[The 493th case: recurrent edema of bilateral lower extremeties].

A 43-year-old male patient with onset of edema caused by nephrotic proteinuria and low titer of anti-M type phospholipase-A2-receptor (PLA2R) antibody was diagnosed as idiopathic membranous nephropathy by renal biopsy. Administrated with prednisone 40 mg once a day and cyclosporine 100 mg twice a day as front-line regimen, the patient relapsed after transient partial remission. When treatment was combined with cyclophosphamide 100 mg once a day, the 24-hour total urine protein and titer of anti-PLA2R antibody were even elevated. Therefore, the patient received rituximab 1 g intravenously in April 2019, October 2019 and October 2020 respectively. CD19 positive B lymphocytes in peripheral blood were eliminated from 71/µl to zero. Immunosuppressants and corticosteroids were withdrawn successively. On the last follow-up in November 2020, the anti-PLA2R antibody was negative, and the 24-hour total urine protein and serum albumin was 4.4 g and 34 g/L, respectively. This case suggested the potential efficacy of rituximab for refractory membranous nephropathy. Further studies should explore whether the titer of anti-PLA2R antibody indicates the dose of rituximab.

[The 489th case: acute kidney injury, gross hematuria, nephrotic syndrome].

Onset with fever and back pain, an 81-year-old man had sudden oliguria and progressively elevated serum creatine from normal range to 660 µmol/L within 1 week after receiving contrast agents, various antibiotics, and several nonsteroidal anti-inflammation drugs. Urine output recovered after supportive treatment. However, his serum creatinine level rose again soon after a temporary decline accompanied by gross hematuria with almost normal morphology, nephrotic proteinuria, and hypoalbuminemia. Renal biopsy revealed necrotizing glomerulonephritis. Methylprednisolone was intravenously administrated 500 mg per day for 3 days, followed by oral glucocorticoids and cyclophosphamide. Gradually the patient's serum creatinine descended to 144 µmol/L.

[Veracity of using a visual chart with a testing distance of 2.5 meters for measurement of distance visual acuity in teenagers].

Objective: To compare the results of visual acuity testing for teenagers with visual acuity charts designed at 2.5-meter and 5-meter distances, and to investigate the accuracy of the 2.5-meter visual acuity chart. Methods: It was a self-control study. A total of 227 teenagers (454 eyes) with ametropia who came to the ophthalmic clinic of Peking Union Medical College Hospital from March 2019 to September 2019 were included. There were 123 males and 104 females aged (11.74±3.26) years. The vision examiners were trained in the same testing environment and passed the consistency test. Distance visual acuity of all participants was tested with charts designed at 2.5 meters and 5 meters in a 10-minute interval. According to the age (7-9, 10-12, 13-15 and 16-18 years old) and visual acuity (1.00-0.52, 0.40-0.30 and 0.22-0.10), the results of two kinds of visual acuity charts were compared. The testing order of the two visual charts was randomly determined. The visual acuity results were converted into logMAR values and compared. Paried t-student test was used to compare the difference between two charts, and Pearson correlation test was used to explore the correlation between the results of two charts. Results: The visual acuity of the right eye was 0.37±0.24 (logMAR) at 2.5 meters and 0.50±0.26 (logMAR) at 5 meters. The distance visual acuity measured with the 2.5-meter visual acuity chart was better (t=16.19, P<0.01). The visual acuity of the left eye was 0.36±0.23 (logMAR) at 2.5 meters and 0.45±0.23 (logMAR) at 5 meters (t=11.71, P<0.01). The differences between two charts were larger among teenagers with lower visual acuity (r=0.387,P<0.01). Conclusion: Under the same test conditions, the distance visual acuity measured with a 2.5-meter chart was significantly better than a 5-meter chart. The visual acuity chart designed at 2.5 meters was not an appropriate tool to measure distance vision in adolescents. (Chin J Ophthalmol, 2021, 57: 122-125).

Association of IGF1R polymorphisms with the development of HBV-related hepatocellular carcinoma.

Although the involvement of insulin-like signaling in cancer has been well documented in various types of cancers, the association between the genetic variants in the insulin-like signaling and the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains unclear. In this study, a total of 498 individuals including 173 HBV related cirrhosis patients, 171 HBV-related HCC patients, and 154 healthy controls were enrolled. Sixteen single nucleotide polymorphisms (SNPs) in IGF1, IGF2, IGF1R and IGF2R have been genotyped by employing SNaPshot assays. We found A/A genotype at rs3743251 of IGF1R was negatively associated with HBV related HCC [odds ratio (OR) = 0.38, 95% confidence interval (CI) = 0.20-0.72, P = 0.037]; A/G genotype decreased the risk of portal vein thrombosis (OR = 0.38, 95%CI = 0.18-0.82, P = 0.01). These results indicate that rs3743251 polymorphism in IGF1R is associated with the susceptibility of HBV-related HCC.

Development of an improved rat model of dual graft liver transplantation with long-term survival.

Dual graft liver transplantation has been demonstrated to be feasible as well as effective in increasing the donor pool and in preventing the potential for small-for-size syndrome. However, little is known about the pathophysiological and immune processes following dual graft liver transplantation due to the lack of appropriate animal models. The aim of this study, therefore, was to establish an improved rat model of dual graft liver transplantation, with long-term survival. Male inbred rats were used as both donors and recipients. One middle lobe together with another right middle lobe from the livers of two different donors were used as the dual grafts. The "basin-shaped anastomosis" technique was used to connect the suprahepatic inferior vena cava; "Y-shaped bridge" and "three-cuff" techniques were adopted for the anastomosis of the portal veins; and the "two-stent" technique was used for the anastomosis of the bile ducts. Six of the ten recipients survived for more than 100 days after dual graft liver transplantation. There was no difference in graft survival between dual and whole liver transplantation. The long-term survivors with dual grafts from two different donors had unobstructed portal vein flow, unobstructed biliary tract dilatation, normal graft function, and well-preserved hepatic structure. Therefore, this improved model will be potentially useful for evaluating the pathophysiological processes, immune responses between dual grafts and recipient, and mechanisms underlying the liver regeneration in dual grafts after dual graft liver transplantation.

Liver regeneration after liver transplantation.

BACKGROUND/PURPOSE: The liver has a remarkable capacity to regenerate after injury or resection. The aim of this review is to outline the mechanisms and factors affecting liver regeneration after liver transplantation. METHODS: Relevant studies were reviewed using Medline, PubMed and Springer databases. RESULTS: A variety of cytokines (such as interleukin-6 and tumor necrosis factor-α), growth factors (like hepatocyte growth factor and transforming growth factor-α) and cells are involved in liver regeneration. Several factors affect liver regeneration after transplantation such as ischemic injury, graft size, immunosuppression, steatosis, donor age and viral hepatitis. CONCLUSION: Liver regeneration has been studied for many years. However, further research is essential to reveal the complex processes affecting liver regeneration, which may provide novel strategies in the management of liver transplantation recipients and donors.

[Smoking relapse rate and influencing factors in smokers aged 15 years and above in China].

Objective: To describe the relapse status of smokers aged ≥15 years in China, and investigate the main factors influencing their relapse behavior. Methods: The data of this study cames from the 2018 China Adult Tobacco Survey, covering 400 committees or villages in 200 districts or counties in 31 provinces (autonomous regions and municipalities) of China. The relevant data of smoking and relapsing in residents aged ≥15 years were collected by face-to-face interview. Software SAS 9.4 was used to clean and analyze the data, and logistic regression model was used to analyze the influencing factors for relapse rate. Results: A total of 19 376 questionnaires were completed, with a response rate of 91.50%. In 2018, 66.05% of smokers aged ≥15 years in China had smoking relapse, in whom 66.59% were males and 55.79% were females. In all age groups, the age group 15-24 years had the highest smoking relapse rate (82.63%). Multivariate analysis showed that the younger age 15-24 years (OR=4.618,95%CI:1.981-10.763), e-cigarette use (OR=9.782,95%CI:3.139-30.490), and tobacco advertising, promotion and sponsorship in the past 30 days (OR=1.710,95%CI:1.291-2.265) were associated with higher smoking relapse rate. Compared with people who were allowed smoking at home or those without smoking limit, the smoking relapse rate in people who were not allowed to smoke at home (OR=0.562, 95%CI: 0.439-0.719) or those with smoking limit (OR=0.487, 95%CI: 0.366-0.647) was lower. Conclusion: The smoking relapse rate in Chinese smokers is high, especially in young people. It is suggested to conduct targeted intervention based on the results of this study to reduce the smoking relapse rate and help achieve the smoking control goal in Healthy China 2030.

[Quit intention and smoking cessation behavior of current smokers aged 15 years and above in China, 2018].

Objective: To describe the current situation on quit intention and quit attempts among current smokers aged 15 years and above in China and explore the main factors affecting their smoking cessation behaviors. Methods: The subjects were people aged 15 years and above in China. A stratified multi-phased randomized cluster sampling method was used. The survey covered 200 districts/counties in 31 provinces (autonomous regions and municipalities directly under the central government), including 19 376 people under investigation. The data were weighted with SAS 9.4 for complex sampling analysis, and the frequency and weighted component ratio were used for description. The influencing factors were studied using the Rao Scott χ2 test and unconditional multivariate logistic regression. Results: Among current smokers, 6.63% reported that they planned to quit smoking in the next month, 5.44% for men and 8.49% for women. 17.96% of the current smokers made quit attempts in the past 12 months, with 17.80% men and 22.15% women. People who smoke occasionally (OR=3.42, 95%CI: 2.09-5.59) made quit attempts in the past (OR=4.91, 95%CI: 0.33-0.52) and knew that smoking would lead to three diseases (OR=2.24, 95%CI: 1.48-3.40) had higher quit intention. People who occasionally smoke (OR=1.68, 95%CI: 1.20-2.34) received smoking cessation suggestions from medical staff (OR=1.96, 95%CI: 1.62-2.37), were a ware of that smoking causes three diseases (OR=1.63, 95%CI: 1.32-2.02) and had smoking prohibition regulations in indoor areas of their homes, had a higher rate of quit attempts. Conclusion: Providing effective smoking cessation intervention services and creating a supportive tobacco control environment are significant in promoting smoking cessation behavior.

[Secondhand smoke exposure and its influencing factors among Chinese people aged 15 years and above in 2010 and 2018].

Objective: To investigate secondhand smoke (SHS) exposure among non-current smokers aged 15 and over and its influencing factors in China in 2010 and 2018. Methods: The 2010 and 2018 China Adult Tobacco Surveys used multistage stratified cluster random sampling methods to obtain national representative samples across 31 provinces (autonomous regions and municipalities) in China. This study selected non-current smokers aged 15 and over as the research subjects to describe the general demographic characteristics, perceptions of SHS hazards, attitudes towards smoking bans in indoor areas in public places, and SHS exposure and the smoking restriction regulations in different places. The Rao-Scott χ2 test was used to compare the rates, and the unconditional logistic regression was used to analyze the influencing factors of SHS exposure. All the subjects in the analysis were weighted based on a complex sampling design. Results: Among non-current smokers aged 15 and over, from 2010 to 2018, the percentage of indoor smokers had decreased from 84.7% to 71.9%. The rates of people who saw smoking in various places declined with a statistically significant difference (P<0.001). The exposure to SHS for females, people aged 45-64, teachers, medical staff, workers in enterprises, businesses, waiters, and people who live in rural and central areas declined. The differences were statistically significant (P<0.05). In 2018, the percentage of SHS exposure for those who reported a comprehensive smoking ban in households or allowed smoking in certain areas was lower than for those who reported allowed or without a smoking ban. Among those aged 25-44, SHS exposure for those who reported complete smoking prohibition in workplaces was lower than those who reported allowed or without smoking prohibition (OR=0.65, 95%CI: 0.49-0.87). The proportion of SHS exposure for those who believe that smoking should not be allowed in indoor areas of 0-5 public places is higher than that smoking should not be allowed in the indoor spaces of 8 public places (OR=2.13, 95%CI: 1.35-3.36). Among people aged 45-64, the proportions of SHS exposure for teachers (OR=0.37, 95%CI: 0.17-0.78) and medical staff (OR=0.35, 95%CI: 0.16-0.76) and staff working in agriculture, forestry, animal husbandry, fishery, and water conservancy were lower than governmental staff (OR=0.49, 95%CI: 0.29-0.84). Conclusions: Data from the decreased exposure of Chinese non-current smokers aged 15 and over to SHS suggests the initial achievement through constructing a smoke-free environment, but continued efforts are needed. It is necessary to actively advocate for smoke-free families, strengthen publicity and education on the hazards of tobacco and SHS, and improve the skills of non-smokers in rejecting SHS exposure.

[Analysis of exposure to tobacco advertisement and promotion among Chinese adults aged 15 years and above, 2010 and 2018].

Objective: To investigate the exposure to tobacco advertisements and promotions among Chinese adults aged 15 and above, identify the publicity channels and forms of tobacco advertising and promotion in 2010 and 2018, and implicate further tobacco control measures in China. Methods: A multistage, stratified, randomized cluster sampling design was used in 2010 and 2018 China Adult Tobacco Survey, with national representativeness. 13 354 and 19 376 permanent residents were selected in the 2010 and 2018 surveys. SAS 9.4 software was applied for data analysis, and all the data were weighted based on a complex sampling design. Rao Scott χ2 test was used for group comparison of a single factor. Results: In 2010 and 2018, 19.61% and 18.14% of the survey respondents did see tobacco advertising and promotion. From 2010 to 2018, there was no significant change in the situation of tobacco advertisements and promotions. Among those who had gone to a cigarette shop, the proportion of seeing tobacco advertisements increased from 29.28% in 2010 to 43.28% in 2018. Among those who had seen tobacco advertisements on TV, the rate fell from 50.93% in 2010 to 28.58% in 2018. Among those who had gone to movie theaters, the proportion of seeing tobacco advertisements increased from 2.17% in 2010 to 9.89% in 2018. Among those who had used the Internet, the proportion of seeing tobacco advertisements online rose from 19.20% in 2010 to 42.30% in 2018. In terms of tobacco promotion, the percentages of people who had seen tobacco promotions, cigarette price discounts, cigarette discount coupons, gifts, and other preferential activities in various places in the past 30 days were 4.99% vs. 9.30%, 0.78% vs. 4.09%, 0.04% vs. 0.33% and 0.98% vs. 3.33% in 2010 and 2018, respectively (P<0.001). Conclusions: Tobacco advertising and promotion are still prevalent in China, with no significant change in 2010 and 2018. Tobacco advertising and promotion have been with the constant changes and development of media platforms. It is necessary to improve the implementation of relevant policies, comprehensively ban tobacco advertisements and promotions, and strengthen the supervision of ads and promotions.

Sevoflurane protects against acute kidney injury in a small-size liver transplantation model.

BACKGROUND: Living donor liver transplantation (LDLT) patients run the risk of developing acute kidney injury (AKI) and subsequent chronic kidney disease, affecting morbidity and mortality. Sevoflurane has anti-inflammation properties, and renal ischemia/reperfusion under sevoflurane anesthesia resulted in drastic improvements in renal function. Extrahepatic metabolism of sevoflurane has been reported in patients undergoing liver transplantation, and might lead to nephrotoxicity. However, whether sevoflurane anesthesia is safe with regard to renal function in small-size liver transplantation needs further investigation. As neutrophil gelatinase-associated lipocalin (NGAL) is an early predictive biomarker of AKI, we looked at the renal effects of sevoflurane in a rat liver transplantation model using small-for-size grafts to investigate the changes of NGAL level and kidney histology. METHODS: Sixty male Sprague-Dawley rats were randomly divided into 2 groups after 50% size liver transplantation. Rats were anesthetized with chloral hydrate or with sevoflurane and subjected to liver transplantation. Twelve rats in each group were used for the survival study and 6 rats were used for the hemodynamic study. Six rats in each group were sacrificed 2 or 24 h after reperfusion. We harvested kidneys and serum for further analysis, including histological and functional parameters; TNF-α, IL-6 and NGAL immunoassay; expressions of myeloperoxidase (MPO) activity; and NF-κB in renal tissues. RESULTS: Rats in the sevoflurane group had significantly lower Scr 24 h after reperfusion compared with those in the chloral hydrate group. Rats in the sevoflurane group demonstrated significantly reduced NGAL concentrations compared with rats in the chloral hydrate group 2 h after reperfusion. Epithelial necrosis in the chloral hydrate group (3.2 ± 0.8) was greater than that in the sevoflurane group (1.5 ± 1.1; p < 0.05). Sevoflurane anesthesia resulted in significantly lower plasma TNF-α and IL-6 concentrations and reduced MPO concentrations 2 h after reperfusion (p < 0.05). NF-κB protein levels 2 h after reperfusion increased by at least 110% in the chloral hydrate group relative to the sevoflurane group 2 h after reperfusion (p < 0.05). However, the urine inorganic fluoride concentrations increased significantly (p < 0.001) 2 h after reperfusion in the sevoflurane group (6.1 ± 1.5 µmol·l⁻¹) compared with the chloral hydrate group. CONCLUSIONS: Sevoflurane anesthesia can attenuate renal injury and modulate inflammatory cascades in small-size liver transplantation using rat models.

Proteomic analysis of differentially expressed proteins in rat liver allografts developed acute rejection.

BACKGROUND: Acute rejection (AR) after liver transplantation is a cell-mediated immune response that takes place within the allograft and results in graft dysfunction and failure, but the molecular mechanisms about hepatocyte dysfunction remain poorly understood. Here we characterized global protein expression changes in liver allograft during AR. METHODS: The effect of an alloantigen-dependent immunological response was evaluated by syngeneic and allogeneic rat orthotopic liver transplantation (OLT). Using a combination of two-dimensional gel electrophoresis and mass spectrometry, we identified 18 differentially expressed proteins in AR allograft compared with matched tolerance allograft. Serum chemistry and allograft histology were determined. RESULTS: Allogeneic OLT recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allograft compared with syngeneic OLT. 18 protein expressions altered by AR play important roles in metabolism, oxidative stress defense, signal transduction, biotransformation and transport. Decreased expression of protein disulfide isomerase in AR allograft was confirmed by Western blotting and immunohistochemistry. CONCLUSIONS: This study uncovered new mechanistic insights into graft dysfunction in AR of liver allograft. Several significantly altered protein expressions act coordinately in hepatocyte dysfunction by depressed energy, enhanced oxidative stress-induced molecular damage and restrained biotransformation. The present findings may open new avenues for the understanding and prevention of graft dysfunction and failure during AR.

MMP2 promoter polymorphism (C-1306T) and risk of recurrence in patients with hepatocellular carcinoma after transplantation.

Genetic variants in matrix metalloproteinase (MMP) gene may influence the biological function of these enzymes and change their role in carcinogenesis and progression. The effect of MMP2 C-1306T and MMP9 C-1562T polymorphisms on genetic susceptibility has been investigated in various kinds of cancer. However, the relationship between these polymorphisms and risk of recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) has not been reported. The present study was designed to investigate the association of these two loci with the risk of HCC recurrence in 93 HCC patients treated with LT. Genotyping was performed using direct DNA sequencing. For MMP2 C-1306T variant, patients with CT heterozygous conferred a 58% reduction in recurrence risk (risk ratio: 0.419; 95% confidence interval: 0.177-0.994). The mean recurrence-free survival for CT genotype was significantly longer than that for homozygous CC patients (30.4 vs 19.3 months, p = 0.019). However, no association was found between MMP9 C-1562T polymorphisms and recurrence of HCC (p = 0.259). These findings suggest that MMP2 promoter polymorphisms may provide some predictive value for HCC recurrence after LT.

Role of reactive oxygen species in mediating hepatic ischemia-reperfusion injury and its therapeutic applications in liver transplantation.

Increasing evidence has shown that reactive oxygen species (ROS) are important mediators in liver ischemia/reperfusion injury(IRI). ROS include hydrogen peroxide (H(2)O(2)), superoxide anion (O(-2)), and hydroxyl radical (HO(-)), which may be generated by activated Kupffer cells in the liver, contributing to reperfusion injury. Hepatic IRI is a multistep process that damages liver graft function. To establish a series of therapeutic strategies to improve the outcome of liver transplantation, a good understanding of the mechanisms of IRI is essential. However, the detail mechanisms of how ROS lead to hepatocyte damage in IRI remains unclear. The aim of this review was to describe recent developments in the field of oxidative stress research. The first part of this review focused on the key roles and possible mechanisms of ROS in hepatic IRI. The second part of this review summarizes some findings including novel and classic antioxidant methods to ameliorate the hepatocyte damage during IRI.

The role of toll-like receptors 2 and 4 in acute allograft rejection after liver transplantation.

Toll-like receptors (TLRs) are germline-encoded receptors expressed on antigen-presenting cells (APCs) that identify a variety of microbial and endogenous ligands and activate the innate immune responses to the presence of danger. However, their role in the development of allograft rejection after liver transplantation remains unknown. In this study, we used flow cytometry to assess TLR-4 and TLR-2 expression among circulating CD14+ monocytes in 64 liver transplant patients and 24 healthy volunteers. We demonstrated significantly higher TLR-2 and TLR-4 expression on circulating monocytes among conditioned liver transplantation recipients with acute rejection compared with those in clinically stable with normal liver function. Steroid pulse therapy significantly reduced the expression of TLR-4 and TLR-2 on the monocytes of recipients with acute rejection. Based on these data, we have suggested that activation of innate immunity in liver transplant recipients through TLR-4 and TLR-2 contributes to the development of acute allograft rejection after liver transplantation. The reduced expression of TLR-4 and TLR-2 may be one of the mechanisms by which steroid pulse therapy inhibits the development of acute rejection. Estimation of TLR expression on APCs may be predictive of in acute rejection after liver transplantation.

Hypothermic Machine Perfusion's Protection on Porcine Kidney Graft Uncovers Greater Akt-Erk Phosphorylation.

BACKGROUND: To investigate the potential mechanisms of hypothermic machine perfusion (HMP)'s beneficial effects on kidney graft over static cold storage (SCS) in vitro. METHODS: Ten kidneys of 5 Bama miniature male pigs were paired into 2 groups: SCS group and HMP group. Preservation solutions were taken at 0, 1, 3, and 6 hours for the measurement of K+, Na+, Cl-, blood urea nitrogen (BUN), creatinine (Cr), and lactate dehydrogenase (LDH) using the standard laboratory methods. Renal cortex were harvested at 6 hours for the following measurement: lactic acid (LD), adenosine triphosphate (ATP), malondialdehyde (MDA), neutrophil accumulation (MPO), interleukin-10 (IL-10), and transforming growth factor-ß (TGF-ß). Ischemia-induced apoptosis and the protein expression levels of total Akt, phospho-Akt, total Erk, and phospho-Erk were analyzed by Western blotting. RESULTS: Almost all of the tested metabolites in preservation solutions were reduced with time in the HMP group. Levels of Na+, Cl-, BUN, Cr, K+, and LDH were lower in the HMP group compared with the SCS group, with differences in the first 4 reaching statistical significance. HMP alleviated ATP degradation and LD accumulation, diminished the MDA (P < .05) and MPO (P = .227) levels, and greatly raised IL-10 and TGF-ß (P < .05) expression. A marked decrease of proapoptotic and a large increase of antiapoptotic markers (P < .05) along with greatly raised Akt (P < .05) and Erk (P < .01) phosphorylation was observed in the kidney of the HMP group compared with the SCS group. CONCLUSION: HMP's kidney graft protection involves inhibition of accumulation of toxic metabolites, oxidative damage, and apoptosis along with upregulation of the Akt and Erk signaling pathway.

The apoptosis of HEL cells induced by hydroxyurea.

Hydroxyurea has been used to synchronize cultured cells to S-phase and used to treat patients with sickle-cell anemia. Recently, we found that hydroxyurea can induce the apoptosis of HEL (human erythroleukemia) cells. The induced HEL cells showed ultrastructurally chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. However, the cells of K562, another human erythroleukemia cell line, did not show such morphological changes. Under fluoroscope, the HEL cells after induction often displayed a clear reduction in nuclear diameter and nuclear chromatin cleavage and condensation and the presence of nuclear ring and apoptotic bodies. Analysis with flow cytometry showed that the percentage of apoptotic cells is about 30-40% after HEL cells were induced by hydroxyurea for 3 days. DNA ladder can be observed by electrophoretic analysis.

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM).

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native environment. Reproducible high-resolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of approximately 15-20 nm, height of approximately 2-3 nm for each individual nucleosome) can be consistently observed. Furthermore, superbeads (width of approximately 40 nm, height of approximately 7 nm) are demonstrated. Visualization of the solenoid conformation at the level of 30 nm chromatin fiber is attained either by using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (approximately 50-60 nm and approximately 90-110 nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.

[Yeast (Saccharomyces cerevisiae) secretory expression vector maintained stably in Pro3+ transformants in rich medium].

A yeast (Saccharomyces cerevisiae) secretory expression vector containing PRO3 gene was constructed (pCBy310). beta HCG(Human choriogonadotropin beta subunit)-cDNA was inserted into pCBy310 to form a recombinant plasmid pCBy314. Since yeast proline auxotroph will not survive in rich medium (YPD), YPD could be used as a selection pressure, and pCBy314 could be maintained mitotically stable in transformants of yeast Pro3- auxotroph (MB299-7A) in rich medium. At an improved, yet not optimized cultural condition, the expression of beta HCG in culture medium was 650 micrograms/L. Our results showed not only that YPD could be used as a selection medium, but also that yeast grew better in YPD so as to increase the gene expression product, and that the component of YPD was simple and cheap. Our data suggested that PRO genes might be used widely in constructing vectors to clone and express foreign genes in yeast so that rich medium can be used as a selection pressure for direct selection.