ABSTRACT
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.
Subject(s)
Child , Male , Female , Humans , Osteopetrosis/therapy , Retrospective Studies , Prognosis , Genes, Recessive , Phosphorus , Chloride Channels/genetics , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
INTRODUCCIÓN: Osteopetrosis Infantil Maligna (OIM) es un grave e inusual desorden genético debi do a una actividad osteoclástica anormal. OBJETIVO: Reportar lactante en quien se documentó una Osteopetrosis Infantil Maligna, revisando aspectos diagnósticos y terapéuticos más relevantes. CASO CLÍNICO: Reportamos un lactante de 10 meses de sexo masculino en quien se confirmó OIM tras presentar plaquetopenia y visceromegalias. En su historial destacó ser primer hijo de padres no consanguíneos, y entre sus hallazgos presentó hepatoesplenomegalia, plaquetopenia y anemia graves, compromiso sensorial visual y auditivo e infecciones a repetición. El diagnóstico fue confirmado mediante estudio genético, el cual identificó 2 mutaciones heterocigotas en el gen TCIRG1. Se rea lizó trasplante de precursores hematopoyéticos, sin haber presentado recuperación hematológica, falleciendo por enfermedad veno oclusiva. DISCUSIÓN: La OIM es una enfermedad inusual, grave y de inicio temprano, siendo necesario un elevado índice de sospecha ante hepatoesplenomegalia y falla medular. El diagnóstico temprano y el trasplante de precursores hematopoyéticos son las únicas intervenciones potencialmente curativas de esta entidad letal.
INTRODUCCIÓN: Malignant Infantile Osteopetrosis (MIOP) is a rare and severe genetic disorder due to abnormal osteoclast activity. OBJECTIVE: To report an infant who presented Malignant Infantile Osteopetrosis, reviewing the most relevant diagnostic and therapeutic aspects. CLINICAL CASE: A ten- month-old male infant with diagnosis of MIOP confirmed after presenting thrombocytopenia and visceromegaly. He was the first child of non-consanguineous parents, and among the findings, he presented severe hepatosplenomegaly, thrombocytopenia, and anemia; visual and hearing impairment, and repeated infections. The diagnosis was confirmed by genetic study, which identified two heterozygous mutations in the TCIRG1 gene. Hematopoietic stem cells were transplanted without hematological recovery. The patient died due to occlusive venous disease. DISCUSSION: MIOP is a rare, severe, and early-onset disease, with a high rate of suspicion necessary in the presence of hepatosplenomegaly and bone marrow failure. Early diagnosis and hematopoietic stem cells transplanta tion are the only potentially therapeutic interventions of this lethal entity.
Subject(s)
Humans , Male , Infant , Osteopetrosis/diagnosis , Hematopoietic Stem Cell Transplantation/methods , Vacuolar Proton-Translocating ATPases/genetics , Osteoporosis/physiopathology , Osteoporosis/genetics , Fatal Outcome , MutationABSTRACT
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.
Subject(s)
Humans , Male , Beijing , Cell Line, Tumor , Cell Movement , Cell Proliferation , Membrane Proteins/genetics , Mutation , Neoplasm Invasiveness , Prostatic Neoplasms/genetics , Sphingosine N-Acyltransferase/genetics , Transfection , Tumor Suppressor Proteins/genetics , Vacuolar Proton-Translocating ATPasesABSTRACT
Abstract Background: Autosomal malignant osteopetrosis is a rare condition arising from dysfunction of bone-resorbing osteoclasts, in which diagnosis requires a high suspicion index. Treatment of choice is allogeneic stem cell transplantation. Best outcomes occur if the procedure is carried out before damage to cranial nerves ensues; nonetheless, patients improve their clinical condition. Case report: An 8-month-old infant was referred for hematology consultation for cytopenias, hepatomegaly, and growth failure. Autosomal malignant osteopetrosis was diagnosed on the basis of physical findings, alteration in calcium and phosphorus metabolism, and hyperdensity of bone. DNA was obtained from the patient and parents; compound heterozygosity of the TCIRG1 gene with a previously non-described deletion (c.1809_1818del) was identified. Conclusions: A new pathogenic mutation of TCIRG1 was identified in a Mexican osteopetrotic patient. Hematopoietic stem cell transplantation was offered as the best available treatment but declined by the parents. An early recognition and wider access to this procedure should be implemented.
Resumen Introducción: La osteopetrosis infantil maligna es una condición rara cuyo origen es la deficiente reabsorción ósea por parte de los osteoclastos. Su diagnóstico requiere un alto índice de sospecha. El tratamiento de elección es el trasplante alogénico de células hematopoyéticas. Los mejores desenlaces ocurren si el procedimiento se lleva a cabo antes de que ocurra daño a los nervios craneales. Caso clínico: Paciente masculino de 8 meses de edad fue referido a la consulta de hematología por citopenias, hepatomegalia y falla para crecer. Se diagnosticó osteopetrosis infantil maligna basándose en los hallazgos de la exploración física, la alteración del metabolismo del calcio y el fósforo y la hiperdensidad del hueso. Se obtuvo ADN del paciente y ambos padres; se demostró un heterocigosidad compuesta del gen TCIRG1 con una deleción (c.1809_1818del) no descrita previamente. Conclusiones: Una nueva mutación patogénica de TCIRG1 se identificó en un paciente mexicano con osteopetrosis. Se ofreció trasplante de células progenitoras hematopoyéticas como el mejor tratamiento disponible, pero fue rechazado por los padres. Se necesita un reconocimiento temprano y la implementación del acceso generalizado a este procedimiento.
Subject(s)
Humans , Infant , Male , Osteopetrosis/congenital , Hematopoietic Stem Cell Transplantation , Vacuolar Proton-Translocating ATPases/genetics , Osteopetrosis/diagnosis , Osteopetrosis/genetics , Osteopetrosis/therapy , Treatment Refusal , Sequence Deletion , Mexico , MutationABSTRACT
PURPOSE: This study was conducted to investigate whether a proton pump inhibitor (PPI) could enhance chemosensitivity via the inhibition of vacuolar-type H⁺ ATPase (V-ATPase) in cervical cancer. MATERIALS AND METHODS: The expression of V-ATPase was evaluated in 351 formalin-fixed, paraffin-embedded human cervical cancer tissues using immunohistochemistry and compared with clinicopathologic risk factors for disease prognosis. The influence of cell proliferation and apoptosis following V-ATPase siRNA transfection or esomeprazole pretreatment was assessed in cervical cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and enzyme-linked immunosorbent assay, respectively. RESULTS: Immunohistochemical analysis revealed that V-ATPase was expressed in about 60% of cervical cancer tissue samples (211/351), and the expression was predominantly found in adenocarcinoma histology (p=0.016). Among patients with initially bulky cervical cancer (n=89), those with V-ATPase expression had shorter disease-free survival (p=0.005) and overall survival (p=0.023). Co-treatment with V-ATPase siRNA or esomeprazole with paclitaxel significantly decreased the cell proliferation of cervical cancer cell lines, including HeLa and INT407, compared to cell lines treated with paclitaxel alone (p < 0.01). Moreover, V-ATPase siRNA or esomeprazole followed by paclitaxel significantly increased the expression of active caspase-3 in these cells compared to cells treated with paclitaxel alone (both, p < 0.05). CONCLUSION: V-ATPase was predominantly expressed in cervical adenocarcinoma, and the expression of V-ATPases was associated with poor prognosis. The inhibition of V-ATPase via siRNA or PPI (esomeprazole) might enhance the chemosensitivity of paclitaxel in cervical cancer cells.
Subject(s)
Humans , Adenocarcinoma , Adenosine Triphosphatases , Antineoplastic Agents , Apoptosis , Caspase 3 , Cell Line , Cell Proliferation , Disease-Free Survival , Enzyme-Linked Immunosorbent Assay , Esomeprazole , Immunohistochemistry , Paclitaxel , Prognosis , Proton Pump Inhibitors , Proton Pumps , Protons , Risk Factors , RNA, Small Interfering , Transfection , Uterine Cervical Neoplasms , Vacuolar Proton-Translocating ATPasesABSTRACT
<p><b>OBJECTIVE</b>To detect potential mutation of the TCIRG1 gene in a boy with infantile malignant osteopetrosis.</p><p><b>METHODS</b>Target 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>
Subject(s)
Adult , Humans , Infant , Infant, Newborn , Male , Middle Aged , Asian People , Base Sequence , Infant, Newborn, Diseases , Genetics , Molecular Sequence Data , Mutation , Osteopetrosis , Genetics , Pedigree , Vacuolar Proton-Translocating ATPases , GeneticsABSTRACT
Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.
Subject(s)
Animals , Bombyx , Cloning, Molecular , Escherichia coli , Metabolism , Insect Proteins , Genetics , Metabolism , Larva , Recombinant Proteins , Genetics , Metabolism , Silk , Vacuolar Proton-Translocating ATPases , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase (a2NTD) and macrophage colony-stimulating factor (M-CSF) on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells.</p><p><b>METHODS</b>Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups: the control group (RPMI 1640), the experimental group I (M-CSF 100 μg/L), the experimental group II (a2NTD 500 μg/L) and the experimental group III (a2NTD 500 μg/L plus M-CSF 100 μg/L). After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901.</p><p><b>RESULTS</b>The expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups (+). The mean absorbance (A) was 0.092 ± 0.005 in control group, 0.095 ± 0.006 in group I, 0.094 ± 0.005 in group II, 0.094 ± 0.005 in group III, and no significant differences were observed among 4 groups (all P>0.05). Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group (-) with A 0.025 ± 0.004; it was normal in groupI and group II (+) with A 0.191 ± 0.012 in group I and 0.197 ± 0.136 in group II (P=0.212), and it was up-regulated significantly in group III (+++) with A 0.285 ± 0.011. There were significant differences between either two groups except group I and group II (all P<0.01). Secretion of IL-10 in group I and group II [(85.65 ± 13.64) ng/L and (87.77 ± 14.25) ng/L] was significantly higher compared with control group [(71.67 ± 7.56) ng/L, P<0.01]. Secretion of IL-12 in group I and group II [(9.91 ± 1.50) ng/L and (10.15 ± 1.80) ng/L] was significantly lower compared with control group [(16.87 ± 1.10) ng/L, P<0.01]. Secretion of IL-10 in group III [(116.98 ± 14.27) ng/L] was the highest, and secretion of IL-12 [(5.31 ± 0.88) ng/L] was the lowest (all P<0.01). There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group III showed the greatest impact compared with other 3 groups (P<0.01).</p><p><b>CONCLUSIONS</b>a2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.</p>
Subject(s)
Humans , Cell Proliferation , Interleukin-10 , Metabolism , Interleukin-12 , Metabolism , Macrophage Colony-Stimulating Factor , Pharmacology , Macrophages , Cell Biology , Phenotype , Stomach Neoplasms , Pathology , Tumor Cells, Cultured , Vacuolar Proton-Translocating ATPases , PharmacologyABSTRACT
Resumo Objetivo A acidose tubular renal distal (ATRd) é caracterizada por acidose metabólica devido à excreção renal de ácido prejudicada. O objetivo deste artigo é apresentar o diagnóstico genético de quatro crianças com ATRd com uso do sequenciamento total do exoma. Métodos Selecionamos duas famílias não relacionadas, quatro crianças com ATRd e seus pais, para fazer o sequenciamento total do exoma. A audição foi preservada em ambas as crianças da família um, porém em nenhuma criança da família dois, na qual um par de gêmeas teve perda auditiva severa. Fizemos o sequenciamento total do exoma em dois conjuntos de amostras e confirmamos os achados com o método de sequenciamento de Sanger. Resultados Duas mutações foram identificadas nos genes ATP6V0A4 e ATP6V1B1. Na família um, detectamos uma nova mutação no éxon 13 do gene ATP6V0A4 com uma alteração em um nucleotídeo único GAC → TAC (c.1232G>T) que causou substituição de ácido aspártico por tirosina na posição 411. Na família dois, detectamos uma mutação recorrente do homozigoto com inserção de um par de bases (c.1149_1155insC) no éxon 12 do gene ATP6V1B1. Conclusão Nossos resultados confirmam o valor do sequenciamento total do exoma para o estudo de nefropatias genéticas complexas e permitem a identificação de mutações novas e recorrentes. Adicionalmente, demonstramos claramente pela primeira vez a aplicação desse método molecular em doenças tubulares renais.
Abstract Objective Distal renal tubular acidosis (dRTA) is characterized by metabolic acidosis due to impaired renal acid excretion. The aim of this study was to demonstrate the genetic diagnosis of four children with dRTA through use of whole-exome sequencing. Methods Two unrelated families were selected; a total of four children with dRTA and their parents, in order to perform whole-exome sequencing. Hearing was preserved in both children from the first family, but not in the second, wherein a twin pair had severe deafness. Whole-exome sequencing was performed in two pooled samples and findings were confirmed with Sanger sequencing method. Results Two mutations were identified in the ATP6V0A4 and ATP6V1B1 genes. In the first family, a novel mutation in the exon 13 of the ATP6V0A4 gene with a single nucleotide change GAC → TAC (c.1232G>T) was found, which caused a substitution of aspartic acid to tyrosine in position 411. In the second family, a homozygous recurrent mutation with one base-pair insertion (c.1149_1155insC) in exon 12 of the ATP6V1B1 gene was detected. Conclusion These results confirm the value of whole-exome sequencing for the study of rare and complex genetic nephropathies, allowing the identification of novel and recurrent mutations. Furthermore, for the first time the application of this molecular method in renal tubular diseases has been clearly demonstrated.
Subject(s)
Adolescent , Child , Female , Humans , Infant , Male , Acidosis, Renal Tubular/diagnosis , Exons/genetics , Hearing Loss, Sensorineural/diagnosis , Vacuolar Proton-Translocating ATPases/genetics , Acidosis, Renal Tubular/genetics , DNA Mutational Analysis , Hearing Loss, Sensorineural/genetics , Severity of Illness IndexABSTRACT
<p><b>OBJECTIVE</b>To explore the application of preimplantation genetic diagnosis (PGD) for infantile malignant osteopetrosis (IMO).</p><p><b>METHODS</b>For a family affected with IMO, PGD was provided using combined parental mutation detection and haplotype constructions with microsatellite markers spanning the TCIRG1 gene. Prenatal diagnosis was performed on the chorionic villus and amniocentesis samples by direct sequencing.</p><p><b>RESULTS</b>Prenatal diagnosis showed that the fetus by the third pregnancy has carried the parental mutations [c.242delC (p.Pro81Argfs*85) and c.1114C>T (p.Gln372*)], and the pregnancy was terminated. PGD was subsequently performed through mutations detection and haplotype analyses following whole genome amplification (WGA) of each of 13 cells. The results showed that 6 of the 13 embryos were unaffected, 3 were carriers and 4 were affected. Well developed unaffected/carrier embryos were selected and transferred into the uterus. A single pregnancy was confirmed. Subsequently pre- and post-natal diagnoses have confirmed development of a healthy child.</p><p><b>CONCLUSION</b>The study demonstrated the advantage of PGD over prenatal diagnosis when natural pregnancies have repeatedly produced IMO children/fetuses.</p>
Subject(s)
Adult , Female , Humans , Infant , Male , Pregnancy , Base Sequence , Fertilization in Vitro , Fetus , Genetic Carrier Screening , Microsatellite Repeats , Molecular Sequence Data , Osteopetrosis , Diagnosis , Embryology , Genetics , Pedigree , Point Mutation , Preimplantation Diagnosis , Vacuolar Proton-Translocating ATPases , GeneticsABSTRACT
HCO3(-) reabsorption in the renal tubules plays a critically important role in maintaining the global acid-base balance. Loss of HCO3(-) causes metabolic acidosis. Proximal renal tubule is the major site for HCO3(-) reabsorption, accounting for more than 80% of total HCO3(-) reabsorption along the nephron. Over the past more than half centuries, tremendous progresses have been made on understanding the molecular mechanisms underlying the HCO3(-) reabsorption in proximal tubules. The transepithelial movement of HCO3(-) involves the coordinated operation of machineries on both the apical and the basolateral membranes of the epithelial cells. On the apical domain, Na(+)-H(+) exchanger NHE3 and the vacuolar H(+)-ATPase are two major pathways mediating the apical uptake of HCO3(-)-related species. Taken together, NHE3 and H(+)-ATPase are responsible for about 80% of HCO3(-) reabsorption in the proximal tubule. The remaining 20% is likely mediated by pathways yet to be characterized. On the basolateral membrane, NBCe1 represents the only major known pathway mediating the extrusion of HCO3(-) coupled with Na(+) into the interstitial space. In the present article, we provide a historical view about the studies on the mechanisms of HCO3(-) reabsorption since 1940s. Moreover, we summarize the latest progresses emerging over the past decade in the physiological as well as pathological roles of acid-base transporters underlying the HCO3(-) reabsorption in proximal tubules.
Subject(s)
Animals , Humans , Acidosis , Bicarbonates , Metabolism , Kidney Tubules, Proximal , Sodium-Hydrogen Exchangers , Physiology , Vacuolar Proton-Translocating ATPases , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of LASS2/TMSG1 silencing on the growth, invasion and metastasis of prostate carcinoma cells and to investigate the related molecular mechanisms.</p><p><b>METHODS</b>LASS2/TMSG1 expression of human prostate carcinoma cell line with low metastatic potentiality (PC-3M-2B4 cells) was knocked down using DNA vector-based small interfering RNA (shRNA), followed by evaluations of tumor cell invasion and metastasis.</p><p><b>RESULTS</b>A stable PC-3M-2B4 cell line with expression of LASS2/TMSG1-shRNA was successfully established. MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSG1-shRNA.LASS2/TMSG1-shRNA transfected clones demonstrated an increased clonogenicity by soft agar colony formation assay and a significant increase of tumor cell invasion by matrigel invasion study.Flow cytometry showed that after LASS2/TMSG1 gene silencing, the apoptotic rate of PC-3M-2B4 cell significantly decreased (P<0.01) without significant cell cycle change (P>0.05).Eight weeks after implantation into subcutaneous tissues in BAL B/c (nu+) mice, the size and weight of sh-LASS2/TMSG1 xenografts were significantly larger than those of the control group (P<0.05).Nuclear proliferation index of the subcutaneous tumor was also higher in the LASS2/TMSG1 shRNA group than those in the control group. Lymph node metastasis was observed in 5 of 6 mice of LASS2/TMSG1 shRNA group and only 1 of 6 of the control group. V-ATPase activity, activities of secreted MMP-2 and MMP-9 and extracellular hydrogen ion concentration were significantly increased in LASS2/TMSG1-shRNA group compared with the control group (P<0.05).</p><p><b>CONCLUSION</b>Silencing of LASS2/TMSG1 promotes the growth, invasion and metastasis of prostate cancer cells through up-regulation of V-ATPase activity, indicating that LASS2/TMSG1 is a tumor metastasis suppressor gene.</p>
Subject(s)
Animals , Humans , Male , Mice , Apoptosis , Cell Cycle , Cell Line, Tumor , Gene Silencing , Hydrogen-Ion Concentration , Lymphatic Metastasis , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Membrane Proteins , Genetics , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA, Small Interfering , Genetics , Sphingosine N-Acyltransferase , Genetics , Metabolism , Transfection , Tumor Burden , Tumor Suppressor Proteins , Genetics , Metabolism , Up-Regulation , Vacuolar Proton-Translocating ATPases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore effects of Yougui recipe (see text) and salmon calcitonin acetate in preventing osteolysis surrounding artificial prosthesis.</p><p><b>METHODS</b>Thirty-two SD male rats with weighted (250 +/- 20) g, aged 8 weeks, were randomly divided into four groups: blank group, model group, salmon calcitonin acetate group and Yougui recipe (see text) group, and 8 rats in each group. Blank group did not undergo any process, other 24 rats underwent anesthesia by chloral hydrate, their knee joints were exposed through medial patellar side,drilling from fermoral condyle nest to marrow cavity,high density of polythlene particles were injected into hole, titanium nail were put into, bone wax closed the window, then suturing step by step. After the molding, saline were used to gavaged in blank group and model group, Yougui recipe (see text) for Yougui recipe (see text) group, salmon calcitonin maximus injection for calcitonin group. After 10 weeks' mediation, rats were executed, and arterial blood and bilateral femoral organization were collected to biochemical, imaging morphology, tissue pathology and molecular biology detection.</p><p><b>RESULTS</b>The key gene expression of activiting osteoclast were inhibited in Yougui recipe (see text) group and calcitonin group. The level of OPG, Ca, ALP in Yougui recipe group were higher than calcitonin group (P<0.01); the content of RANKL were lower (P<0.01). There were no significance meaning in RANK, Trap5b, P between two groups.</p><p><b>CONCLUSION</b>Both of Yougui recipe (see text) and calcitonin can slow and treat surrounding osteolysis of artificial joint prosthesis, and Yougui recipe (see text) has better effect in promoting bone formation. The effect of Yougui recipe (see text) in promoting bone formation, inhibiting osteoclasts to provide a new method to treating surrounding osteolysis of artificial joint prosthesis.</p>
Subject(s)
Animals , Male , Rats , Biomarkers , Metabolism , Drugs, Chinese Herbal , Pharmacology , Gene Expression Regulation , Interleukin-1 , Genetics , Interleukin-6 , Genetics , Osteolysis , Metabolism , Pathology , Prostheses and Implants , Rats, Sprague-Dawley , Tomography, X-Ray Computed , Vacuolar Proton-Translocating ATPases , GeneticsABSTRACT
Studies on cell signaling pay more attention to spatial dynamics and how such diverse organization can relate to high order of cellular capabilities. To overview the specificity of cell signaling, we integrated human receptome data with proteome spatial expression profiles to systematically investigate the specificity of receptors and receptor-triggered transduction networks across 62 normal cell types and 14 cancer types. Six percent receptors showed cell-type-specific expression, and 4% signaling networks presented enriched cell-specific proteins induced by the receptors. We introduced a concept of "response context" to annotate the cell-type dependent signaling networks. We found that most cells respond similarly to the same stimulus, as the "response contexts" presented high functional similarity. Despite this, the subtle spatial diversity can be observed from the difference in network architectures. The architecture of the signaling networks in nerve cells displayed less completeness than that in glandular cells, which indicated cellular-context dependent signaling patterns are elaborately spatially organized. Likewise, in cancer cells most signaling networks were generally dysfunctional and less complete than that in normal cells. However, glioma emerged hyper-activated transduction mechanism in malignant state. Receptor ATP6AP2 and TNFRSF21 induced rennin-angiotensin and apoptosis signaling were found likely to explain the glioma-specific mechanism. This work represents an effort to decipher context-specific signaling network from spatial dimension. Our results indicated that although a majority of cells engage general signaling response with subtle differences, the spatial dynamics of cell signaling can not only deepen our insights into different signaling mechanisms, but also help understand cell signaling in disease.
Subject(s)
Humans , Cell Line , Databases, Protein , Gene Expression Profiling , Metabolic Networks and Pathways , Neoplasms , Metabolism , Pathology , Proteome , Receptors, Cell Surface , Metabolism , Receptors, Tumor Necrosis Factor , Metabolism , Signal Transduction , Vacuolar Proton-Translocating ATPases , MetabolismABSTRACT
Steroid cell tumor, not otherwise specified (NOS), are rare ovarian tumor, in addition, it is more rare in children. The majority of these tumors produce several steroid hormones, particularly testosterone. Estrogen also secreted by steroid cell tumor, NOS, but it is uncommon. Furthermore, hypertension is an infrequent sign in steroid cell tumor, NOS. An 8.5-yr-old girl with hypertension and frequent vaginal spotting visited at our clinic. On laboratory evaluation, secondary hypertension due to an elevated plasma renin level and isosexual pseudoprecocious puberty was diagnosed. Right solid ovarian mass was detected in radiologic tests. She underwent a right ooporectomy and it revealed renin and progesterone receptor positive steroid cell tumor, NOS. After operation, her blood pressure returned to normal level and vaginal bleeding disappeared. Even though this case is very rare, when hypertension coincides with virilization or feminization, a renin-secreting ovarian steroid cell tumor, NOS, should be considered.
Subject(s)
Child , Female , Humans , Hypertension/etiology , Ovarian Neoplasms/complications , Puberty, Precocious/enzymology , Receptors, Cell Surface/metabolism , Receptors, Progesterone/metabolism , Renin/blood , Sex Cord-Gonadal Stromal Tumors/complications , Steroids/biosynthesis , Tomography, X-Ray Computed , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.</p><p><b>METHODS</b>The localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.</p><p><b>RESULTS</b>H(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.</p><p><b>CONCLUSION</b>This finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.</p>
Subject(s)
Animals , Male , Rats , Cell Membrane , Hepatocytes , Cell Biology , Histocytochemistry , Methods , Kidney , Cell Biology , Lysosomes , Organelles , Rats, Wistar , Vacuolar Proton-Translocating ATPases , MetabolismABSTRACT
Background & objectives: Although clinical reports have described infantile malignant autosomal recessive osteopetrosis (ARO) in Indian patients, no published data are available about the genetic causes of ARO in this population. We investigated the main genetic causes of ARO in eight Indian patients with early postnatal onset and the typical severe clinical course including visual impairment and anaemia. Methods: Mutation screening in the genes CLCN7 and TCIRG1 was done on genomic DNA from 8 affected individuals (diagnosed on the basis of clinical and haematological parameters and characteristic radiological changes of increased bone density) and their parents. In one family, after detection of both mutations in the proband, targeted mutation analysis was also done in chorionic villus samples for prenatal diagnosis. Results: Six patients had mutations in TCIRG1 and two patients harboured mutations in CLCN7 gene. Three of the five different TCIRG1 mutations identified and both CLCN7 mutations were novel mutations. Except for the already known mutation p.Ile720del, all TCIRG1 mutations disrupt conserved splice consensus sequences or lead to premature stop codons. In contrast, both CLCN7 mutations only lead to missense changes of conserved amino acids. In a foetus harbouring TCIRG1 mutations osteopetrosis was visible radiologically at 23 wk of gestation. Interpretation & conclusions: That the CLCN7 mutations provoke a phenotype as severe as the one caused by TCIRG1 loss of function suggests the affected residues to be crucial for the function of the ClC-7 chloride channel or chloride/proton-exchanger. Our data also show that ARO can manifest as early as in the second trimester of pregnancy.
Subject(s)
Chloride Channels/genetics , Female , Genes, Recessive , Humans , India , Infant , Male , Mutation , Osteopetrosis/genetics , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
Homo sapiens longevity assurance homologue 2 (LASS2) is a novel gene isolated from a human liver cDNA library by our laboratory, and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene). According to our previous results, LASS2 could interact with subunit c of vacuolar type H(+)-ATPase (V-ATPase), and the overexpression of LASS2 could inhibit the cell growth of a human hepatocellular carcinoma (HCC) cell line, SMMC-7721. In order to understand the role of the interaction between LASS2 and V-ATPase in HCC cell growth, we transiently transfected plasmid pCMV-HA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The pH-sensitive fluorescence probes, BCECF and BCECF-AM, were used to measure the intracellular and extracellular H(+) concentrations of HCCLM3 cells respectively. The effect of LASS2 gene on apoptosis was evaluated with Annexin-V/FITC and propidium iodide (PI) by flow cytometry. Western blot was used to detect cytochrome c (Cyt c) in the cytosol and mitochondria, as well as pro-caspase-3 in cytosol. The results showed that the cell growth of LASS2-transfected HCCLM3 cells was significantly inhibited compared with that of the mock control. LASS2 transfection increased intracellular H(+) concentration of HCCLM3 cells, while decreased extracellular H(+) concentration. Moreover, LASS2 transfection significantly enhanced the apoptosis of HCCLM3 cells. In LASS2-transfected cells, the amounts of Cyt c increased in the cytosol, while decreased in the mitochondria. Meanwhile, the expression of pro-caspase-3 in the cytosolic extracts was decreased. These results implicate that LASS2 gene might increase intracellular H(+) of HCC cells via the interaction with V-ATPase, thereby inducing cell apoptosis through mitochondrial pathway.
Subject(s)
Humans , Apoptosis , Carcinoma, Hepatocellular , Pathology , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Liver Neoplasms , Pathology , Membrane Proteins , Metabolism , RNA, Small Interfering , Sphingosine N-Acyltransferase , Metabolism , Transfection , Tumor Suppressor Proteins , Metabolism , Vacuolar Proton-Translocating ATPases , MetabolismABSTRACT
TMSG-1 is a newly discovered tumor metastasis suppressor gene, which plays important roles in promoting apoptosis and inhibiting invasion and metastasis of tumor cells. The inhibitory function of TMSG-1 in tumor cells may be related to vacuolar H+-ATPase and ceramide, but the underlying mechanism remains unknown. Studies on TMSG-1 are limited worldwide, and only a research group in Shanghai and our group have recently studied on it. As a new research field, the function of TMSG-1 remains to be explored. This review discusses the discovery of TMSG-1, structure of its encoded protein, its roles and possible mechanism in inhibiting tumor invasion and metastasis.
Subject(s)
Animals , Humans , Apoptosis , Cell Cycle , Ceramides , Pharmacology , Enzyme Activation , Membrane Proteins , Metabolism , Physiology , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasms , Metabolism , Pathology , Phosphorylation , Sphingosine N-Acyltransferase , Metabolism , Physiology , Tumor Suppressor Proteins , Metabolism , Physiology , Vacuolar Proton-Translocating ATPases , MetabolismABSTRACT
Distal renal tubular acidosis (RTA) with sensorineural deafness is a rare entity, inherited in an autosomal recessive manner. It is caused by mutations in the ATP6V1B1 gene, leading to defective function of H+-ATPase pump in the distal nephron, cochlea and endolymphatic sac. We report two siblings with distal RTA and sensorineural deafness having mutation C>T in the first coding exon of the gene, resulting in a non functional protein. The parents were found to be carriers for the mutation.