Numerical simulation and testing of laser-MIG hybrid-welding angle-structure sheets.

Numerical simulation and experimental investigation of laser-MIG hybrid angle-welding low-carbon 1.5-mm-thin SPCC steel sheets are presented in this work. The transient simulation analysis provides an access to the thermal-fluid phenomena prediction by employing a hybrid three-dimensional heat source model. Special attention is paid to the melt dynamic behaviors within the triangular molten pool affected by the Marangoni convection. The simulation results show that the temperature and its gradient distribution are symmetrical with respect to the laser beam, which is validated well by the experimental study. The microstructure of the welded joints was analyzed by scanning electron microscopy and transmission electron microscopy. The results show that the cross-section microstructures of welded joint are mainly composed of the weld zone, narrow heat-affected zone, and substrate. The semielliptic-like molten pool shape is consistent with that of the simulated results. The finer microstructure in the weld bead results from the rapid cooling rate of laser welding confirmed by the FEM calculation. The columnar and equiaxed dendrites are formed in the peripheral and central region of the molten pool, which is beneficial for the improvement of the microhardness.

Enhanced 1,3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect.

AIMS: This study aimed to strengthen the reducing equivalent generation in Klebsiella pneumoniae for improving 1,3-propanediol (PDO) production. METHODS AND RESULTS: Disruption of the arcA gene activated the transcription levels of the TCA cycle genes and thus increased the NADH/NAD+ ratio by 54·2%, leading to the improved PDO titre and yield per cell from 16·1 g l-1 and 4·0 g gDCW-1 to 18·8 g l-1 and 6·4 g gDCW-1 respectively. Further ldhA gene deletion eliminated lactate accumulation and promoted the PDO titre to 19·9 g l-1 . Finally, the glucose effect was weakened by deleting the crr gene to enhance the co-utilization of glucose and glycerol, resulting in the increased PDO production to 23·8 g l-1 with the glycerol conversion rate of 59·5%. The PDO titre in bioreactor was promoted from 61·2 to 78·1 g l-1 . CONCLUSIONS: Deletions of the arcA and the crr genes showed positive effects on the TCA cycle activity and the co-utilization of glucose and glycerol, leading to the strengthened reducing equivalent generation and the improved PDO titre by 47·8% in shaker. The PDO titre in the bioreactor was enhanced to 78·1 g l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided novel information on generating reducing equivalent for the PDO biosynthesis by strengthening the TCA cycle and weakening the glucose effect in K. pneumoniae.

Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds.

Neu differentiation factor (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-2/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal growth factor (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-2/neu, and HER-3 are present in skin, and other EGF family members can influence wound keratinocytes in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha 2 (rhNDF-alpha 2), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm2. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta 2 had no apparent biologic effects in this model. rhNDF-alpha 2 also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha 2-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-2/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-2/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha 2 may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Radioimmunotherapy for hepatocellular carcinoma (HCC) using 131I-anti HCC isoferritin IgG: preliminary results of experimental and clinical studies.

Based on radioimmunoimaging for HCC using 131I-anti HCC isoferritin IgG, the experimental and clinical studies on radioimmunotherapy for HCC were reported. Thirty-six nude mice bearing human HCC were used for the study of labeled IgG, pure 131NaI and pure IgG. In the labeled IgG group, the tumor inhibition rate was significantly higher than that in other groups (81%, 60%, and 18%, respectively, p less than 0.05). The tumor cell DNA analysis showed the tumor cell was inhibited in the S stage of the cell cycle. Twenty pathologically proven unresectable HCC patient were treated by 131I-antihuman HCC isoferritin IgG 20-55mCi monthly for 1-3 times (via hepatic arterial catheter or intravenously). The short-term response was promising, a decline in AFP level and shrinkage of tumor were observed in 80% (12/15) and 65% (13/20) of patients respectively. Sequence resection was successful in five patients (5/20) after radioimmunotherapy. No marked toxic effects were noted in our limited experience, but some problems remain to be discussed.

Autologous bone marrow mixed with HLA-haploidentical allogeneic marrow transplantation for treatment of patients with malignant blood diseases.

We have previously demonstrated that syngeneic marrow mixed with H-2 haploidentical marrow transplantation could provide not only protection against graft-versus-host disease (GVHD) but also anti-leukemic (GVL) effects in mice. In the present studies, we report clinical observations using autologous marrow mixed with HLA-haploidentical allogeneic marrow transplantation for treatment of patients with malignant blood diseases. Sixteen cases, including 12 with acute leukemia and four with advanced malignant lymphoma, were treated by autologous marrow, which was purged in vitro by hyperthemia (42.5 degrees C for 70 min) following incubation for 5 days with interleukin 2 (IL-2) in liquid culture and mixed with HLA haploidentical marrow cells from their sibling or parent. Acute GVHD was not observed in any patient after transplantation. Hematological rescue in the clinical setting was demonstrated in all cases but one who died early from hepatic veno-occlusive disease (VOD). Five cases who were transplanted at the time of CR2 or CR3 and in advanced phase of lymphoma, relapsed 4 to 7 months after transplantation. The relapse rate was 31.3%. None of eight patients who received allogeneic BMT within 2 h after ABMT relapsed with median follow-up of 12 months and two of them died from procedure-related complications. Seven cases are still alive and disease-free with a median follow-up of 12 months. Mixed chimerism was found in 3/6 cases, who had different sex donors, by analysis of sex chromosomes. These results show that mixed transplantation is a safe, effective and new approach to treating patients with malignant tumors. In order to detect the effects of GVL, studies are now in progress in our clinic.

Improved long-term survival for unresectable hepatocellular carcinoma (HCC) with a combination of surgery and intrahepatic arterial infusion of 131I-anti-HCC mAb. Phase I/II clinical trials.

Resectional therapy has been accepted as the only curative therapy for hepatocellular carcinoma (HCC). Unfortunately, it is estimated that only 10% of HCC are resectable at the time of diagnosis. Cytoreduction and sequential resection offer a new hope for patients with unresectable HCC. Radioimmunotherapy (RIT) is an attractive approach for cytoreduction. We have previously shown that intrahepatic arterial 131I-labelled anti-HCC monoclonal antibody (131I-Hepama-1 mAb) could be used safely in combination with hepatic artery ligation for treatment of unresectable HCC, and encouraging results have been achieved. In this paper, the long-term survival and the prognostic factors in HCC patients treated with radioimmunotherapy will be analysed. Sixty-five patients with surgically verified unresectable HCC were treated with hepatic artery ligation plus hepatic artery cannulation and infusion from 1990 to 1992. Thirty-two patients were enrolled in a phase I-II clinical trial with infusion of 131I-radiolabelled anti-HCC monoclonal antibody (Hepama-1 mAb) via the hepatic artery (the RIT group). Another 33 patients formed the group treated with intrahepatic-arterial chemotherapy (the non-RIT group). T cell subsets were measured in 24 patients and human anti-(murine Ig) antibody (HAMA) were monitored in the RIT group. The 5-year survival rate was significantly higher in the RIT group than in the chemotherapy group, being 28.1% compared to 9.1% (P < 0.05); this was mainly a result of better cytoreduction and a higher sequential resection rate (53.1% compared to 9.1%). Significant prognostic factors in the RIT group included tumour capsule status and the number of tumour nodules. HAMA incidence and CD4+ T lymphocytes influenced short-term, but not long-term survival. It is suggested that intrahepatic-arterial RIT, using 131I-Hepama-1 mAb, combined with hepatic artery ligation might be an effective approach to improve long-term survival in some patients with unresectable HCC, which may successfully be made resectable by intra-arterial infusion of 131I-Hepama-1 mAb.

Long-term results of surgery for small primary liver cancer in 514 adults.

During 1958-1993, 2030 patients with pathologically proven primary liver cancer (PLC) were retrospectively reviewed. Comparison between small PLC (< or = 5 cm, n = 514) and large PLC (> 5 cm, n = 1516) revealed that small PLC had a higher resection rate (92.4% versus 49.1%), lower operative mortality (1.7% versus 5.2%), a higher percentage of single tumour nodules (78.0% versus 53.4%), a higher percentage of well encapsulated tumour (74.5% versus 35.8%) and higher survival rates after resection (5-year, 63.8% versus 36.6%; 10-year, 46.8% versus 28.5%). No significant difference was found between survival following limited resection (n = 440) and lobectomy (n = 34) in patients with small PLC. Re-resection of any subclinical recurrence or solitary pulmonary metastasis after small PLC resection was done in 70 cases. These results indicate that resection is still the modality of choice for treatment of small PLC; limited resection instead of lobectomy was the key to increasing resectability and decreasing operative mortality; re-resection of subclinical recurrence was important to prolong survival further.

Radioimmunotherapy for unresectable hepatocellular carcinoma using 131I-Hepama-1 mAb: preliminary results.

Twenty-three patients with surgically verified unresectable hepatocellular carcinoma (HCC) have been treated by intrahepatic arterial administration of 131I-labeled anti-HCC monoclonal antibody (Hepama-1) combined with hepatic artery ligation. Radioimmunoimaging demonstrated that the median tumor/liver ratio was 2.1 (1.1-3.6) at day 5. A decline in alpha-fetoprotein level and shrinkage of tumor were observed in 75% (12/16) and 78% (18/23) of patients respectively. Sequential resection was done in 11 patients (48%) after treatment. The surgical specimens revealed massive necrosis of tumor, but residual cancer cells were found at the edge of the specimens. Anti-antibody was determined in 43% (10/23) of patients 2-4 weeks after the administration of 131I-Hepama-1 mAb. No marked toxic effects were noted. It is suggested that 131I-Hepama-1 mAb might be of value as one of the multimodality treatments for unresectable HCC.

Recurrence after resection of alpha-fetoprotein-positive hepatocellular carcinoma.

The long-term prognosis of surgery for hepatocellular carcinoma (HCC) is not yet satisfactory, the main reason being the high recurrence rate. The authors report the results of a long-term follow-up of 308 patients with HCC who became alpha-fetoprotein-(AFP)-negative after resection between 1975 and 1991. By March 1992, there was recurrence in 134 patients (43.5%). The 1-, 3-, 5- and 10-year recurrence rates were 9.2%, 38.8%, 54.9% and 85.0%, respectively. The 5-year survival rate was 49.7% for patients who had undergone a second hepatic resection (n = 48). Analysis of factors influencing postoperative recurrence indicated that patients subjected to mass survey, with a lower gamma-glutamyltransferase level, at an early stage of TNM classification, with a tumour of less than 5 cm, without tumour embolus, and with postoperative immunotherapy had a lower incidence of recurrence. It is concluded that the earlier the disease is diagnosed, the less the recurrence rate; adjuvant immunotherapy may reduce postoperative recurrence, and the early detection and resection of a recurrent tumour are important to prolonging survival further after curative resection of HCC.

The behaviors of Ca(2+)-ATPase embedded in interdigitated bilayer.

We investigated the behavior of a membrane protein, Ca(2+)-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca(2+)-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca(2+)-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions.

Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures.

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.

Characteristics and prognosis of primary liver cancer in young patients in China.

To investigate the clinicopathologic characteristics of primary liver cancer (PLC) in young adults, 77 patients aged 35 or younger were compared with 603 patients older than 35 years during the same period. In the young patients, PLC showed: (1) a low incidence detected at mass survey (young 15.6% vs older 28.7%, P < 0.05); (2) a low level of history of hepatitis (young 36.8% vs older 66.3%, P < 0.01); (3) a high incidence of positivity for hepatitis B surface antigen (HBsAg) (young 79.2% vs older 67.6%, P < 0.05); (4) a relatively low incidence of associated cirrhosis (young 64.9% vs older 90.7%, P < 0.01); (5) larger tumor size (PLC > 5 cm; young 87.0% vs older 73.0%, P < 0.01); and (6) a more advanced stage of the disease according to the TNM classification (stage III; young 29.9% vs older 18.2%, P < 0.05). It is suggested that hepatitis B virus (HBV) may play an important role in the development of PLC without associated liver cirrhosis in young patients. Close periodic surveillance of young adults who are positive for HBsAg is important to detect PLC at an early stage.

Interaction of the interdigitated DPPG or DPPG/DMPC bilayer with human erythrocyte band 3: differential scanning calorimetry and fluorescence studies.

Human erythrocyte band 3 reconstituted into phospholipid vesicles has been used for studying the interaction of interdigitated lipid bilayer with an integral membrane protein. The interdigitated gel phase in DPPG/band 3 or DPPG/DMPC/band 3 systems was induced with polymyxin B (PMB) or Tris+. The phase transitions of the vesicles were detected with high-sensitivity differential scanning calorimetry (DSC). The results indicated that band 3 does not cause significant alterations in the interdigitated phase of phospholipids, with only a little decrease of the phase transition enthalpies. Fluorescence measurements showed that the transition of phospholipid/band 3 systems from the non-interdigitated to interdigitated phase is accompanied by marked intrinsic fluorescence changes of band 3. The interdigitated phase of DPPG or DPPG/DMPC vesicles increases the intrinsic fluorescence intensity of band 3 and significantly decreases the accessibility of certain tryptophan residues on the protein to Cs+.

Surgical treatment of recurrent hepatocellular carcinoma.

During the last 16 years, radical resection was performed in 392 patients with hepatocellular carcinoma (HCC). Eight patients died within 30 days after resection. The other 384 patients were discharged from hospital and closely followed for 6 to 195 months. By December 1991, 185 patients had developed a recurrent tumor, the 1-, 3-, and 5-year recurrence rates being 15.0%, 45.4%, and 55.3%, respectively. Ninety of the 185 patients underwent reoperation, including second hepatic resection (65 cases), cryosurgery (8 cases), resection of lung metastasis (6 cases), hepatic artery ligation and infusion chemotherapy (2 cases), intratumor ethanol injection (3 cases), microwave coagulation plus intratumor ethanol injection (2 cases), and exploration (4 cases). The survival rate of these 90 patients was significantly better than that of 95 patients who were treated by other palliative methods, the 5-year survival rate being 63.4% as compared with 28.6% after the first resection (P < 0.01), and 40.8% vs. 2.2% after recurrence (P < 0.01), respectively. These results suggest that reoperation for recurrent HCC might be an important approach to prolonging survival further after hepatic resection.

Neonatal hearing impairment complicated by neonatal pneumonia. Application of auditory evoked potentials in neonatology.

A group of 433 neonates with pneumonia were studied through the monitoring of audiology on the basis of epidemiology in perineonatal hearing impairment and applied auditory evoked potentials. The results showed that 29 cases had a threshold of more than 30 dBnHL in primary auditory brainstem response test (6.7%), and 19 cases with threshold, more than 40 dBnHL in primary 40Hz-auditory event related potential (40Hz-AERP) test (4.4%). The follow-up between the 3rd and 12th month after the initial test demonstrated that 12 cases had hearing impairment to various degrees.

Combined traditional Chinese medicine and Western medicine. Relieving effects of Chinese herbs, ear-acupuncture and epidural morphine on postoperative pain in liver cancer.

In the evaluation of Chinese herbs (A), ear-acupuncture (B) and epidural morphine (C) to relieve postoperative pain and abdominal distension, sixteen male patients with primary liver cancer were observed. This study was conducted by means of orthogonal experiment and double blind, randomized design. The patients received various treatments according to the display of the orthogonal table L16(2)15 which corresponds to 2(3) factorial experiment design. C+ (morphine 2 mg) was given before the peritoneum was sutured. A+ (orally administered) and B+ were given 24 hours after operation. 50-100 mg of pethidine was given when the pain intensity VAS (0-100) exceeded 50-70. The observation parameters included plasma leucine enkephalin (LEK), postoperative total dosage of narcotics administered for 5 days, VAS for pain and pain reliever, abdominal distension, urinary retention, constipation, etc. The results were as follows: a. Patients who had received A (A+B+C+, A+B+C-, A+B-C-, A+B-C+); C (C+A+B+, C+A+B-, C+A-B+, C+A-B-), or B (B+A+C+, B+A+C-, B+A-C+, B+A-C-) produced better analgesic effects than those who had received placebo. The A, B, and C reduced narcotics 650, 450 and 550 mg respectively when compared with placebo. The effects of A and C were of statistical significance (P < 0.05), while AB, BC, and AC interactions were not found; b. A and B minimized abdominal distension and urinary retention, while C prolonged them. As compared with the placebo, A and B accelerated restoration of bowel peristalsis (P < 0.05, ANOVA). Both A and B decreased it for 165 hours, while epidural morphine prolonged it for 49 hours; and c.(ABSTRACT TRUNCATED AT 250 WORDS)

[Randomized clinical trial of cis-platinum diamminedichloride (PDD) in the treatment of hepatocellular carcinoma (HCC)].

From Oct. 1982 to Apr. 1985, 82 patients with HCC proven by pathology were treated in our hospital. 43 treated by hepatic arterial perfusion, were randomized into PDD group: PDD 10 mg per day X 10, every 3 weeks; control group: fluorouracil (5-Fu) 250 mg per day X 4, every week and thio-tepa (TSPA) 10 mg, twice a week. The other 39 treated by intravenous chemotherapy, were also randomized into PDD group: PDD 20 mg per day X 5, every 3 weeks; control group: 5-Fu 500 mg and TSPA 10 mg, twice a week. The objective response rates were 31.8% (7/22) in PDD group and 23.8% (5/21) in control group by hepatic arterial perfusion, and 20.0% (4/20) in the former and 0% (0/19) in the latter who were treated intravenously. The median survivals were 8 months for all the patients receiving hepatic arterial perfusion, and 6 and 5 months for the intravenous PDD and its control group, respectively. The side effects and kidney toxicity of PDD were tolerable to the patients. It is observed that PDD is better than 5-Fu and TSPA in the treatment of HCC.

[Otoacoustic emission cochleogram evoked by bone conducted stimulation].

As bone conducted stimulation, tone bursts of different frequencies were applied through the forehead in 7 normal-hearing subjects. Binaural evoked otoacoustic emissions (EOAE) were then recorded simultaneously, which saved one half of the time required for conventional monaural recording. Analysed with autoregressive modeling, the main echo of EOAE was a narrow-band sound with a stimulus dependent central frequency. It was suggested that the generation site of EOAE was near to that cochlear portion stimulated by the corresponding frequency. The latency of EOAE, although independent of the stimulus intensity, tended to be shorter at higher stimulus frequencies. This was possibly due to the differences in the distances from the generation sites of the otoacoustic emissions to the tympanic membrane. Recordable otoacoustic emissions were evoked by tone bursts of 1.0, 2.0, 3.0 and 4.0 kHz in all the 14 normal ears except one at 4.0 kHz, and 10 and 7 ears by tone bursts of 0.5 and 6.0 kHz, respectively. Emission cochleogram was obtained when the means of EOAE detection thresholds were plotted in an audiogram format. The lowest threshold was found at 1.0 kHz. This might be related to the middle ear resonance frequency of 1100 +/- 230 Hz. The technique of simultaneous recording of binaural EOAE and plotting of emission cochleogram described in this paper is clinically useful as a means of objective evaluation of hearing.

[High level expression of human atrial natriuretic peptide in fusion form in E. coli system].

OBJECTIVE: To construct recombinant plasmid with human atrial natriuretic peptide (ANP) gene in fusion form for stable and high level expression of genetic engineering product ANP in E. coli system. METHODS: Plasmids with ANP fusion gene were constructed by PCR and sub-cloning and fusion protein was expressed in E. coli system. High level expression of the fusion genes was enhanced by linking operons in tandem. ANP was released from the fusion protein with thrombin treatment and purified by chromatography. Genetic-engineered ANP was evaluated with the drug production requirement, and produced ANP was tested for its effects of lowering blood pressure and diuresis in vivo and vasodilation in vitro. RESULTS: A series of 4 plasmid pCW111-114, containing 1 to 4 gene operons respectively, were constructed, and the yields of fusion protein were 46%, 54.8%, 56.1% and 60.1% of total cell protein. Fusion protein in the form of inclusion body was isolated and purified, and then treated with thrombin to release ANP. After purification using chromatography columns, at least 3 mg/L culture of ANP with the purity higher than 96% was produced in shaking flask. Primary pharmacological evaluation showed the produced ANP had the effects of blood pressure lowering in vivo and diuresis in vivo and vasodilation in vitro, which was similar to the activity of standard ANP. CONCLUSIONS: By the protocol of fusion gene cloning and expression, the human ANP was produced successfully in E. coli system.