In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.
Fermentation ; Hepatitis B Antibodies ; biosynthesis ; isolation & purification ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; isolation & purification ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo study the expression of NFkappaB p65 and its target genes in intestinal metaplasia (IM), dysplasia (Dys), gastric cancer (GC) infected with Helicobacter pylori (Hp) and explore the mechanism of infection by cytotoxin-associated antigen A expressing Hp (CagA(+)Hp) in the development of gastric cancer.

METHODSCagA antibody in blood sample of 289 patients was determined by ELISA. Hp was detected by rapid urease test and Warthin starry staining. Expression of NFkappaB p65 and its target genes in IM, Dys and GC was examined by immunohistochemistry.

RESULTSIn IMI approximately II, IMIII, DysI, DysII approximately III and GC, the expression of NFkappaB p65 was significantly higher in patients with CagA(+)Hp infection than those without CagA Hp infection. In IMIII and DysII approximately III, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in patients with CagA Hp infection than those without CagA Hp infection. In gastric cancer infected with CagA(+)Hp, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in intestinal type than in diffuse type.

CONCLUSIONThere are different mechanisms in intestinal type and diffuse type in the development of gastric cancer. The occurrence of intestinal type gastric cancer is associated with CagA(+)Hp infection which by NFkappaB p65 upregulating the expression of c-myc, CyclinD(1),bcl-xl in patients with IMIII, DysII approximately III. It may be an effective method to prevent gastric cancer by inhibiting NFkappaB p65.

Adult ; Aged ; Antigens, Bacterial ; analysis ; Bacterial Proteins ; analysis ; Cyclin D1 ; metabolism ; Female ; Helicobacter Infections ; complications ; metabolism ; microbiology ; Helicobacter pylori ; Humans ; Male ; Middle Aged ; Precancerous Conditions ; metabolism ; microbiology ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Stomach Neoplasms ; metabolism ; microbiology ; pathology ; Transcription Factor RelA ; genetics ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo investigate the anti-damage effect of iminoethyl-lysine on noise-induced cochlea damage in guinea pig.

METHODS40 healthy red eye guinea pigs were randomly divided into 4 groups: Group A, normal control group; Group B, noise group; Group C, noise and drug group; Group D, iminoethyl-lysine group. Guinea pigs of both group B and group C were exposed to 115 dB white noise for 6 hours/day for 6 days. Group C and Group D were treated with iminoethyl-lysine(10 mg/kg) intraperitoneally, while Group B were treated with the same volume of physiological saline. ABR hearing threshold was measured in all animals before and after experiment. The expression of inducible nitric oxide synthase(NOS II) in the cochlea was examined by the method of immunohistochemistry. Also the cochlea of four groups were examined with scanning electronic microscope. ABR hearing threshold, expression of NOS II and structure of cochlea of four groups were compared with each other.

RESULTSThere was no significant difference in ABR hearing threshold among four groups before the experiment(P > 0.05). There was no significant ABR hearing threshold shift in Group A and Group D after the experiment(P > 0.05), and there was significant ABR hearing threshold shift in Group B and Group C(P < 0.05). After the experiment, the ABR hearing threshold of group B(60.23 +/- 11.23) dB, was higher than that of Group C(38.46 +/- 7.24) dB, P < 0.05. The expression of NOS II was negative in the cochlea of Group A and Group D, but was stronger in Group B than that in Group C. The damage of outer hair cells of cochlea of Group B was more severe than that of Group C.

CONCLUSIONSThe expression of NOS II in the guinea pig's cochlea damaged by noise is positive. Iminoethyl-lysine could inhibit the activity of NOS II and has the anti-damage effect on noise-induced cochlea damage. It is indicated that nitric oxide plays an important role in the pathogenesis of noise-induced hearing loss.

Animals ; Auditory Threshold ; Cochlea ; pathology ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Immunohistochemistry ; Lysine ; analogs & derivatives ; pharmacology ; Microscopy, Electron, Scanning ; Nitric Oxide ; physiology ; Nitric Oxide Synthase Type II ; analysis ; Noise ; adverse effects

OBJECTIVE: To study the disability identification for cases with clinical diagnosis of diffuse axonal injury (DAI) due to traffic accidents, and to explore the possible effects of DAI on identification results. METHODS: Five hundred and fifty-six cases of cerebral injury due to traffic accidents were collected, including 467 cases diagnosed with cerebral contusion or laceration and 89 cases diagnosed with DAI. The identification results of different groups with diagnosis of DAI diagnosis, diagnosis of DAI with cerebral contusion (laceration), and diagnosis of cerebral contusion or laceration without DAI were compared and statistically analyzed, based on the results of CT and MRI re-review. RESULTS: The disability identification levels in DAI group (20 cases), DAI group (69 cases) with cerebral contusion (laceration) and DAI group (467 cases) not complicated by cerebral contusion (laceration) were 7.72 +/- 1.09, 7.78 +/- 1.11, and 8.86 +/- 0.66, respectively. The disability levels of the two groups diagnosed with DAI were higher than those of the group without DAI diagnosis (P < 0.05). CONCLUSION Patients with DAI diagnosis might have more severe cerebral injury. In the identification process, one should pay attention to the possible missed diagnosis and misdiagnosis, and meanwhile avoid relying on those evidences provided only by CT and MRI.
Accidents, Traffic ; Brain Injuries/diagnosis* ; Diagnostic Errors ; Diffuse Axonal Injury/etiology* ; Disability Evaluation ; Forensic Pathology ; Humans ; Magnetic Resonance Imaging ; Resin Cements ; Tomography, X-Ray Computed

OBJECTIVEBenign familial infantile convulsions (BFIC) is a recently recognized autosomal dominant inherited disorder. This epileptic syndrome typically begins between 3 and 12 months of age with clusters of partial seizures in most cases and carries a good prognosis. So far, three loci have been linked to chromosome 19q12.1-13.1, chromosome 2q24 and chromosome 16p12-q12. The authors performed linkage analysis on this pedigree.

METHODSA four-generation Chinese family was investigated. The total number of members was 32 in this family and two neurologists in Xiangya Hospital gave systemic physical examinations and interictal neurological examinations to nineteen members of this family. Venous blood samples were taken for genetic analysis. DNA was extracted from peripheral blood leukocytes using phenol-chloroform method. Seventeen microsatellite markers spanning the critical regions on chromosomes 19q12-13.1, 2q24, and 16p12-q12 were genotyped. These markers included D19S49, D19S250, D19S414, D19S416 and D19S245 for the 19q region, D2S2380, D2S399, D2S111, D2S2195, D2S2330 and D2S2345 for the 2q region, D16S401, D16S3131, D16S3093, D16S517, D16S3120 and D16S415 for the 16p-q region. The DNA from each sample was amplified for the 17 markers. After polymerase chain reactions (PCR), PCR products of chromosome 19 with markers D19S49, D19S250, D19S414, D19S416 and D19S245 were subjected to electrophoresis on 8% denatured polyacrylamide gel for at least 2 hours and 20 minutes. Then the length of the PCR products was judged in the Strategene Eagle Eye II automated gel image analyzer. For the markers from chromosome 2 and 16, PCR products were scanned at ABI 377 autosequencer. The data of PCR products were analyzed using the software Genescan v3.1, Genetyper v2.1 (Applied Biosystem, CA. USA) and GenoDB v1.0. After Mendelian checking, the eligible genotyping data were used for linkage analysis. LOD scores were calculated by using MLINK program of LINKAGE v5.1, under an assumption of autosomal dominant inheritance and the estimated penetrance was 0.9. The allele frequencies of each marker were assumed to be equal and the disease-allele frequencies were designated to be 1/10,000. The LOD scores were calculated at combination rate (theta) 0.0, 0.1, 0.2, 0.3, and 0.4.

RESULTSAmong the 17 selected microsatellite markers, which cover the previously reported regions, seven markers' data (D16S3131, D16S517, D16S3120, D16S3093, D2S2380, D19S250 and D19S414) were omitted due to failed genotyping, low genetic heterogeneity, or failure to pass Mendelian checking. Omission of these markers was to ensure the reliability of our raw data. The two-point LOD scores were below zero for all the markers and the maximum LOD scores at theta = 0.0 were less than -2 for markers D19S49, D19S416, D19S245, D16S401, D16S415, D2S399, D2S111, D2S2195, D2S2330 and D2S2345. Thus, the linkage result showed no evidence that the disease locus is linked to any of these selected markers, which excludes the previously reported candidate regions found in other ethnic families.

CONCLUSIONThere is no evidence that this Chinese family was linked to one of the following loci: 19q12.1-13.1, 16p12-q12 and 2q24. The results indicated that BFIC showed genetic heterogeneity and the Chinese BFIC families might be mapped on another new locus.

China ; Epilepsy, Benign Neonatal ; genetics ; Family Health ; Female ; Gene Frequency ; Genetic Heterogeneity ; Genetic Linkage ; Genetic Markers ; Humans ; Infant ; Lod Score ; Male ; Microsatellite Repeats ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo evaluate the clinical outcome of reconstruction of maxillary defects with vascularized iliac crest flap and simultaneous osseointegrated implant embedding.

METHODSDuring September to October 2003, two patients with maxillary defects from tumor resection underwent microsurgical reconstruction. The free iliac osteomuscular flap transferring and simultaneous osseointegrated implant embedding were performed to repair the defects. Three months after the reconstructive surgery, an abutment operation was preformed and denture was applied in both cases.

RESULTSThe flaps survived well. Postoperative follow-up for 8 to 9 months showed that the patients obtained good zygomaxillary appearance, normal occlusion, and satisfactory pronunciation, without oronasal fistula or other serious complications.

CONCLUSIONSThe free iliac crest osteomuscular flap with simultaneous osseointegrated implant embedding is an ideal, effective and cosmetically acceptable method for maxilla reconstruction.

Adult ; Bone Transplantation ; methods ; Female ; Humans ; Ilium ; transplantation ; Male ; Maxilla ; surgery ; Middle Aged ; Transplantation, Homologous ; Treatment Outcome

OBJECTIVETo study the anatomy characters of renal artery and the treatment of multiple arteries in living donor renal grafts.

METHODSRecords of 142 living donors were analyzed in our center. We analyzed the anatomic structure of renal arteries by DSA and CTA pre-transplantation. Thirty-one kidneys with multiple arteries were transplanted after reconstruction. Then clinical effects were compared between multiple-renal-arteries group (n=31) and single-renal-artery group (n=111).

RESULTSThe incidence of multiple renal artery was 30.99%, and there was no difference between both sides (left kidney 22.54%, right kidney 22.13%). If the multiple artery occurred in left or right kidney, the incidence of the multiple artery occurred in the other side was 56.25% and 60.00%, respectively. The diameter of left main renal artery was more magnanimous (P=0.001) and the first branch was more closed to abdominal aorta (P=0.004). Operation time and warm/cool ischemia time were longer in the multiple-renal-arteries group. However, estimated blood loss, delayed graft function, acute rejection and flow rate of arcuate artery were similar in both groups, the same as serum creatinine and serum creatinine clearance rate on day 7, 1 month and 3 month post-operation. It was shown by repeated measures ANOVA that graft with multiple arteries didn't affect the tendency of renal function at early time post-operation.

CONCLUSIONComprehending the character of renal artery and accurate treatment of multiple artery anastomosis are critical for the effect of the living kidney transplantation.

Arteries ; anatomy & histology ; surgery ; Female ; Follow-Up Studies ; Humans ; Kidney ; blood supply ; Kidney Transplantation ; Living Donors ; Male ; Treatment Outcome

OBJECTIVETo investigate the prevalence, age and gender distribution and clinical features of HIV/AIDS oral lesions in patients in Guangxi province, and to provide the epidemiological information for prevention and treatment of these diseases in the certain population.

METHODSA total of 64 HIV/AIDS patients were included in this study. All patients HIV serum-status was confirmed in Guangxi Center of Disease Control (GXCDC). Oral examination was carried out by standardized specialists. HIV/AIDS orofacial lesions were recorded and diagnosed using the EC Clearing House Criteria on Oral Problems related to HIV Infection (1992).

RESULTSAmong the total of 64 HIV/AIDS patients included in this study, there were 53 males and 11 females, with mean age of 36.1 years. Candidiasis was the most common lesion with the pseudomembranous type predominating. High prevalences of xerostomia, 11 oral ulceration and 7 HIV related periodontitis were noted. 6 Herpetic stomatitis and 3 herpes zoster, 2 oral hairy leukoplakia and 1 Kaposi's sarcoma and 1 lymphadentitis also were found.

CONCLUSIONThis study shows a high prevalence of candidiasis, salivary gland disease. Maybe oral ulceration prevalence is not increased, but lesion severity is increased with more severe heperiform or major RAU. It suggested that HIV/AIDS usually shows oral lesion and partly can appear in early phase.

Acquired Immunodeficiency Syndrome ; Adult ; Candidiasis, Oral ; China ; Female ; HIV Infections ; Humans ; Leukoplakia, Hairy ; Male ; Mouth Diseases ; Periodontitis ; Prevalence ; Sarcoma, Kaposi

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.
Adolescent ; Adult ; Aged ; Anesthetics, Dissociative ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Heart Atria ; cytology ; Humans ; Ketamine ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; Young Adult

Objective To investigate the linear relationship and standard curve equation between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,and to apply it in personalized dialysis.Methods The speed of concentrated liquid pump of Fresenius 4008S hemodialysis machine was calibrated,the ratio of the concentration solution to the reverse osmosis water was determined,KCl was added to the concentrated A solution by an equal increment method to detect K+ concentration in the corresponding dialysate,and the K+ concentration standard curve of dialysate was mapped.This study is based on blood K+ concentration of adams-stokes syndrome patients before dialysis,referring to the standard curve,the most suitable dialysate K+ concentration was selected to personalized dialysis,the blood K+ concentration of the patients was measured after dialysis,and ECG monitoring and clinical symptoms observation were carried out.Results There was a linear relationship between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,the curve equation was y =0.384 lx + 0.002 3,R2 =0.999 4.There was no obvious change in the concentration of other electrolyte ions in the dialysate.Referring to the standard curve,the concentration of dialysate K+ could be adjusted accurately.The blood K+ concentration of adams-stokes syndrome patients could be corrected in time after several times of K+ concentration of personalized dialysis,and ECG recovered eventually,and arrhythmia,syncope,chest tightness and other symptoms disappeared.Conclusion There is a linear relationship between the concentration of dialysate K + and the concentration of KCl added in acidic concentrated solution in the Fresenius 4008S hemodialysis machine.Personalized dialysis is performed by the standard curve with obvious clinical application value,and references are provided for precise regulation of dialysate ion concentration.