Screening for hereditary tyrosinemia and genotype analysis in newborns / 浙江大学学报·医学版

OBJECTIVE@#To analyze the results of screening for hereditary tyrosinemia (HT) in newborns and its clinical features and genotype.@*METHODS@#The HT screening was conducted among 2 188 784 newborns from November 2013 to November 2018. The tyrosine (TYR)/ succinylacetone (SA) levels were detected by tandem mass spectrometry (MS-MS). The clinical characteristics, genetic results and following up data of identified patients were analyzed.@*RESULTS@#The normal ranges (0.5%-95.5%) of TYR and SA were 34.5-280.0 μmol/L and 0.16-2.58 μmol/L, respectively. Three HT cases were confirmed with a detection rate of 1∶729 595. There was 1 case of tyrosinemia type Ⅰ (HTⅠ) (homozygous variations of c.455G>A in gene), 1 case of tyrosinemia type Ⅱ(HTⅡ) (heterozygous variations of c.890G>T and c.408+1G>A in gene), and 1 case of tyrosinemia type Ⅲ (HT Ⅲ) (homozygous variations of c.257T>C in gene). The variations of c.890G>T, c.4081G>A of and c.257T>C of were novel. The positive predictive value of the screening was 3.4%. Case 1 (HTⅠ) with TYR and SA values of 666.9 μmol/L and 3.87 μmol/L respectively, presented cholestasis, mild elevated of liver enzyme and lactic acid, who were although fed with TYR and phenylalanine free milk, but died at 2 months of age. Case 2 (HTⅡ) with higher TYR (625.6 μmol/L) and normal SA at screening, received medical milk treatment; during the 7 months of follow-up the baby showed normal score of Bayley assessment and normal TYR without eye and skin symptoms. Case 3 (HT Ⅲ) with TYR of 1035.3 μmol/L and normal SA at screening; during the 29 months of follow-up the value of TYR fluctuated from 532.1 μmol/L to 1060.3 μmol/L due to irregular medical milk treatment, while the score of Bayley assessment was normal.@*CONCLUSIONS@#HT is rare in the southern Chinese population, and the gene spectrum is scattered. Early treatment with nitisinone is recommended in children with HTⅠ, otherwise the prognosis is poor; the prognosis of children with HTⅡ is good when early treated with special diet; the prognosis of children with HTⅢ needs to be determined with more data.

Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp: ONBA-17 and deduction on its metabolic pathway

A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation.

Synthesis and biological evaluation of 2-(3-butynoicamidophenyl) benzothiazole derivatives as antitumor agents / 药学学报

A series of 2-(3-butynoicamidophenyl)benzothiazole derivatives were synthesized starting from 4-fluoro-3-nitrobenzoic acid. Structures of all the synthesized compounds were confirmed by 1H NMR and HR-MS. Their antitumor activities against human tumor cells lines (HCT116, Mia-PaCa2, U87-MG, A549, NCI-H1975) were evaluated by MTT assay. The results revealed that most of the synthesized compounds showed potent activities against HCT116, Mia-PaCa2, U87-MG tumor cells lines. Particularly, compounds 14c and 14h exhibited better activity with IC50 values of 1 x 10(-8) mol x L(-1) against U87-MG and HCT116 respectively. The structure-activity relationship of compounds was also discussed preliminarily.

Evolución clínica de pacientes chilenos con tirosinemia tipo I tratados con 2-(2-nitro-4-trifluorometilbenzoil)-1,3-ciclohexanediona (NTBC) / Clinical follow up of Chilean patients with tyrosinemia type 1 treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-ciclohexanedione (NTBC)

Background: Tyrosinemia type I is an inborn error of metabolism due to deficiency of fumarilacetoacetase. Acute presentation is with liver failure, hypophosphatemic rickets and peripheral neuropathy. Chronic presentation is with visceromegaly and subclinical rickets. The most severe complications are hepatic cancer and acute neurological crises. Without treatment, tyrosinemia type 1 is fatal. In 1992 treatment for tyrosinemia type 1 with 2-(2-nitro-4-trifluoromethybenzoyl)-1,3-ciclohexanedione (NTBC) was proposed. A clinical response was reported in 90% of patients. In cases that did not respond, a successful liver transplantation was performed, reducing mortality to 5%. Aim: To report the follow up of 12 patients treated with NTBC. Patients and Methods: Review of clinical records of 12 Chilean cases treated with NTBC at the Instituto de Nutrición y Tecnología de los Alimentos (INTA) from January 2004 until June 2010. Results: In all patients, a rapid metabolic control was achieved. Two patients developed hepatocarcinoma. One of these patients died and one was successfully treated with liver transplantation. One patient died after receiving a liver transplantation. Nine patients have at present good liver function, but 2 had peripheral neuropathy due to late diagnosis and discontinuing NTBC treatment. Conclusions: Treatment with NTBC allows metabolic normalization in tyrosinemia type 1, prevents liver cirrhosis and hepatic cancer, improving survival rates and quality of life in the patients. Neonatal screening is essential for the early diagnosis of this treatable disease, that otherwise may be lethal.

Berberine activates volume-sensitive chloride channel in human colorectal carcinoma cells / 生理学报

The present study aimed to clarify the effect of berberine on the chloride channels in human colorectal carcinoma cells (SW480). The whole-cell patch clamp technique was used to detect the Cl(-) current activated by berberine. The physiological and pharmacological characteristics of the current were clarified by changing the osmotic pressure of extracellular perfusate and applying chloride channel blockers. The results showed that, under isotonic conditions, the background current of SW480 cells was weak and stable. A large current was induced by perfusing the cells with the isotonic solution containing berberine (10 nmol/L), current density being (85.8 ± 4.6) pA/pF at +80 mV, (-71.9 ± 3.5) pA/pF at -80 mV, with a latency of (115.6 ± 21.7) s. The chloride current showed weak outward rectification and negligible time- and voltage-dependent inactivation. The reversal potential (-5.5 mV ± 1.2 mV) of the current was close to the calculated equilibrium potential for Cl(-) (ECl = -0.9 mV). Experiments under different osmotic pressures showed that the properties of hypotonicity-activated current recorded in SW480 cells were similar to those of the current induced by berberine, and hypertonic solutions suppressed the berberine-induced current by (98.6 ± 2.3)%. On the other hand, berberine-induced Cl(-) current was significantly inhibited by the chloride channel blockers NPPB (100 µmol/L) and tamoxifen (20 μmol/L), with the inhibition ratios of (83.1 ± 3.6)% and (95.6 ± 1.2)% respectively. These results suggest that berberine can activate the chloride channels that are sensitive to NPPB and tamoxifen, as well as the changes of cell volume in human colorectal carcinoma cells.

Time-course Transcriptional Profiling of Human Amniotic Fluid-derived Stem Cells Using Microarray / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To maintain the homeostasis of stem cells and prevent their ability to initiate tumorigenesis, it is important to identify and modify factors that prevent or accelerate stem cell senescence. We used microarrays to attempt to identify such factors in human amniotic fluid (HAF)-derived stem cells. MATERIALS AND METHODS: To identify gene expression changes over a time course, we compared gene expression profiles of HAF-derived stem cells in different passages (1st, 2nd, 4th, 6th, 8th, and 10th) using a Sentrix Human illumina microarray. RESULTS: Of the 25,804 genes in the microarray chip, 1,970 showed an over 2-fold change relative to the control (the 1st passage)-either upregulated or downregulated. Quantitative real-time PCR validated the microarray data for selected genes: markedly increased genes were CXCL12, cadherin 6 (CDH6), and folate receptor 3 (FOLR3). Downregulated genes included cyclin D2, keratin 8, insulin-like growth factor 2 (IGF2), natriuretic peptide precursor B (NPPB) and cellular retinoic acid binding protein 2 (CRABP2). The expression pattern of the selected genes was consistent with the microarray data except for CXCL12 and IGF2. Interestingly, the expression of NPPB was dramatically downregulated along the time course; it was almost completely shut-down by the 10th passage. In contrast, FOLR3 mRNA expression was dramatically increased. CONCLUSION: Taken together, although a function for NPPB and FOLR3 in stem cell senescence has not been reported, our results strongly suggest that NPPB and/or FOLR3 play a significant role in the regulation of stem cell senescence.

Isolation and preliminary characterization of a o-nitrobenzaldehyde-degrading Alcaligenes sp. ND1 / Isolamento e caracterização preliminar de lcaligenes SP ND1 degradador de o-nitrobenzaldeido

This paper reports the isolation and characterization of a new o-nitrobenzaldehyde (ONBA)-degrading bacterium, Alcaligenes sp. ND1. ND1 degraded almost all ONBA (100 mg L-1) in M9 medium within 36 hours. The key enzyme(s) involved in the initial biodegradation was a constitutively intracellular enzyme(s). This bacterium has great potential utility for bioremediation.

Esse trabalho relata o isolamento e a caracterização de uma nova bactéria degradadora de o-nitrobenzaldeido (ONBA), Alcaligenes sp ND1. A bactéria ND1 decompôs todo o ONBA (100 mg.L-1) do meio M9 em 36 horas. A enzima-chave envolvida na biodegradação inicial foi uma enzima constitutiva intracelular. Esta bactéria apresenta um potencial de aplicação para bioremediação.

Calcium-dependent chloride channels in plasma membrane of oocytes from toad, Bufo bufo gargarizans / 生理学报

In this paper, membrane current properties of the fully-grown oocytes from toad, Bufo bufo gargarizans, were studied by using two-microelectrode voltage clamp technique. Axion of adult female toad was destroyed, and then ovarian lobes containing oocytes in stage I to VI were removed and incubated in Ca(2+)-free ND96 solution with collagenase (1.5 mg/ml) for 1 h. Subsequently, the oocytes were washed in Ca(2+)-free ND96 solution for 10 min to completely remove the follicular layer. For the experiments only the oocytes in stage V and VI were selected and used during 1 to 5 d. The membrane was depolarized from a holding potential of -80 mV to +60 mV in 10 mV step. It was found that a sustained outward current was elicited by depolarization. Potassium channel blockers (tetraethylammonium chloride, TEA, 10 mmol/L and 4-aminopyridine, 4-AP, 10 mmol/L) reduced the outward current to (23.4+/-0.72)% of the maximum. However, further addition of chloride channel blocker (5-nitro-2, 3-phenypropylamino benzoate, NPPB, 30 micromol/L) could almost completely block the outward current to (2.1+/-0.08)% of the maximum. In the presence of TEA and 4-AP, removal of extracellular Ca(2+) or adding verapamil (40 micromol/L), could also reduce the outward current to (2.2+/-0.04) % and (3.1+/-0.15) % of the maximum, respectively. It is concluded that calcium-dependent chloride channels exist in plasma membrane of Bufo bufo gargarizans oocytes, besides potassium channels.

Background chloride currents in fetal human nasopharyngeal epithelial cells / 生理学报

To characterize the background current in fetal human nasopharyngeal epithelial cells and clarify its relationship with volume activated Cl(-) currents (I(Cl,vol)), whole-cell patch clamp and cell imaging techniques were employed. Under isotonic conditions, a background current [(5.9+/-2.1) pA/pF at +80 mV, n=21] was detected. The current presented a weak outward rectification and a negligible time-dependent inactivation. The current-voltage relationship showed that the reversal potential of the background current [(-0.73+/-1.7) mV, n=21] was close to the calculated equilibrium potential for Cl(-)(-0.9 mV). Application of extracellular hypertonic stimulation (440 mOsmol/L) suppressed the current by (59.6+/-7.1)% and the inhibition was reversible after returned to isotonic conditions. Bathing the cells in hypotonic solution (160 mOsmol/L) induced a volume-sensitive Cl(-) current. The Cl(-) channel blockers, tamoxifen (20 micromol/L) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (100 micromol/L), inhibited the background current by (74.0+/-5.2)% (P<0.01, n=5) and (60.9+/-8.9)% (P<0.01, n=6) at +80 mV and increased basal cell volume by (107.7+/-2.9)% (P<0.01, n=25) and (104.4+/-2.4)% (P<0.01, n=19), respectively. The data indicate that Cl(-) current is an important component of the background current in fetal human nasopharyngeal epithelial cells. The background Cl(-) current is involved in volume activated Cl(-) current and basal cell volume regulation.

Inhibitory effects of chloride channel blockers NPPB on proliferation of human glomerular mesangial cells / 药学学报

<p><b>AIM</b>To investigate the effects of NPPB, a chloride channel blocker, on proliferation of mesangial cells.</p><p><b>METHODS</b>Cell proliferation was determined by measuring cell number and 3H-thymidine incorporation. The LDH activity released from these cells was measured as evaluation of cell viability. The phase of cell cycle was detected by flow cytometry.</p><p><b>RESULTS</b>Cell proliferation assays showed that treatment with both NPPB (50 and 25 micromol x L(-1)) and in hypertonic media (100% increased osmolarity with D-mannitol ) significantly reduced the number of human MC and 3H-thymidine incorporation in a dose-dependent manner. But the LDH activity was not significantly altered in the treatment with 50 micromol x L(-1) NPPB. Flow cytometry experiments showed that 50 and 25 micromol x L(-1) NPPB arrested (84.2 +/- 2.4) % and (80.8 +/- 2.9) % of cells at G0/G1 stage, versus (70.5 +/- 1.4) % of control cells. Conclusion NPPB suppresses cell proliferation and produces growth arrest at G0/G1 phase in human MC by a mechanism probably associated with changes in cell volume.</p>

Volume-activated Cl- current in migrated nasopharyngeal carcinoma cells / 生理学报

The transwell chamber migration assay and the patch-clamp technique were used to investigate the volume-activated Cl(-) current (I(Cl.vol)) in migrated nasopharyngeal carcinoma cells (CNE-2Z). 47% hypotonic solution activated a ICl.vol in the migrated CNE-2Z cells. Compared with the control cells (non-migrated), the properties of this current and the sensitivity to Cl(-) channel blockers were changed. The current density in migrated CNE-2Z cells was higher than that in non-migrated cells. The current was almost completely inhibited by extracellular application of adenosine-5'-triphosphate (ATP, 10 mmol/L), 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB, 100 mmol/L) and tamoxifen (30 mmol/L) in all voltage steps applied. The inhibition of NPPB and tamoxifen on the current was stronger in migrated cells than that in non-migrated cells. The permeability sequence of the four anions was Br(-)>Cl(-)> I (-)>Gluconate. The sequence was different from that of the non-migrated cells (I(-)> Br(-)> Cl(-)> Gluconate). The results suggest that volume-activated chloride channels may be involved in the CNE-2Z cell migration.

Activation of chloride current and decrease of cell volume by ATP in nasopharyngeal carcinoma cells / 生理学报

Whole-cell patch clamp and cell volume measurement techniques were used to investigate the ATP-activated chloride current and the ATP effect on cell volume in nasopharyngeal carcinoma cells. Extracellular application of ATP in micromolar concentrations activated a current with the properties of modest outward rectification and negligible time-dependent inactivation in a dose-dependent manner. The current reversed at a potential [(-0.05+/-0.03) mV] close to the Cl- equilibrium potential (-0.9 mV). Substitution of Cl- with gluconate in the extracellular solution decreased the ATP-activated current and shifted the reversal potential positively. NPPB, one of the chloride channel blockers, inhibited the current by (81.03+/-9.36)%. The current was also depressed by the P2Y purinoceptor antagonist, reactive blue 2, by (67.39+/-5.06)%. ATP (50 micromol/L) decreased the cell volume under the isotonic condition. Depletion of extracellular and intracellular Cl- abolished the ATP effect on cell volume. The results suggest that extracellular ATP of micromolar scales can induce a chloride current associated with cell volume regulation by activation of chloride channel through binding to purinoceptor P2Y.

Synthesis of a novel starch derivative and its adsorption property for creatinine / 药学学报

<p><b>AIM</b>To prepare a novel creatinine adsorbent with anti-renal failure activity.</p><p><b>METHODS</b>A novel starch ester was prepared under heterogeneous condition by the reaction of starch (ST) and 3, 5-dinitrobenzoyl chloride (DNBZ-Cl). The products were characterized by means of elemental analysis, FTIR, 13CNMR and UV-Vis spectra. The adsorption properties of DNBZ-ST were examined.</p><p><b>RESULTS</b>The adsorption equilibrium is reached after 4 h, the adsorption capacity increases with the degree of substitution (DS) of adsorbent and creatinine concentration. When creatinine concentration is higher than 300 mg.L-1, concentration showed no apparent effect on adsorption capacity. As the adsorption temperature varied from 19 degrees C to 49 degrees C, adsorption capacity first decreases and then increases, and is lowest at 37 degrees C. The adsorption capacity first increases and then decreases as the pH value increase from 4 to 11 and is highest at pH 8. The highest adsorption capacity reached 25 mg.g-1 at 37 degrees C pH 7 and the creatinine concentration is 100 mg.L-1.</p><p><b>CONCLUSION</b>Starch 3,5-dinitrobenzoate showed a better adsorption property for creatinine, and it is worthy to be studied more deeply.</p>

Oxidative stress in cucumber (Cucumis sativus L) seedlings treated with acifluorfen.

Treatment of diphenyl ether herbicide acifluorfen-Na (AF-Na) to intact cucumber (Cucumis sativus L cv Poinsette) seedlings induced overaccumulation of protoporphyrin IX in light (75 mumole m-2 s-1). The extra-plastidic protoporphyrin IX accumulated during the light exposure disappeared within two hours of transfer of acifluorofen-treated seedlings to darkness. The dark disappearance was due to re-entry of migrated protoporphyrin IX into the plastid and its subsequent conversion to protochlorophyllide. In light, protoporphyrin IX acted as a photosensitizer and caused generation of active oxygen species. The latter caused damage to the cellular membranes by peroxidation of membrane lipids that resulted in production of malondialdehyde. Damage to the plastidic membranes resulted in damage to photosystem I and photosystem II reactions. Dark-incubation of herbicide-sprayed plants before their exposure to light enhanced photodynamic damage due to diffusion of the herbicide to the site of action. Compared to control, in treated samples the cation-induced increases in variable fluorescence/maximum fluorescence ratio and increase in photosystem II activity was lower due to reduced grana stacking in herbicide-treated and light-exposed plants.

Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.

Early results from indirect drug susceptibility test for tubercle bacilli.

The indirect susceptibility test results on L-J medium for tubercle bacilli against streptomycin, isoniazid and rifampicin were read at the end of 2 wk and compared with the results at 4 wk. It was found that drug resistance could be correctly predicted in over 70 per cent of cultures including multi-drug resistant tuberculosis (MDR TB) strains at the end of 2 wk. The susceptibility to para-nitrobenzoic acid (PNB) read at 2 wk was able to distinguish non-tuberculous mycobacteria from Mycobacterium tuberculosis cultures. The early detection of resistance by this procedure requires only minimum inputs, and can benefit the majority of patients harbouring drug resistant tubercle bacilli.