[Applied research of ultrasound attenuation parameter in the diagnosis of metabolic dysfunction-associated fatty liver disease].

Objective: To evaluate the efficacy, establish a diagnostic model, and value of ultrasound attenuation parameters (UAP) to diagnose hepatic steatosis in metabolic dysfunction-associated fatty liver disease (MAFLD) and its relevant disorders. Methods: 3770 cases were selected from the Health Examination Center of the Third Hospital of Hebei Medical University between October to December 2020. MAFLD diagnosis was based on the Asia-Pacific region MAFLD clinical diagnosis and treatment guidelines. The degree of hepatic steatosis was divided into mild, moderate and severe according to ultrasound imaging. UAP, clinical characteristic indexes, serum biochemical indexes, characteristics of hepatic steatosis and related factors were compared and analyzed in MAFLD patients and healthy controls. Logistic regression method was used to analyze the independent risk factors affecting the progression of hepatic steatosis in MAFLD to establish the diagnostic model. The clinical efficacy of UAP and the new model in diagnosing MAFLD was evaluated by the receiver operating characteristic curve (ROC). One-way ANOVA was used to compare means among multiple groups. Mann-Whitney U test was used to compare non-normally distributed measurement data between the two groups, and rank-sum test was used to compare multiple groups. χ2 test was used to compare count data between groups. Results: Among the 3 770 cases, 650 were MAFLD, with a prevalence rate of 17.24%, and the highest prevalence was 37.23% in the age group of 60-69. The prevalence rate was significantly higher in male than female (30.34% vs. 9.17%). Age-sex analysis showed that the prevalence rate in males aged 30-69 years was 38.26%, and that in females aged over 60 years was 31.94%. UAP was significantly higher in patients with MAFLD than healthy controls (278.55 dB/m vs. 220.90 dB/m, Z=-12.592, P＜0.001), and an increasing trend with increased degree of hepatic steatosis (mild:257.20 dB/m, moderate:286.20 dB/m, and severe: 315.00 dB/m) were observed. The cut-off values of UAP for the diagnosis of mild, moderate and severe hepatic steatosis were 243≤UAP＜258 dB/m, 258≤UAP＜293 dB/m, ≥293 dB/m in MAFLD. The sensitivity and specificity were 67.20%, 93.60%, 95.90%, and 82.10%, 72.00%, and 84.80%, respectively. UAP, alanine aminotransferase and fasting blood glucose were independent risk factors for the progression of hepatic steatosis in MAFLD. The combined MAFLD classification model (UAG model) was established. The AUC of mild, moderate and severe hepatic steatosis in MAFLD were 0.906, 0.907, and 0.946, respectively, and the sensitivity and specificity were 76.50%, 82.10%, 98.00%, and 90.80%, 83.30% and 76.10%, respectively. Conclusion: MAFLD is a common disease in the general population, with a higher incidence in male and elderly female over 30 years of age. UAP can be used as a new noninvasive diagnostic technique to evaluate hepatic steatosis in MAFLD. The UAG model has a good diagnostic efficacy on MAFLD and its relevant disorders, and thus can be used as a guide for evaluating clinical diagnosis and prognosis.

[The correlation between coagulation function and prognosis in patients with acute respiratory distress syndrome caused by extrapulmonary sepsis or pulmonary infection].

Objective: To explore the difference of coagulation function and its correlation with prognosis in patients with acute respiratory distress syndrome (ARDS) caused by extrapulmonary sepsis and pulmonary infection. Methods: ARDS patients caused by extrapulmonary sepsis and pulmonary infection admitted to the ICU were retrospectively analyzed at the First Affiliated Hospital of China Medical University from July 2017 to June 2019. The clinical characteristics were collected including sequential organ failure assessment (SOFA), coagulation parameters [prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), fibrinogen (Fib), D-Dimer (D-D), fibrinogen degradation product (FDP), antithrombin Ⅲ(AT-Ⅲ), platelet (PLT)], duration of mechanical ventilation, length of stay (LOS) in ICU and 28-day mortality. According to the risk factors, the patients were divided into extrapulmonary sepsis group and pulmonary infection group. The correlation analysis between coagulation parameters and the prognosis of ARDS patients were analyzed by multivariate logistic regression analysis. Results: A total of 268 ARDS patients were screened and 28 cases were excluded. Finally, 240 ARDS patients were enrolled, including 145 caused by extrapulmonary sepsis and 95 by pulmonary infection. PT, INR and APTT in the extrapulmonary sepsis group were significantly higher than those in pulmonary infection group (P<0.05). AT-Ⅲ level was lower than that in pulmonary infection group (P<0.05). Ninty-three patients survived at 28 days in the non-pulmonary sepsis group, the mortality rate was 35.9% (52/145). PT, INR, APTT in patients who died at 28 days were significantly higher than those of the patients who survived (P<0.05), while AT-Ⅲ level was lower than those of the patients who survived (P<0.05). 49 patients survived at 28 days in the pulmonary infection group, the mortality rate was 48.4% (46/95). There was no significant difference in the coagulation parameters between two groups. Multivariate logistic regression analysis showed that SOFA score without PLT(OR=1.210,95%CI 1.067-1.372,P=0.003) and INR (OR=2.408,95%CI 1.007-5.760,P=0.048) were independent risk factors for 28-day mortality in extrapulmonary sepsis group. Coagulation parameters are not independent risk factors for 28-day mortality in ARDS patients related to pulmonary infection. Conclusion: There are significant differences in coagulation function between ARDS patients caused by extrapulmonary sepsis or pulmonary infection. INR is an independent risk factor for 28-day mortality in extrapulmonary sepsis group.

[The value of brachial artery peak velocity variation during the Valsalva maneuver to predict fluid responsiveness].

Objective: To evaluate whether brachial artery peak velocity variation(ΔVp) during a Valsalva maneuver(VM) could predict fluid responsiveness in spontaneously breathing patients. Methods: Ninety-six patients required radial artery catheter for elective surgery of Ningbo Yinzhou People's Hospital from December 2014 to June 2016 were enrolled. The brachial artery Doppler signal was recorded to measure the ΔVp while the VM was performed.Then doing the volume expansion (VE) , the cardiac output variation (ΔCO) before and after VE were measured.Pearson correlational analyses were conducted between ΔVp and ΔCO. Also the sensitivity and specificity of ΔVp were determined in predicting fluid responsiveness by the receiver operating characteristic (ROC) curve. Results: Patients were classified as group responders (n=24) and group non-responders (n=72). Responder was defined as cardiac output increased≥15% after VE.The ΔVp correlated well with ΔCO (r=0.792, P<0.01). The area under ROC curve was 0.903, with the ΔVp cut-off of 33%, the sensitivity of 87% and the specificity of 82%(P<0.01). Conclusion: Brachial artery peak velocity variation during a valsalva maneuver is a feasible method for predicting fluid responsiveness in spontaneously breathing patients.

[Treatment strategies of complex lesions in patients with acute Stanford type A dissection of important branches involvement].

Acute Stanford type A aortic dissection with important branches involved is more complex, could lead to organ malperfusion syndrome even organ failure. The understanding of pathological anatomy, classification, staging, and the pathophysiological change has increasingly mature, but not complete. In addition, the treatment strategy for complex lesions is diversified, some questions may not reach consensus. Fully understanding of the anatomical and pathophysiology is very important for surgeons to choose reasonable treatment strategy. As the rapid development of the basic research, imaging techniques and the concept of surgery procedures, the manage technique of Stanfrod type A dissection and branch vessels at the same time is getting seriously, the related issues also need further discussions.

Investigation of genes in chronic and acute morphine-treated mice using microarray datasets.

Morphine is a psychoactive medication that is used as a standard analgesic treatment to relieve pain in clinics. Many patients rely on chronic or acute treatment of morphine to treat pain. However, morphine is a narcotic and has a reverse effect when inappropriately used. Therefore, it is necessary to study chronic and acute morphine treatment to improve pain relief. In this study, differentially expressed genes of acute and chronic morphine-treated mice were identified using Array Express datasets. The genes that were associated with these two types of morphine treatment are discussed. A co-expression network was constructed, and the hub genes were identified. Gene ontology enrichment analysis and pathway analysis were performed using the Gene Ontology website and Kyoto Encyclopedia of Genes and Genomes, respectively. Our study revealed genes that are associated with acute and chronic morphine treatment. Therefore, this study is potentially useful for improving pain relief of patients by acute and chronic morphine treatment.

Atherogenic dyslipidaemic profiles associated with the development of Type 2 diabetes: a 3.1-year longitudinal study.

AIMS: While there is thought to be an association between glucose and lipid metabolism, it is largely unknown whether apolipoprotein B and non-high density lipoprotein (HDL) cholesterol are associated with the development of Type 2 diabetes. It is also unknown whether these atherogenic dyslipidaemic profiles have a stronger association with diabetes risk compared with conventional lipid measurements. METHODS: A total of 118 429 subjects without diabetes (70 980 men and 47 449 women), aged 17-90 years (mean age 39.6 years), were enrolled in this study and followed for a mean duration of 3.1 years. RESULTS: Apolipoprotein B and non-HDL cholesterol levels showed a strong association with the development of Type 2 diabetes compared with conventional lipid measurements and their ratios [hazard ratio per 1 sd; 1.39 (95% CI 1.37-1.42) and 1.38 (95% CI 1.35-1.40), respectively; both P < 0.001]. The Kaplan-Meier survival curve demonstrated that Type 2 diabetes developed more frequently as apolipoprotein B or non-HDL cholesterol levels increased across quartiles (both P < 0.001). In multivariate Cox regression analyses, both apolipoprotein B and non-HDL cholesterol were associated with the development of Type 2 diabetes, independent of other risk factor including age, sex, waist circumference, family history of diabetes, fasting serum glucose and insulin levels, HbA1c , systolic blood pressure and other conventional lipid measurements [hazard ratio per 1 sd; 1.14 (95% CI 1.11-1.18) and 1.13 (95% CI 1.10-1.16), respectively; both P < 0.001]. CONCLUSIONS: Atherogenic dyslipidaemia was more strongly associated with the development of Type 2 diabetes than conventional lipid measurements, and this effect was independent of other well-established risk factor for diabetes.

Induction of cell cycle arrest and apoptosis by tomentosin in hepatocellular carcinoma HepG2 and Huh7 cells.

Tomentosin, a sesquiterpene lactone, is known to possess various biological activities. However, its anticarcinogenic activity against human hepatocellular carcinoma (HCC) cells has not been investigated in detail. Thus, this study aimed to elucidate the cytotoxic mechanism of tomentosin in human HCC cell lines HepG2 and Huh7. WST-1, cell counting, and colony formation assay results showed that treatment with tomentosin decreased the viability and suppressed the proliferation rate of HepG2 and Huh7 cells in a dose- and time-dependent manner. Cell cycle analysis revealed increased population of cells at the SubG1 and G2/M stage, and decreased population of cells at the G0/1 stage in HepG2 and Huh7 cells treated with tomentosin. Annexin V/propidium iodide double staining and TUNEL assay results showed increased apoptotic cell population and DNA fragmentation in HepG2 and Huh7 cells treated with tomentosin. Western blotting analysis results showed that tomentosin treatment significantly increased the expression level of Bax, Bim (short form), cleaved PARP1, FOXO3, p53, pSer15p53, pSer20p53, pSer46p53, p21, and p27, but decreased the expression of Bcl2, caspase3, caspase7, caspase9, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclinB1, cyclinD1, cyclinD2, cyclinD3, and cyclinE in a dose-dependent manner. Taken together, this study revealed that tomentosin, which acted through cell cycle arrest and apoptosis, may be a useful therapeutic option against HCC.

Grain Discoloration of Rice Caused by Pantoea ananatis (synonym Erwinia uredovora) in China.

In the autumn of 2008, a new bacterial disease of rice was noted in paddy fields near Hangzhou, Zhejiang Province, China. The disease caused severe discoloration of rice grains on cv. Zhong-zhe-you 1 (Oryza sativa L.). It often occurred at early flowering of hybrid rice. Initially, light, rusty, water-soaked lesions appeared on the lemma or palea and then turned brown. More immature and lighter grains were observed on panicles at harvest. No bacterial ooze was observed. Ten bacterial isolates were recovered from eight samples of discolored rice grains (1). Six isolates were selected for identification. They were similar to those of the reference strain of Pantoea ananatis (Serrano, synonym Erwinia uredovora) LMG 2665T (ATCC 33244) from Belgium in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with the aerobic bacterial library (TAB 5.0), and electron microscopy (TEM,KYKY-1000B, Japan). All isolates were facultatively anaerobic, gram-negative rods that measured 1.6 to 2.5 × 0.5 to 0.7 µm and had three to six peritrichous flagella. Colonies on nutrient agar were yellow and raised with smooth margins. A hypersensitive reaction was observed on tobacco (Nicotiana tobacum cv. Benshi) 24 h after inoculation. All isolates were identified as P. ananatis with Biolog similarity indices of 0.716 to 0.852 and FAME similarity indices of 0.783 to 0.903. Further identification as P. ananatis was done by 16S rDNA sequence analysis. Amplicons were produced from three strains using the universal primers (3) fD2: 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer and rP1: 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer and then sequenced (GenBank Accession Nos. GU324769, GU324770, and GU338399). A BlastN search of GenBank revealed that they had 97 to 98% nt identity with P. ananatis strain 3Pe76 (GenBank Accession No. EF178449). Koch's postulates were completed by spray inoculating panicles of rice cv. Zhong-zhe-you 1 at booting stage, grown in pots, with cell suspensions containing 108 CFU/ml of the six strains at 25 to 29°C. Three plants were inoculated with each strain, controls were sprayed with water, and the experiment was repeated once. Three weeks after inoculation, all strains produced symptoms on panicles similar to those observed in the field. Yellow pigmented bacteria were reisolated from symptomatic panicles and their identity was confirmed by FAMEs. These results indicate that the pathogen is P. ananatis (2), which also causes leaf blight and bulb decay of onion. To our knowledge, this is the first report of rice grain discoloration caused by P. ananatis in China. The disease cycle on rice and the control strategies in the regions are being further studied. References: (1) J. Y. Luo et al. Plant Dis. 91:1363, 2007. (2) H. G. Truper and L. de Clari. Int. J. Syst. Bacteriol. 47:908, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Maresin 1 Promotes Wound Healing and Socket Bone Regeneration for Alveolar Ridge Preservation.

Tooth extraction results in alveolar bone resorption and is accompanied by postoperative swelling and pain. Maresin 1 (MaR1) is a proresolving lipid mediator produced by macrophages during the resolution phase of inflammation, bridging healing and tissue regeneration. The aim of this study was to examine the effects of MaR1 on tooth extraction socket wound healing in a preclinical rat model. The maxillary right first molars of Sprague-Dawley rats were extracted, and gelatin scaffolds were placed into the sockets with or without MaR1. Topical application was also given twice a week until complete socket wound closure up to 14 d. Immediate postoperative pain was assessed by 3 scores. Histology and microcomputed tomography were used to assess socket bone fill and alveolar ridge dimensional changes at selected dates. The assessments of coded specimens were performed by masked, calibrated examiners. Local application of MaR1 potently accelerated extraction socket healing. Macroscopic and histologic analysis revealed a reduced soft tissue wound opening and more rapid re-epithelialization with MaR1 delivery versus vehicle on socket healing. Under micro-computed tomography analysis, MaR1 (especially at 0.05 µg/µL) stimulated greater socket bone fill at day 10 as compared with the vehicle-treated animals, resulting in less buccal plate resorption and a wider alveolar ridge by day 21. Interestingly, an increased ratio of CD206+:CD68+ macrophages was identified in the sockets with MaR1 application under immunohistochemistry and immunofluorescence analysis. As compared with the vehicle therapy, local delivery of MaR1 reduced immediate postoperative surrogate pain score panels. In summary, MaR1 accelerated extraction wound healing, promoted socket bone fill, preserved alveolar ridge bone, and reduced postoperative pain in vivo with a rodent preclinical model. Local administration of MaR1 offers clinical potential to accelerate extraction socket wound healing for more predictable dental implant reconstruction.

Effect of Saccharomyces cerevisiae fermentation product on immune functions of broilers challenged with Eimeria tenella.

Three hundred sixty 1-d-old male Arbor Acres broilers were randomly allotted to 6 groups with a 2x3 factorial arrangement of treatments. Three supplemental levels (0, 0.25, and 0.50%) of Saccharomyces cerevisiae fermentation product (XP) were fed to control and Eimeria tenella-infected broilers. Growth performance and immune response criteria were measured after coccidian infection. Broiler ADG was lowered (P<0.01) by coccidian infection and improved by XP supplementation (P=0.04). Supplementation of XP increased CD3+, CD4+, and CD8+ T-lymphocyte counts (P<0.05) and the ratio CD4+:CD8+ in blood (P=0.06) and spleen (P=0.04) as well as ileum intraepithelial lymphocyte count, cecal tonsil secretory IgA counts, serum lysozyme content (P<0.01), and albumin:globulin ratio (P=0.02). These results suggest that dietary XP supplementation could improve immune function and growth performance in coccidia-infected broilers.

Bacterial Stem Rot of Poinsettia Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in China.

In December 2006, a rot symptom of unknown etiology was observed on stems of plants (Euphorbia pulcherrima cv. Fu-xing) at a flower nursery in the Zhejiang Province of China where we had previously reported leaf spot of poinsettia caused by Xanthomonas campestris (2). Chlorotic spots anywhere along the stem and purplish black petioles were the first noticeable symptoms. The spots rapidly coalesced, forming large irregular chlorotic areas. Petioles turned black and shriveled and affected leaves wilted. Infected tissues were soft and water soaked. Ten bacterial strains were isolated from the diseased samples and five were selected for identification. They were similar to those of the standard reference strains of Pectobacterium chrysanthemi (Dickeya sp.), LMG 2804 from Belgium and ZUPB20056 from China, in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with aerobic bacterial library (TABA50), and transmission electron microscopy (TEM,KYKY-1000B, Japan). All strains tested were gram-negative facultative anaerobic rods measuring 1.5 to 3.6 × 0.6 to 1.1 µm, with peritrichous flagella. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They were negative for trehalose and positive for phosphatase production and reducing substances from sucrose. A hypersensitive reaction was observed on tobacco cv. Benshi, 24 h after inoculation. All five isolates, LMG 2804, and ZUPB20056 were identified as P. chrysanthemi (Dickeya sp.) with a Biolog similarity index of 0.58 to 0.83, 0.68, and 0.72 and a FAME similarity index of 0.52 to 0.80, 0.59, and 0.70, respectively. Identification as P. chrysanthemi (Dickeya sp.) was confirmed by PCR with specific primers used by Nassar et al (3). Koch's postulates were completed with the inoculation of 12 4-month-old intact poinsettia plants of cv. Fu-xing with cell suspensions containing 108 CFU/ml by a pinprick at the base of the stem. All five strains induced stem infection similar to those observed in natural infections. No symptoms were noted on the two control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of poinsettia plants. P. chrysanthemi (Dickeya sp.) was first reported in United States as the cause of bacterial stem rot of poinsettia in 1972 (1). To our knowledge, this is the first report of poinsettia stem rot caused by P. chrysanthemi (Dickeya sp.) in China. The disease cycle and the control strategies of the bacterial stem rot of poinsettia in the regions are being further studied. References: (1) H. A. J. Hoitink et al. Plant Dis. Rep. 56:480, 1972. (2) B. Li et al. Plant Pathol. 55:293, 2006. (3) A. A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996.

Effects of yeast culture in broiler diets on performance and immunomodulatory functions.

A study was conducted to evaluate the effect of supplemental yeast culture (Diamond V XP Yeast Culture; YC) in broiler diets on performance, digestibility, mucosal development, and immunomodulatory functions. One-day-old Arbor Acres chicks (n = 960) were randomly assigned to 1 of 4 dietary treatments based on corn and soybean meal and containing 0, 2.5, 5.0, and 7.5 g/kg of YC in the diet for 42 d. Each treatment consisted of 12 replicates of 20 broilers each. Nutrient digestibility was determined on d 15 and 35 by total fecal collection. On d 21 and 42, 12 birds per treatment were sacrificed to evaluate gut morphology and secretory IgA. Broilers were vaccinated with Newcastle disease vaccine by eye drop on d 7 and 28 and antibody titer was determined on d 14, 21, 35, and 42. Dietary supplemental YC at 2.5 g/kg improved average daily gain and feed conversion during grower and overall periods (P 0.05) protein retention and energy digestibility. Villus height to crypt depth ratios in the duodenum and jejunum (d 42) and ileum (d 21) were increased (P

[Emotional and behavioral problems associated with sleep problems in preschool aged children].

Objective: To examine whether sleep problems are related to both emotional and behavioral problems in children aged 3-6 years. Methods: A large cross-sectional study was conducted in Anqing, Wuhu, Tongling and Yangzhou from March to June 2015. A total of 8 900 preschool aged children were included. Sleep problems were obtained by using adapted BISQ completed by the parents or the people who took care of children. Emotional and behavioral problems of the children were accessed by using Strengths and Difficulties Questionnaire (SDQ), and multivariate logistic regression model was used for statistical analyses. Results: The detected rates of emotional symptoms, conduct problems, hyperactivity problems, peer problems, total difficulties and prosocial behavior in preschool aged children were 9.0%, 13.9%, 18.9%, 25.5%, 13.6% and 16.2% respectively. All the detected rates were higher in boys than in girls except the higher rate of emotional symptoms. The proportions of children with high sleep quality, moderate sleep quality and poor or worse sleep quality were 3.9%, 52.9% and 43.2% respectively. After controlling the confounding factors of demographic variables, including gender, age, delivery mode, birth weight, birth height and patent's educational level, multinomial logistic regression analysis showed that the risk of emotional symptoms, conduct problems, hyperactivity problems, peer problems, total difficulties and prosocial behavior in children with longer sleep duration was lower than that in children with shorter sleep duration, the ORs were 0.86 (95%CI: 0.77-0.95), 0.85 (95%CI: 0.78-0.93), 0.85 (95%CI: 0.79-0.92), 0.87(95%CI: 0.81-0.93), 0.83 (95%CI: 0.76-0.91) and 0.82 (95%CI: 0.76-0.89) respectively. Compared with the children with good sleep quality, the risk of emotional symptoms, conduct problems, hyperactivity problems, peer problems, total difficulties and prosocial behavior were higher in children with poor or worse sleep quality, the ORs were 3.26 (95%CI: 2.40-4.42), 2.86 (95%CI: 2.16-3.78), 2.60 (95%CI: 2.00-3.38), 1.96 (95%CI: 1.52-2.54), 4.02 (95%CI: 3.06-5.27) and 2.56 (95%CI: 1.96-3.35) respectively. Conclusion: There was a negative impact of shorter sleep and poor or worse sleep on emotional and behavioral problems of preschool aged children.

Characterization of high- and low-metastatic clones derived from a methylcholanthrene-induced murine fibrosarcoma.

A methylcholanthrene-induced fibrosarcoma (3AM) and several of its clones were evaluated for pulmonary metastasis, growth rate, and chromosome composition. Heterogeneity was observed in the three parameters, and no correlation was found between growth rate and metastatic potential. Furthermore, three clones (10, 34, and 27) were identified with distinctive, high or low, metastatic potential and marker chromosomes. The marker chromosomes characteristic for each clone were identified in the early passages of the parental 3AM line, indicating the preexistence of the different cell types in the original neoplasm. The three clones were then characterized as to tendency to adhere to vascular endothelium, immunogenicity, and antigenic specificity. Clone 10, with two large metacentric markers (T2, 4 and T10, 15) and the highest metastatic potential (221 foci/lung), expressed the highest endothelial attachment and immunogenicity. Clone 27 was characterized with an extremely low rate of metastasis (nine foci/lung) and a T2, 7 large acrocentric marker, while clone 34 was characterized with a moderate rate of metastasis (107.5 foci/lung) and a T4, 16 acrocentric marker. Antigenically, clone 10 cross-reacted with clones 34 and 27 and 3AM, while clones 27 and 34 cross-reacted with clone 10 and 3AM but not with each other, suggesting that, within the original tumor, there were common tumor antigens shared by some cells but no universal antigen shared by all cells.

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant.

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)

Adsorption, compression and stability of surface films from natural, lipid extract and reconstituted pulmonary surfactants.

A pulsating bubble surfactometer was used to study the surface activities and surface film stabilities of bovine pulmonary surfactants (10 mg/ml) and a reconstituted surfactant (10 mg/ml). Pulmonary surfactants were natural surfactant (NS), lipid extract surfactant [LES(chol)] and lipid extract surfactant without neutral lipids (LES). NS is composed of phospholipids, neutral lipids and surfactant-associated proteins (SP-A, SP-B and SP-C). Both LES(chol) and LES are organic solvent extracts of NS. LES(chol) retains all the components of NS except SP-A. Reconstituted surfactant was dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG): SP-B/7:3:1%. All three pulmonary surfactants attained the equilibrium surface tension almost instantaneously at 37 degrees C. The adsorption rates of NS and LES(chol) at 24 degrees C were similar to those at 37 degrees C, while LES exhibited a lower adsorption rate at 24 degrees C. Reconstituted surfactant adsorbed slower than any of the pulmonary surfactants. Film stability was studied by recording the spontaneous increase in the pressure gradient of a static bubble at the minimum size (Rmin) once near zero surface tension was attained. The order of surface film stabilities were: reconstituted surfactant > > NS > LES > LES(chol). Surface films of NS and LES could be stabilized by prolonged pulsation, while film stability of LES(chol) was only moderately affected by pulsation. These results indicate that SP-A in NS promotes formation of some unique structure, possibly tubular myelin, which induces selective adsorption of lipids into the surface.

Effect of pulmonary surfactant protein B (SP-B) and calcium on phospholipid adsorption and squeeze-out of phosphatidylglycerol from binary phospholipid monolayers containing dipalmitoylphosphatidylcholine.

The pulsating bubble technique was used to study the surface activity of binary phospholipid mixtures containing dipalmitoyl-phosphatidylcholine (DPPC) and an unsaturated acidic phospholipid such as egg phosphatidylglycerol (egg PG), 1-palmitoyl-2-oleoyl-PG (POPG) or egg phosphatidic acid (egg PA) in the presence of surfactant-associated protein B (SP-B) and calcium. The relative surface activities were DPPC/egg PG/SP-B (7:3:1%) greater than DPPC/POPG/SP-B (7:3:1%) greater than DPPC/egg PA/SP-B (7:3:1%). The Wilhelmy surface plate technique was utilized to investigate the interaction between pure SP-B in the bulk phase (0.9% NaCl/1.5 mM CaCl2) and preformed DPPC or phosphatidylglycerol (PG) monolayers. Although SP-B injected into the bulk phase reduces the surface tension of a clean surface, no evidence was obtained for the insertion of SP-B into surface monolayers at equilibrium surface tension. Surface radioactivity measurements and the Wilhelmy surface plate technique were also used to study the potential interactions between liposomes of DPPC/POPG (7:3) with or without SP-B and surface monolayers of [14C]DPPC or [14C]POPG. No exchange of phosphatidylcholine (PC) or PG was found between the monolayer and liposomes. We also compared the adsorption of pure POPG or 1-palmitoyl-2-oleoyl- phosphatidylcholine (POPC) and binary mixed liposomes with DPPC in the presence or absence of SP-B and calcium. DPPC/POPG/SP-B (7:3:1%) was found to be more surface active than pure POPG plus 1% SP-B in the presence of calcium. Injection of SP-B into the bulk phase promoted the adsorption of DPPC/POPG liposomes to a greater extent than POPG liposomes. The enhanced adsorption was dependent on the presence of calcium. In contrast to PG, DPPC/POPC/SP-B (7:3:1%) was less surface active than pure POPC plus 1% SP-B either in the presence or absence of calcium. Our findings suggested that the molecular composition and organization of mixed monolayers play an important role in the surface activity of the surfactant.

The interaction of nonhistone chromosomal proteins HMG1 and HMG2 with subfractions of H1 histone immobilized on agarose.

Chromatographically isolated subfractions of calf thymus H1 histone have been covalently coupled to agarose beads and tested for their ability to form complexes with the non-histone proteins HMG1 and HMG2 (High Mobility Group proteins, Walker, J.M., Goodwin, G.H. and Johns, E.W. (1976) Eur. J. Biochem. 62, 461-469). When a mixture of HMG1 and HMG2 is passed through a column of H1 histone-agarose, the HMG2 does not bind. HMG1 does bind and can be eluted from the column with NaCl in the range of 0.05 M--0.15 M. The NaCl concentration required to elute HMG1 from each of the three H1 histone subfraction coulmns is different, suggesting that HMG1 has a different binding affinity for the three H1 histone subfractions.

Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures.

The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.