EFFECT OF VL AND VH CONSENSUS SEQUENCE-SPECIFIC PRIMERS ON THE BINDING AND EXPRESSION OF A MINI- MOLECULE ANTIBODY DIRECTED TOWARDS HUMAN GASTRIC CANCER

Objective. To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody(mAb) 3H11.Methods. At first,3H11 ScFv and Fab were constructed with Ⅴ genes PCR amplified by degenerate primers for FR1 .The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybfidoma cells and panning selection was performed. Again the i-dentification of positive clone was failed. Finally the N-terminal sequences of Ⅴ regions were resumed to 3H11 original sequences by site-directed mutagenesis via PCR.Restdts. Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab, though the expression of the Ab fragments was not affected. Correction of either VL or VH N-terminal se-quences could partially resume the antigen binding activity. Conclusion. Sequence changes of Ⅴ region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.     

Disease type-associated increases of the plasma levels and ligand expression for natural alpha- or beta-galactoside-binding immunoglobulin G subfractions in patients with lung cancer

The presence of natural carbohydrate-binding antibodies may play a role in host defence against malignant cells in addition to elicitation of an immune response by artificial carbohydrate antigens. Human serum contains immunoglobulin G(2) (IgG) fractions with selectivity to alpha- and to beta-galactosides, respectively, irrespective of the type of blood group of the donor. To determine whether these naturally occurring subfractions may have any relevance for tumor disease control, their binding to malignant cells was ascertained by cytofluorimetric assays in vitro with a number of human tumor cell lines of different histogenetic origin. The affinity of cell binding was comparable to that of binding to lactosylated or melibiosylated neoglycoconjugates as model ligands in solid-phase assays and K-D values were found to be in the range of 5-300 nM. Cross-reactivity of the anomer-selective subfractions to the other type of ligand was observed to be rather low. When the IgG contents of plasma samples of patients with diverse types of lung cancer were assessed, the concentrations of both galactoside-binding immunoglobulin G subfractions were significantly increased in association with presence of small cell lung carcinoma and of metastatic lesions to the lung without any marked change in the overall IgG plasma level. Such an apparently general enhancement was seen for patients with adenocarcinoma and included both subfractions with no impact on their percentage in the total IEC content. When detergent extracts of tumor and tumor-free specimens of the same patient were analyzed with the affinity purified antibody subfractions to comparatively determine ligand presentation, increases in sugar-inhibitable binding were especially noted for the tumor tissue of small cell lung carcinomas and apparently tumor-free samples of cases with lung metastasis. Material from other types of lung cancer revealed no significant indication for disease-related alterations with the exception of carcinoids. These data demonstrate that plasma levels and ligand expression for two types of natural galactoside-binding immunoglobulin G fractions can show nonuniform responses in patients within the class of lung cancer. They encourage to deliberately monitor these parameters of the natural carbohydrate-directed antibody fractions in cancer patients with various types of disease to clarify the clinical significance of respective malignancy-associated changes.     

Effect of hepatic cirrhosis on the pharmacokinetics of theophylline in rats

The experimental hepatic cirrhosis was induced either by bile duct ligation (BDL) or by pretreatment with dimethylnitrosamine (DMNA). The pharmacokinetics of theophylline were studied after a single intravenous or a single oral administration. Using the ultrafiltration method, protein-drug binding experiments were also carried out. The bilirubin level was several-fold increased by BDL, but not by DMNA treatment. The albumin content was decreased in both cirrhotic groups. The total clearance (Clt, ml/kg/hr) of theophylline in both hepatic cirrhosis groups significantly decreased and the terminal half-life (t_1/2) in the cirrhotic rats was increased about two-fold after intravenous and oral administration. The volume of distribution at steady state (Vdss, ml/kg) was increased slightly in the cirrhotic groups. Protein binding in BDL (8.67±4.85%) decreased about four-folds, but in DMNA (73.00±9.85%) similar result, was observed as compared with the control. Increased free fraction of theophylline did not increase the volume of distribution in BDL. Therefore decreased total body clearance of theophylline was mainly due to decreased intrinsic clearance of theophylline in the liver. The absolute bioavailability of theophylline in these experiments was between 63.8 and 72.8%(66.1% in BDL, 63.8% in Sham operated and Control, 72.8% in DMNA). These results suggest that in the experimental hepatic cirrhosis model, administration route does not affect the disposition of theophylline.     

Determination of microviscosity and location of 1,3-Di(1-pyrenyl)propane in brain membranes

We determined the microviscosity of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex and liposomes of total lipids (SPMTL) and phospholipids (SPMPL) extracted from SPMV. Changes in the microviscosity induced by the range and rate of lateral diffusion were measured by the intramolecular excimerization of 1,3-di(1-pyrenyl)-propane (Py-3-Py). The microviscosity values of the direct probe environment in SPMV, SPMTL and SPMPL were 38.17, 31.11 and 27.64 cP, respectively, at 37°C and the activation energies (E_a) of the excimer formation of Py-3-Py in SPMV, SPMTL and SPMPL were 8.236, 7.448 amd 7.025 kcal/mol, respectively. Probe location was measured by polarity and polarizability parameters of the probe Py-3-Py and probe analogues, pyrene, 1-pyrenenonanol and 1-pyrenemethyl-3β-hydroxy-22,23-bisnor-5-cholenate (PMC), incorporated into membranes or solubilized in reference solvents. There existed a good linear relationship between the first absorption peak of the¹L_a band and the polarizability parameter (n ²−1)/(2n ²+1). The calculated refractive index values for SPMV, SPMTL and SPMPL were close to 1.50, which is higher than that of liquid paraffin (n=1.475). The probe location was also determined by using a polarity parameter (f−1/2f¹). Here f=(ε−1)/(2ε+1) is the dielectric constant function and f'=(n ²−1)/(2n ²+1) is the refractive index function. A correlation existed between the monomer fluorescence intensity ratio and the solvent polarity parameter. The probes incorporated in SPMV, SPMTL, and SPMPL report a polarity value close to that of 1-hexanol (ε=13.29). In conclusion, Py-3-Py is located completely inside the membrane, not in the very hydrophobic core, but displaced toward the polar head groups of phospholipid molecules, e.g., central methylene region of aliphatic chains of phospholipid molecules.     

Current Status of the VU MFEL Compton X-Ray Program

The Vanderbilt University medical FEL (free electron laser) Compton x-ray program is close to being operational. The FEL modifications necessary for this new capability are near completion. The transport and detection systems for electron and IR beams have been designed, delivered, and tested. We initially expect to produce 108 x-ray photons per second in the 15- to 20-keV region.     

Hydrolysis kinetics of metampicillin by high performance liquid chromatography

The hydrolysis of metampicillin to ampicillin was investigated using high performance liquid chromatography. We developed the simultaneous determination of metampicillin and ampicillin using a Zorbax CN column and 5% acetonitrile and 8% methanol in 0.02 M phosphate buffer (pH 7.0) as mobile phase. Metampicillin was hydrolyzed to ampicillin with half life of 41.5 min at physiological pH and temperature. In acidic pH, metampicillin was rapidly hydrolyzed to ampicillin within a chromatographic separation.     

Epimerization and Hydrolysis of Dalvastatin,a New Hydroxymethylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitor

In aqueous solutions, dalvastatin (1) undergoes epimerization as well as hydrolysis. The transformation of the drug was studied as a function of pH at 25°C in aqueous solutions containing 20% acetonitrile. At all pH values, first-order plots for the conversion are biphasic, indicating rapid equilibration of 1 with its epimer (2) and slower hydrolysis of 1 to the corresponding -hydroxy acid (3). Apparent first-order rate constants for the biexponential equation are given as a function of pH. The alkyl–oxygen cleavage of the lactone ring results in the epimerization of 1 to 2, whereas the acyl–oxygen cleavage results in the hydrolysis of 1 to 3. The epimerization is an S_N1 reaction reaching an equilibrium of [l] _eq/[2] _eq = 1.27. The epimerization rate is increased with an increase in the water content of the solvent. The hydrolysis of 1 to 3 is acid and base catalyzed. The hydrolysis is reversible in acidic media and irreversible in neutral and basic media. At pH values greater than 9, the hydrolysis reaction proceeds more rapidly than the epimerization.     

上海市50岁及以上人群维生素D水平与握力的关联研究

目的 探讨上海市≥50岁人群维生素D水平与握力的关系。方法 数据来源于WHO全球老龄化与成人健康研究我国上海市2018-2019年数据,采用logistic回归模型分析维生素D水平与握力的关系,进一步按照性别、年龄及乳制品摄入情况进行分层;采用限制性立方样条曲线绘制维生素D水平与低握力的剂量-反应曲线。结果 共4 391人纳入研究,其中男性2 054人（46.8%）;年龄（67.02±8.81）岁;低握力1 421人（32.4%）;维生素D不足及缺乏分别为1 533人（34.9%）和401人（9.1%）。在调整相关混杂因素后,logistic回归分析结果显示,维生素D缺乏的人群发生低握力的风险更高（OR=1.41,95%CI：1.09~1.83）;在男性中,调整相关混杂因素后,维生素D缺乏与低握力发生风险呈显著正相关（OR=1.67,95%CI：1.12~2.50）,而女性中两者之间无关联（OR=1.30,95%CI：0.97~1.74）;在60~69岁及≥80岁年龄组中,调整相关混杂因素后,维生素D缺乏与低握力发生风险呈显著正相关（OR=1.57,95%CI：1.05~2.35;OR=2.40,95%CI：1.08~5.31）,在乳制品摄入<250 ml/d的人群中,调整相关混杂因素后,二者之间呈显著正相关（OR=1.57,95%CI：1.17~2.09）,而在乳制品摄入≥250 ml/d的人群中无明显关联。限制性立方条样图显示,低握力的发生风险可能随维生素D含量的上升而降低,但差异无统计学意义（P>0.05）。结论 维生素D水平与握力存在一定的关系,维生素D缺乏人群出现低握力的风险更高。