A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization.

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate.

A novel alcohol dehydrogenase from Bartonella apis (BaADH) was heterologous expressed in Escherichia coli. Its biochemical properties were investigated and used to catalyze the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is a chiral intermediate of the cholesterol-lowering drug atorvastatin. The purified recombinant BaADH displayed 182.4 U/mg of the specific activity using ethyl 4-chloroacetoacetate as substrate under the conditions of 50 °C in pH 7.0 Tris-HCl buffer. It was stable in storage buffers of pH 7 to 9 and retains up to 96.7% of the initial activity after 24 h. The K m and V max values of BaADH were 0.11 mM and 190.4 µmol min-1 mg-1, respectively. Synthesis of (S)-CHBE catalyzed by BaADH was performed with a cofactor regeneration system using a glucose dehydrogenase, and a conversion of 94.9% can be achieved after 1 h reaction. Homology modeling and substrate docking revealed that a typical catalytic triad is in contact with local water molecules to form a catalytic system. The results indicated this ADH could contribute to the further enzymatic synthesis of (S)-CHBE.

Targeting STAT1 in Both Cancer and Insulin Resistance Diseases.

With the rapid development of targeted therapy and our understanding of the underlying molecular mechanisms, drug repurposing is becoming trendy in drug development field. Drugs that display promising therapeutic effects towards multiple diseases tend to target important signaling proteins. Insulin resistance and obesity are strongly associated and both are reported to be correlated with cancer, suggesting they may be linked via some critical pathways. Signal transducer and activator of transcription 1 (STAT1) is an important transcription factor involved in the regulation of multiple cellular processes such as proliferation, survival, inflammation, and angiogenesis. The expression and activity of STAT1 is misregulated in both insulin resistance diseases and cancer. In this review, we summarize recent progress on the function and regulation of STAT1 in both cancer and insulin resistance diseases. Drugs and small molecules that can interfere with STAT1 activity or expression are also discussed.

Amniotic stem cell transplantation therapy for type 1 diabetes: a case report.

This case report presents an evaluation of the clinical effects of an allogeneic amniotic cell transplant for the treatment of type 1 diabetes mellitus. A 26-year-old man with type 1 diabetes was treated with stem cells isolated from his neonatal son's amniotic membrane, collected at birth (2 × 10(7) cells). The cells, which expressed high levels of cluster of differentiation (CD) 133 and CD34 as assessed by flow cytometry, were infused into the pancreatic dorsal artery through the left femoral artery. The main study outcome was the change in exogenous insulin requirements, which began to decrease 3 days after transplantation. At 3 months post-transplantation, the patient was insulin independent and remained so for 6.2 months. During a 36-month follow-up, the patient's blood glucose remained under control and insulin treatment was readjusted to a dosage of 8 IU/day. These preliminary data suggest that amniotic membrane stem cell transplantation can improve islet-cell function in response to glucose in vivo, although an alternative explanation (such as a honeymoon period due to reduced glucose toxicity) also has to be considered.

[Vitreal surgery and etiological diagnosis of bacterial endophthalmitis].

OBJECTIVE: To evaluate the clinical effects of vitreal surgery and the importance of etiological diagnosis in the treatment for bacterial endophthalmitis. METHODS: Retrospective series case study. 20 cases (20 eyes) of bacterial endophthalmitis that were treated in Peking Union Hospital were enrolled. 14 eyes were post-traumatic endophthalmitis, and 6 eyes were postoperative endophthalmitis. Twenty cases of aged 3 to 83 years [mean (40.5 ± 23.9) years] were enrolled, including 12 male and 8 female patients. Pre-operative visual acuity: 2 cases were able to count fingers, 6 cases were able to perform hand movement, 11 cases had light perception, light projection were uncertain in all cases, and there was no light perception in 1 case. Hypopyon was seen in 13 eyes. Severe anterior chamber inflammatory reaction was seen in the other 7 eyes. The fundus could not be observed in all 20 eyes. B-Scan ultrasound examination indicated that all 20 eyes displayed moderate to severe vitreous opacity; proliferation and organization were apparent in 12 eyes, and retinal detachment in 2 eyes. Vitrectomy and intravitreal injection of antibiotics were performed in 18 eyes, and only intravitreal injection of antibiotics was administered in the other 2 eyes. At the beginning of operation, vitreous fluids were smeared and Gram stained. To eyes that showed a positive result in Gram staining, 1 mg of Vancomycin was injected into the vitreous cavity or added in the perfusion fluid (balanced salt solution, BSS) in the eyes. To eyes that showed a negative result in Gram staining, 2 mg or 4 mg of Ceftazidime was injected into the vitreous cavity or added in the perfusion fluid (BSS) in the eyes, respectively. Additionally, we examined the vitreous specimens and performed drug susceptibility testing of the bacteria cultured from the specimens. The antibiotics that the bacteria were susceptible to were chosen according to the drug sensitivity tests. The follow-up period is from 1 to 102 months (average 16.6 months). RESULTS: Thirteen eyes presented with a positive Gram staining result, and 3 eyes presented a negative result; the other 4 eyes were not infected. Bacteria were cultured in 15 eyes. The detection rate of pathogen was 75%. The result for 11 eyes was consistent with the smear results. The bacteria detected were Staphylococcus aureus in 3 eyes, Staphylococcus epidermidis in 3 eyes, and Bacillus spp in 2 eyes. Streptococcus pneumoniae, Streptococcus mitis, Plesiomonas, Pseudomonas cepacia, Klebsiella oxytoca, Loffi Acinetobacter and Pseudomonas fluorescens were detected in 1 eye. The remaining 5 eyes did not have bacterial growth. The intraocular infection of all 20 eyes was controlled, and the intraocular inflammation was relieved. The visual acuity was significantly elevated. Postoperative visual acuity achieved were ≥ 0.3 in 4 eyes, 0.1 to 0.2 in 4 eyes, 0.02 to 0.09 in 6 eyes, CF in 2 eyes, HM in 3 eyes and LP in 1 eye. The retinas of 17 eyes were normal, but recurrent retinal detachment occurred in the other 3 eyes, postoperatively. CONCLUSIONS: Vitrectomy combined with antibiotics and intravitreal injection of antibiotics were an effective treatment of bacterial endophthalmitis. We obtained the vitreous fluid smears at the beginning of surgery to quickly and accurately obtain etiological diagnoses by Gram staining. It is crucial to use etiological diagnosis to choose the susceptible antibiotics.

[Intravitreal injection of Ganciclovir in the treatment of acute retinal necrosis].

OBJECTIVE: To evaluate the indication and clinical effects of intravitreal injection of Ganciclovir in the treatment of acute retinal necrosis (ARN). METHODS: Fourteen cases (14 eyes) of ARN which were consistent with the diagnostic criteria of American Uveitis Society were enrolled. Preoperative visual acuity was: light perception, hand movement and counting fingers (CF), each in 1 eye; 0.08 - 0.1 in 4 eyes; 0.2 - 0.4 in 5 eyes 0.5 in 1 eye and 0.8 in 1 eye. Keratic precipitate and aqueous flare were presented in the anterior segment. Peripheral focal and/or patch retinal necrosis, retinal occlusive arteritis and retinal hemorrhage were observed in the fundus. Acyclovir or Ganciclovir was administrated systematically by intravenous injection. The condition of 14 eyes deteriorated underwent intravitreal injection of Ganciclovir but without retinal detachment. After intravitreal injection 2 eyes became worse and underwent vitrectomy for PVR and retinal detachment. The follow-up time varied from 4 to 74 months (mean 25 months). RESULTS: The inflammation of anterior segment and vitreoretinopathy of 14 cases disappeared after intravitreal injection of Ganciclovir. The visual acuity markedly increased in 12 eyes without surgical intervention. Visual acuity achieved 1.0 - 1.5 in 5 eyes, 0.5 - 0.9 in 5 eyes and 0.3 in 2 eyes after intravitreal injection of Ganciclovir. The retina of the 2 eyes undergone vitrectomy was reattached and their visual acuity improved from CF to 0.4 and LP to CF, respectively. CONCLUSIONS: In ARN patients whose conditions could not be controlled by systemic antivirus medicine treatment, early intravitreal injection of Ganciclovir can yield satisfactory therapeutic effects and better visual prognoses if applied before the occurrence of PVR or retinal detachment.

[Myopic and retinopathy].

OBJECTIVE: To investigate the incidence of myopic retinopathy and its risk factors. METHODS: The fundus of 1449 patients (2879 eyes) with myopia were retrospectively examined. The clinical relationship between myopic retinopathy and diopter, age, and sex was analyzed. RESULTS: Myopic retinopathy was detected in 413 eyes (14.35%). Posterior pole retinal lesions were detected in 22 eyes (0.76%). Peripheral retinal lesions were found in 396 eyes (13.75%). According to their diopters, the myopic patients were divided into four groups: low, medium, high and super high myopia The incidence of peripheral retinal lesions was 4.18%, 8.72%, 19.18%, and 37.44% in these four groups, which significantly different (chi2 = 178.594, P<0.001). By age these patients were divided into three groups: I group, age <25; II group, age 25-34; III group, age >34. The incidences of peripheral retinal lesions in these three groups were 8.11%, 15.34%, and 24.59%, which were significantly different (chi2 = 76.090, P<0.001). The incidence of retinal lesion in male and female was 9.32% and 16.07%, respectively, which was significantly different (chi2 = 24.886, P<0.001). Posteriorpole retinal lesions were only detected in the highly or super highly myopic patients, all of them were more than 25 years. The incidence of posteriorpole retinal lesions in the highly and super highly myopia group was 0.86% and 6.67% respectively, which was significantly different (chi2 = 31.898, P<0.001). The incidence of posteriorpole retinal lesions in group II and group III was 0.55% and 3.55% respectively, which was significantly different (chi2 = 22.523, P<0.001). CONCLUSIONS: The prevalence of retinal lesions in myopic patients is higher than that of emmetropia. The incidence of peripheral retinal lesions increases in patients with deeper diopters. Posterior pole retinal lesions usually occur in the myopic patients whose age are more than 25 years and diopter more than - 6.00 D. Careful examination of fundus is essential for early detection and timely treatment.