobacterium/metabolism* ; Phosphotransferases (Phosphate Group Acceptor)/metabolism* ; Amino Acids ; Adenosine Triphosphate ; Regeneration ; Polyphosphates/metabolism*

OBJECTIVE: To explore the clinical characteristics and genetic basis for a child featuring autosomal recessive cutis laxa (ARCL). METHODS: Clinical data of the patient was collected. Trio-whole exome sequencing (trio-WES) was carried out for the proband, his sister and parents. Candidate variant was verified by Sanger sequencing. RESULTS: The 5 years and 2 month old child, was 109.5 cm tall (40% centile) and 14.2 kg in weight (< 3% centile). Physical examination discovered facial dysmorphisms including downslanting palpebral fissure, hypertelorism, broad nasal bridge, prominent forehead, long philtrum, obvious loose and wrinkled of abdominal and groin skin. He also had previous history of cryptorchidism and umbilical hernia. Trio-WES revealed that the child harbored compound heterozygous variants c.1421_1424delAGTC (p.Val476Thrfs*71) and c.2293+1G>A of the ATP6V0A2 gene, both of which were unreported previously. In addition to our patient, 75 cases of ATP6V0A2 gene-related ARCL have so far been diagnosed, with main features including cutis laxa [100% (75/75)], facial dysmorphism [78.7% (59/75)] and delayed closure/large anterior fontanelle [65.3% (49/75)]. Typical facial features have included downslanting palpebral fissures [57.3% (43/75)], broad nasal bridge [40.0% (30/75)] and long face [34.7% (26/75)]. CONCLUSION Patients presenting with generalized skin wrinkling, facial dysmorphism, delayed closure/large anterior fontanelle, mental retardation, global developmental disabilities and seizures should be considered for ATP6V0A2 gene-related ARCL. Exome sequencing may facilitate the identification of genetic etiology, to confirm the diagnosis.
Child ; Cutis Laxa/genetics* ; Humans ; Infant ; Intellectual Disability ; Male ; Proton-Translocating ATPases/genetics* ; Skin ; Exome Sequencing

OBJECTIVE: To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. METHODS: Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. RESULTS: In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05). CONCLUSION Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.
Animals ; Male ; Mice ; Amino Acids/pharmacology* ; AMP-Activated Protein Kinases/metabolism* ; Cisplatin/pharmacology* ; Epithelial Cells ; Low Density Lipoprotein Receptor-Related Protein-2/metabolism* ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Membrane Proteins/metabolism* ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism* ; RNA, Small Interfering/metabolism* ; Signal Transduction ; Vacuolar Proton-Translocating ATPases/metabolism*

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
Cytidine Monophosphate ; metabolism ; Escherichia coli ; genetics ; Industrial Microbiology ; methods ; Phosphotransferases (Phosphate Group Acceptor) ; metabolism ; Uridine Kinase

The present study was aimed to clarify the signaling molecular mechanism by which fibroblast growth factor 21 (FGF21) regulates leptin gene expression in adipocytes. Differentiated 3T3-F442A adipocytes were used as study object. The mRNA expression level of leptin was detected by fluorescence quantitative RT-PCR. The phosphorylation levels of proteins of signal transduction pathways were detected by Western blot. The results showed that FGF21 significantly down-regulated the mRNA expression level of leptin in adipocytes, and FGF21 receptor inhibitor BGJ-398 could completely block this effect. FGF21 up-regulated the phosphorylation levels of ERK1/2 and AMPK in adipocytes. Either ERK1/2 inhibitor SCH772984 or AMPK inhibitor Compound C could partially block the inhibitory effect of FGF21, and the combined application of these two inhibitors completely blocked the effect of FGF21. Neither PI3K inhibitor LY294002 nor Akt inhibitor AZD5363 affected the inhibitory effect of FGF21 on leptin gene expression. These results suggest that FGF21 may inhibit leptin gene expression by activating ERK1/2 and AMPK signaling pathways in adipocytes.
3T3 Cells ; Adenylate Kinase ; Adipocytes ; metabolism ; Animals ; Down-Regulation ; Fibroblast Growth Factors ; metabolism ; Leptin ; metabolism ; MAP Kinase Signaling System ; Mice ; Phosphorylation ; Signal Transduction

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
Cinnamomum camphora/genetics* ; Cloning, Molecular ; Genes, Plant ; Phosphotransferases (Phosphate Group Acceptor)/genetics* ; Phylogeny ; Sequence Alignment

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.
Adenosine Triphosphate ; metabolism ; Animals ; Caenorhabditis elegans ; radiation effects ; Electromagnetic Radiation ; Energy Metabolism ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

OBJECTIVE: LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by Department of Pathology,Peking University of Basic Medical Sciences. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of vacuolar-ATPase (ATP6V0C). In this study, we explored the effect of LASS2/TMSG1 and its mutants on proliferation, migration and invasion of human prostate cancer cells and its molecular mechanism. METHODS: We constructed four LASS2/TMSG1 mutants and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The stable transfectants were identified by qPCR and Western blot through analyzing the expression of LASS2/TMSG1 and ATP6V0C, the cell biology functions of LASS2/TMSG1 and its four mutants were studied using growth curve,MTT assay, soft agar colony formation assay, wound migration assay, Matrigel invasion study and flow cytometry. Furthermore, immunofluorescence was used to analysis the interaction of LASS2/ TMSG1 mutants and ATP6V0C. RESULTS: LASS2/TMSG1 mRNA and protein in LASS2/TMSG1 group and Mut1-Mut4 groups were higher than that in Vector group; Western blot showed that ATP6V0C protein in LASS2/TMSG1 wild group was lower than that in Vector group, but ATP6V0C protein in LASS2/TMSG1 S248A group was obviously higher than that in Vector group. MTT test and growth curve assay showed growth ability in LASS2/TMSG1 S248A group was increasing compared with other groups from day 5. Soft Agar colony formation experiment showed anchor independent growth ability in LASS2/TMSG1 S248A group was higher than those in the other groups (P<0.05), Cell migrations (from 35.3%±3.2% to 70.3%±3%) in LASS2/TMSG1 S248A group was increasing compared with LASS2/TMSG1 wild group (P<0.01), and more cells passed through Matrigel in LASS2/TMSG1 S248A group compared with LASS2/TMSG1 wild group (from 50±3.2 to 203±6.5, P<0.01), the apoptosis rate in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 7% to 15.1%, P<0.05), and the G0/G1 ratio in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 51.0% to 85.4%). Furthermore, double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, the expression of ATP6V0C in LASS2/TMSG1 S248A group increased significantly compared with the other groups. CONCLUSION LASS2/TMSG1 S248A promotes proliferation, migration and invasion of prostate cancer cells through increasing ATP6V0C expression, suggesting that aa248-250 is an important function site for LASS2/TMSG1 in invasion suppression of prostate cancer cells.
Beijing ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Male ; Membrane Proteins/genetics* ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms/genetics* ; Sphingosine N-Acyltransferase/genetics* ; Transfection ; Tumor Suppressor Proteins/genetics* ; Vacuolar Proton-Translocating ATPases

OBJECTIVETo detect potential mutation of the TCIRG1 gene in a boy with infantile malignant osteopetrosis.

METHODSTarget sequence capture and next-generation sequencing were applied for the proband and his parents to identify the causative mutation, and Sanger sequencing was used to verify the suspected mutation.

RESULTSThe proband manifested at 4 months of age with symptoms including anemia, thrombocytopenia, hepatosplenomegaly, and cephalus quadratus. X-ray revealed generalized increased bone density. A novel compound heterozygous mutation, c.796G to T (p.E266X) and c.1372G to A (p.G458S), were identified in the boy. His father and grandmother also carried the c.796G to T (p.E266X) mutation, and his mother carried the c.1372G to A (p.G458S) mutation. Neither mutation was found in the PubMed and ClinVar databases.

CONCLUSIONThe novel compound heterozygous mutation c.796G to T (p.E266X) and c.1372G to A (p.G458S) probably underlies the disease in the proband. Above results may enrich the mutation spectrum of the TCIRG1 gene and provide new evidence for the molecular basis of infantile malignant osteopetrosis.

Adult ; Asian Continental Ancestry Group ; Base Sequence ; Humans ; Infant ; Infant, Newborn ; Infant, Newborn, Diseases ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Osteopetrosis ; genetics ; Pedigree ; Vacuolar Proton-Translocating ATPases ; genetics