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Jin Wu

Publications and source records attributed to Jin Wu.

At least 19 recordsLinked to original sources
PubMed2025

The functional study of novel KLHL3 missense mutations associated with pseudohypoaldosteronism type II.

BACKGROUND: Pseudohypoaldosteronism type II (PHA II) is an inherited tubulopathy, clinically defined by three hallmark features, including secondary hypertension, hyperchloremic metabolic acidosis, and persistent hyperkalemia occurring despite maintained glomerular filtration function. Herein, we aim to investigate the association of kelch like family member 3 (KLHL3) gene mutations with PHA II. METHODS: Compound heterozygous KLHL3 mutations were identified through whole-exome sequencing and Sanger validation. AlphaFold-based structural modeling, site-directed mutagenesis of Flag-tagged plasmids, and co-immunoprecipitation (Co-IP)/immunoblotting in vivo were combined to analyze mutant protein interactions and ubiquitination effects. RESULTS: A Chinese patient was identified with two previously unreported KLHL3 variants (c.131G > A [p.R44Q] and c.744 C > G [p.Y248*]), exhibiting a biochemical triad of asymptomatic hyperkalemia, mild metabolic acidosis, and borderline hypertension. Administration of thiazide diuretics effectively normalized the patient’s hyperkalemia and hypertension. A p.R44Q missense mutation predicted as variants of uncertain significance (VOUS) by American College of Medical Genetics and Genomics (ACMG) guidelines, and a p.Y248* nonsense mutation predicted as variants of likely pathogenic. Functional study revealed that the two KLHL3 mutations impair its ubiquitination of with-no-lysine kinase 1 (WNK1) and with-no-lysine kinase 4 (WNK4), and further increase phosphorylation of both SPAK (sterile20/sporulation-specific protein-1 related proline/alanine-rich kinase)/OSR1 (oxidative stress response kinase-1) and Na-Cl-cotransporter (NCC). CONCLUSIONS: Our study characterized two previously unreported KLHL3 mutations, followed by comprehensive in vitro functional analyses to elucidate their pathophysiological contributions at the molecular level.

Humans↗
PubMed2007

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I.

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.

Allosteric Regulation↗
PubMed2006

Structure, function, and mechanism of the phenylacetate pathway hot dog-fold thioesterase PaaI.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

Binding Sites↗
PubMed2006

A novel diagnostic strategy for trisomy 21 using short tandem repeats.

Molecular technique with STRs can rapidly diagnose aneuploidy. In order to improve its fidelity, we developed a novel STR-based strategy for fast diagnosis of trisomy 21 and constructed a multimarker diagnostic system according to it. The system is based on nine STRs, of which two were previously known and seven were newly identified from the genomic sequence of the long arm of chromosome 21. They were confirmed to be highly polymorphic in the Chinese population by PCR amplification and gel electrophoresis. The combination of nine STR markers, when applied to DNA from 102 Chinese individuals with normal karyotype, did not yield any false-positives, and clearly revealed three different alleles in DNA from 15 out of 18 trisomy 21 patients. The results show that our new strategy can provide an alternative molecular technique for the rapid detection of aneuploidy.

Alleles↗
PubMed2006

Simultaneous analysis of prostanoids using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry.

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.

Animals↗
PubMed2006

Sequential evaluation of hepatic functional reserve by 99mTechnetium-galactosyl human serum albumin scintigraphy after proton beam therapy: a report of three cases and a review of the literatures.

The treatment strategy for malignant liver tumors should be appropriately determined because post-treatment quality of life greatly depends on the patients' residual hepatic function. In this report, we present three patients with malignant liver tumors treated by proton beam therapy in whom pre- and post-therapeutic hepatic functional reserves were evaluated sequentially for more than a year by 99mTechnetium-galactosyl human serum albumin (99mTc-GSA) scintigraphy. All three patients exhibited the distinctive time course of 99mTc-GSA uptake efficiency, which suggested a transient decline in the ratio of liver activity to heart and liver activity at 15 minutes (LHL15) 3-6 months after proton beam therapy. This change was not in parallel with that expected from a functioning normal liver tissue volume. In a year after proton beam therapy, LHL15 recovered nearly to the pre-treatment level in all three patients. Our observations may be related to the up-regulation of receptor-mediated 99mTc-GSA uptake during hepatic regeneration after proton beam therapy.

Aged↗
PubMed2006

MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) attenuated simulated ischemia/reperfusion injury in cultured rat hippocampal cells.

The reactive oxygen species and Ca2+ overload play a critical role in ischemia/reperfusion (I/R) injury. MCI-186 has potent effects in the brain as a free radical scavenger in ischemia-reperfusion. Acute glucose-oxygen deprivation and subsequent reoxygenation were used to model ischemia/reperfusion injury in cultured hippocampal cells. MCI-186 reduced malondialdehyde level and raised the SOD activity when applied upon reoxygenation in a dose-dependent manner compared with the untreated group. The peak neuroprotective effects occurred at 100 and 300 microM. Intracellular free calcium concentration ([Ca2+]i) was significantly reduced in the 100 microM MCI-186-treated group compared to the untreated group (32.5+/-4.0 versus 50.2+/-3.6, p<0.01). Treatment with 100 microM MCI-186 significantly inhibited the decrease of mitochondria membrane potential after simulated ischemia/reperfusion (204+/-11.6% compared with the untreated group, p<0.01). Cell apoptotic rate was significantly decreased following MCI-186 treatment from 33.7+/-2.3% (untreated group) to 16.6+/-1.4% (100 microM MCI-186 treated group). There was no significantly protective difference between 100 and 300 microM MCI-186. MCI-186 effectively protects neuron injury after simulated ischemia/reperfusion by inhibiting lipid peroxidation, reducing Ca2+ overload, elevating mitochondria membrane potential, and decreasing apoptosis.

Animals↗
PubMed2006

[Constructing of STR slippage model by optimizing some factors].

OBJECTIVE: To construct STR slippage model and study factors involved in this procedure. METHODS: DNA samples were amplified with the technology of Degenerate oligonucleotide- primed PCR, then their products were taken as later DNA template and their STR genotype were analyzed by optimizing several factors. RESULTS: STR slippage model was constructed. CONCLUSION: Several factors were involved in the produce of STR slippage, such as amount of modulate DNA, concentration of MgCl2, property of DNA polymerase, motif sequence of STR loci, sample, etc.

DNA↗
PubMed2006

Study on the genetic mutations of 17 alpha-hydroxylase/17,20-lyase deficiency in Chinese patients.

OBJECTIVE: To investigate the CYP17A1 gene mutations in Chinese patients with 17 alpha-hydroxylase/17, 20-lyase deficiency. METHODS: Clinical data were retrospectively analyzed. The CYP17A1 gene mutations were detected in 5 cases with 17 alpha-hydroxylase/17, 20-lyase deficiency and their relatives. The genomic DNA of the patients was isolated from whole blood. Seven pairs of primers were used to amplify eight exons and exon-intron boundaries of the CYP17A1 gene. The amplified PCR products were purified by agarose gel and then directly sequenced. In order to confirm the DNA sequences of different alleles, some fragments were inserted into pMD 18-T vector and then sequenced. Sequencing results were compared to the established human CYP17A1 sequence. RESULTS: Briefly, we found 2 kinds of compound mutations, of which were: (1) 6436-6438(TAC-->AA), causing amino acid Y329K, 418X; (2) 6531-6532(GC-->A), causing amino acid L361F, 418X. Among the five cases, four were homozygous for 6436-6438(TAC-->AA), whereas one was compound heterozygous for 6436-6438(TAC-->AA)/6531-6532(GC-->A). The clinical characteristics of 5 cases were all completely combined defects of 17 alpha-hydroxylase/17, 20-lyase, and they all carried two alleles of CYP17A1 gene mutations that all shifted the reading frame and resulted in truncated protein which lack of the activity center site of P450C17, of which corresponding with their clinical feature. CONCLUSION: Nine alleles have the mutation of 6436-6438(TAC-->AA), accounting for 90% of total alleles (9/10). That suggests this kind of mutation may have racial specificity. More study should be done to have better understanding of the function of the truncated P450C17 enzymes.

Adolescent↗
PubMed2006

[Polymorphisms of seven short tandem repeat loci: D1S2142, D1S3733, D2S1774, D3S2459, D21S1409, D21S1437 and D21S2055 of Chinese Han population in Chengdu].

OBJECTIVE: To obtain the data in polymorphism distribution of the seven short tandem repeat (STR) loci: D1S2142, D1S3733, D2S1774, D3S2459, D21S1409, D21S1437 and D21S2055 of Chinese Han population in Chengdu, and evaluate the polymorphism data usefulness to the forensic science. METHODS: PCR, polyacrylamide gel electrophoresis (PAGE) and silver staining techniques were used to analyze the DNA samples from unrelated individuals of Chinese Han ethnic group in Chengdu. RESULTS: Eleven alleles and twenty-three genotypes were observed in D1S2142. Eight alleles and nineteen genotypes were observed in D1S3733. Eight alleles and fifteen genotypes were observed in D2S1774. Seven alleles and nineteen genotypes were observed in D3S2459. Six alleles and twelve genotypes were observed in D21S1409. Nine alleles and twenty-six genotypes were observed in D21S1437. Twenty alleles and seventy-seven genotypes were observed in D21S2055. The genotype distributions of the seven STR loci showed no deviation from the Hardy-Weinberg equilibrium. The parentage testing of 50 cases revealed an autosomal codominant inheritances and no mutations happened to seven STR loci. CONCLUSION: These data indicate that D1S2142, D1S3733, D2S1774, D3S2459, D21S1409, D21S1437 and D21S2055 have good polymorphism, with high probability of exclusion and probability of discrimination power as well as being loci available as the candidate genetic markers to forensic parentage testing and personal identification.

Alleles↗
PubMed2006

[Allele frequencies and species specificity of five short tandem repeat loci of Chinese Han population in Chengdu].

OBJECTIVE: To obtain the data in polymorphism distribution of the five short tandem repeat (STR) loci: D18S979, D11S2014, D18S548, D1S1667 and GATA164F07 of Chinese Han population in Chengdu, and to evaluate their usefulness in the field of species specificity in forensic science. METHODS: PCR, polyacrylamide gel electrophoresis (PAGE) and silver staining techniques were used to analyze the DNA samples from 100 unrelated individuals of Chinese Han ethnic group in Chengdu. Twelve different animals: monkey, pig, dog, bull, goat, chicken, duck, eel, mudfish, rabbit, guinea pig and mouse were selected as controls in this study for evaluating the species specificity of the five STR loci. RESULTS: Six alleles and twelve genotypes were observed in D18S979. Five alleles and eleven genotypes were observed in D11S2014. Five alleles and thirteen genotypes were observed in D18S548. Seven alleles and nineteen genotypes were observed in D1S1667. Six alleles and fourteen genotypes were observed in GATA164F07. The genotype distributions of the five loci were analyzed by some related software and no deviation from the Hardy-Weinberg equilibrium was observed. Evaluated by way of using different animals as controls, monkey had amplification products at the extra-typing field of D18S979, D11S2014 and D1S1667. Bull, dog and eel had amplification product at typing field of D18S979, and pig, duck, mouse and rabbit had weak product. Bull had weak product at the typing field of D18S548. Dog, goat and eel had product at the typing field of D1S1667. Dog had weak product at the typing field of GATA164F07. Mudfish, chicken and guinea pig had no amplification product at the five loci. CONCLUSION: These data indicate that D18S979, D18S548, D1S1667 and GATA164F07 are highly polymorphic and D11S2014, D18S548 and GATA164F07 can play a key role in species identification.

Animals↗