The aim of this study is to investigate the anti-inflammatory effect of the adenosine derivative N6-(3-hydroxylaniline) adenosine (WS070117M1) on cigarette smoke plus LPS (lipopolysaccharide)-induced chronic obstructive pulmonary disease (COPD) in mice and its mechanism. COPD model was established by exposing male BALB/c mice to cigarette smoke and challenged with LPS inhalation. Supernatants of bronchoalveolar lavage fluid (BALF) were harvested and IL-1β, IL-6, IL-8 and TGF-β1 levels were measured by ELISA (enzyme-linked immunesorbent assay). The number of total white blood cells and neutrophils in bronchoalveolar lavage fluid was counted separately. Lung tissue was stained with Mayer 's hematoxylin and eosin for histopathologic examination. pAMPKa protein expression and distribution of lung tissue were analyzed by immunohistochemistry method. In vitro, levels of AMPKα phosphorylation in phorbol-12- myristate-13-acetate (PMA) differentiated THP-1 cells was detected by immunohistochemistry, IL-8 level in supernatants of cigarette smoke condensate stimulating PMA differentiated THP-1 cells was measured by ELISA. The results showed that WS070117M1 treatment significantly activated AMPKa in the lung tissue. It also resulted in down regulation of IL-1β, IL-6, IL-8 and TGF-β1 levels in bronchoalveolar lavage fluid and IL-8 level in cigarette smoke condensate stimulating PMA differentiated THP-1 cells. In addition, WS070117M1 could inhibit the recruitment of total white blood cells and neutrophils. These results suggest that WS070117M1 may alleviate the airway inflammation by activating AMPK in the lung tissue.

Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.
Blotting, Western ; Chromatography, Affinity ; Egg Proteins ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism ; Zona Pellucida Glycoproteins

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

The aim of this study is to investigate the anti-inflammatory effect of the adenosine derivative N6-(3-hydroxylaniline) adenosine (WS070117M1) on cigarette smoke plus LPS (lipopolysaccharide)-induced chronic obstructive pulmonary disease (COPD) in mice and its mechanism. COPD model was established by exposing male BALB/c mice to cigarette smoke and challenged with LPS inhalation. Supernatants of bronchoalveolar lavage fluid (BALF) were harvested and IL-1β, IL-6, IL-8 and TGF-β1 levels were measured by ELISA (enzyme-linked immunesorbent assay). The number of total white blood cells and neutrophils in bronchoalveolar lavage fluid was counted separately. Lung tissue was stained with Mayer 's hematoxylin and eosin for histopathologic examination. pAMPKa protein expression and distribution of lung tissue were analyzed by immunohistochemistry method. In vitro, levels of AMPKα phosphorylation in phorbol-12- myristate-13-acetate (PMA) differentiated THP-1 cells was detected by immunohistochemistry, IL-8 level in supernatants of cigarette smoke condensate stimulating PMA differentiated THP-1 cells was measured by ELISA. The results showed that WS070117M1 treatment significantly activated AMPKa in the lung tissue. It also resulted in down regulation of IL-1β, IL-6, IL-8 and TGF-β1 levels in bronchoalveolar lavage fluid and IL-8 level in cigarette smoke condensate stimulating PMA differentiated THP-1 cells. In addition, WS070117M1 could inhibit the recruitment of total white blood cells and neutrophils. These results suggest that WS070117M1 may alleviate the airway inflammation by activating AMPK in the lung tissue.
AMP-Activated Protein Kinases ; metabolism ; Adenosine ; analogs & derivatives ; Animals ; Bronchoalveolar Lavage Fluid ; Cell Line, Tumor ; Disease Models, Animal ; Humans ; Inflammation ; drug therapy ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Leukocyte Count ; Lipopolysaccharides ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Smoke ; adverse effects ; Tobacco ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo find a convenient and exact method for evaluating acrosome reaction in human spermatozoa.

METHODSThe semen of the normal male was mixed and then divided into 6 groups. Coomassie brilliant blue (CBB) staining, chlortetracycline (CTC) fluorescence staining and acid phosphatase (ACP) detection were used for morphological observation and data analysis of the acrosome status of the human sperm treated with or without progesterone.

RESULTSThere were obvious morphological differences between the acrosome-reaction and acrosome-intact spermatozoa in CBB staining and CTC fluorescence staining, and significant differences were observed between the experimental and control spermatozoa by the three methods (P < 0.05).

CONCLUSIONAll the three methods can be used to assess acrosome reaction in human spermatozoa, but Coomassie brilliant blue (CBB) staining is much more convenient and stable.

Acid Phosphatase ; Acrosome Reaction ; drug effects ; Cells, Cultured ; Chlortetracycline ; Humans ; Male ; Progesterone ; pharmacology ; Rosaniline Dyes ; Spermatozoa ; cytology ; Staining and Labeling ; methods

OBJECTIVETo investigate the pathogenesis and treatment of penile necrosis resulting from microwave diathermy following circumcision.

METHODSWe retrospectively analyzed the clinical data about 9 cases of penile necrosis resulting from postoperative microwave diathermy following circumcision. The 9 males, aged 20 - 39 (mean 26) years, underwent traditional circumcision for redundant prepuce or phimosis in other hospitals, followed by microwave diathermy for 30 - 60 minutes daily, which resulted in penile necrosis. With no response to conservative therapy, the patients were referred to our hospital at 3 -30 days postoperatively. Of the 9 patients, 5 presented with dry gangrene and 4 with moist gangrene. Six of the patients underwent partial penectomy, including 1 that received penis lengthening.3 months later, while the other 3 underwent total penectomy for total penile necrosis followed by penile reconstruction 3 months later, with deep inferior epigastric perforator (DIEP) flaps and by implantation of the 12th costal cartilage in 2 cases and with epigastric groin island flaps and by urethroplasty in the other.

RESULTSThe patients were followed up for 2 - 8 years, and all could urinate smoothly in the standing position. Of the 6 men treated by partial penectomy, 1 received penis lengthening and achieved a penile length of 7 cm and 5 had the remaining penile length of 3 -5 cm, 4 with erectile function and the other 2 capable of sexual intercourse. The 3 men treated by total penectomy achieved nearly normal external appearance of the penis, with a finalized length of (11.7 ± 1.3) cm, a circumference of (11.4 ± 2.1) cm, and a normal feel of the skin. Of the 3 cases of penile reconstruction, 2 achieved sufficient erectile hardness of the penis (grade 3) for sexual intercourse, while the other 1 remained impotent.

CONCLUSIONPost-circumcision microwave diathermy may result in penile necrosis, for the management of which, early debridement is necessitated and penile lengthening or reconstruction can be performed according to the severity of the lesion and needs of the patient.

Adult ; Circumcision, Male ; methods ; Coitus ; Costal Cartilage ; transplantation ; Diathermy ; adverse effects ; methods ; Humans ; Male ; Microwaves ; adverse effects ; Penis ; abnormalities ; surgery ; Phimosis ; surgery ; Postoperative Period ; Reconstructive Surgical Procedures ; methods ; Retrospective Studies ; Young Adult

Objective:To summarize the clinical manifestations, electrophysiological, muscle magnetic resonance imaging (MRI), pathological, and genetic characteristics of 8 patients with Emery-Dreifuss muscular dystrophy (EDMD) to improve the recognition and diagnosis of EDMD.Methods:Eight patients with EDMD confirmed by gene analysis admitted to Hebei Medical University Third Hospital from 2011 to 2022 were enrolled. The detailed clinical symptoms, neurophysiological examination, electrophysiological changes (electromyography and electrocardiography), skeletal muscle MRI characters, skeletal muscle pathological features and gene mutations were analyzed retrospectively.Results:The age of onset ranged from 2.0 to 6.0 (3.6±1.2) years. All patients had insidious onset and progressive development. Muscle weakness was the first symptom for 7 cases that manifested as difficulty in squatting and walking up stairs. Later, spinal ankylosis and joint contracture occurred. One patient had scoliosis as the first symptoms. Abnormal electrocardiogram was found in 4 cases. The electromyography of all patients showed myogenic damage. Muscle biopsy demonstrated dystrophic features in 1 patient, and other myopathic features, including a variation in muscle fiber size, a marked increase in internal nuclei, and, smaller diameter of typeⅠfibers. Next-generation sequencing result showed that 6/8 cases carried 4 LMNA heterozygous mutations (c.1583C>G, c.1357C>T, c.148C>T, c.1336A>G); 1/8 case carried EMD hemizygous mutation (c.501C>G); 1/8 carried SYNE1 heterozygous mutation (c.4364G>A). Conclusions:EDMD has highly clinical and genetical heterogeneity. The onset age is usually in childhood. The first symptom is characterized by weakness of lower limbs and abnormal walking posture. Electromyography shows myogenic lesion. Skeletal muscle MRI shows selective fat infiltrations. Muscle biopsy pathology lacks characteristic pathological findings. It is difficult to make diagnosis and differential diagnosis by clinical manifestations and auxiliary examination in the early stage of the disease. The second generation sequencing technology can improve the early diagnosis rate of EDMD.

OBJECTIVETo reconfirm the association of KPNB3 with schizophrenia in Chinese population.

METHODSTwo single nucleotide polymorphisms (SNPs), rs2588014 and rs626716 at the KPNB3 locus, were genotyped in 304 Chinese Han family trios consisting of fathers, mothers, and affected offsprings with schizophrenia. These 2 SNPs were detected by PCR-based restriction fragment length polymorphism (RFLP) analysis. The Hardy-Weinberg equilibrium for genotypic distributions was estimated by the goodness-of-fit test. The UNPHASED program was used to perform transmission disequilibrium test (TDT), haplotype analysis, and pair-wise measure of linkage disequilibrium (LD) between these 2 SNPs.

RESULTSThe genotypic distributions of both rs2588014 and rs626716 were in the Hardy-Weinberg equilibrium (P > 0.05). The TDT revealed allelic association with rs626716 (chi2 = 9.31, P = 0.0023) but not with rs2588014 (chi2 = 3.44, P = 0.064). The global P-value was 0.0099 for 100 permutations. The haplotype analysis also showed a disease association (chi2 = 25.97, df = 3, P = 0.0000097).

CONCLUSIONThe present study provides further evidence in support of the KPNB3 association with schizophrenia in Chinese population.

Adult ; China ; epidemiology ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide ; Schizophrenia ; epidemiology ; genetics ; beta Karyopherins ; genetics

OBJECTIVETo investigate the effect of mesenchymal stem cell (MSC) transplantation in repairing ovarian injury in mice sensitized with porcine ovarian proteins.

METHODSWild-type female mice with ICR background (6-8 weeks old) were divided randomly into groups A, B and C (n=12). In groups B and C, the mice were treated with the total protein extract from porcine ovary to induce immunological injury of the ovary, while those in group A received no treatment. MSCs-derived from GFP transgenic mice were transplanted into the mice of group C, and equal volume of PBS was injected intraperitoneally in mice of the other two groups. PCR was used to detect GFP gene in the genomic DNA of the ovaries to assess MSCs homing in the ovary, and the reparative effect of MSCs on ovarian injury was evaluated using HE staining and TUNEL analysis.

RESULTSAfter transplantation, the MSCs could reach the injured ovaries to promote the repair of the ovarian injury, resulting also in reduced apoptosis of the granulosa cells (GCs) in the injured ovaries.

CONCLUSIONMSCs transplantation can promote the recovery of the immunological injury of the ovary in mice, the mechanism of which may involve reduced apoptosis of the GCs.

Animals ; Apoptosis ; Bone Marrow Cells ; Female ; Granulosa Cells ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred ICR ; Ovarian Diseases ; pathology ; surgery ; Ovary ; cytology ; pathology

AIM:To describe the outcomes of corneal stromal lenticules in repairing of corneal ulcer and/or perforation.METHODS:This was a retrospective chart review of 6 eyes of 6 patients from January to June 2017,who underwent corneal ulcer repair with the corneal,stromal lenticules harvested from femtosecond laser refractive surgery and kept in pure glycerin for use.Three cases of infectious corneal ulcers were bacterial,fungal,and infection with foreign bodies in corneal deep layer,one each.The other 3 were corneal ulcer perforation.Making sure no air bubble between donor graft and Descemet membrane.The mean follow-up time was 3.71 ±1.56mo (range 1-6mo).RESULTS:All eyes were successfully treated under control of infection without intra-operative complications,and early postoperative evaluation showed a clear graft in all cases.The last follow-up visit showed the mean best corrected visual acuity (VA) significantly improved after surgery.There was significant difference from 0.48±0.12 to 1.50±0.08 (P＜0.01).CONCLUSION:The preliminary results suggest that the use of corneal stromal lenticules may be a safe and effective surgical alternative for corneal ulcer,even though the long-term outcome of the graft needs to be further observed.