Self-binding antibodies (autobodies) form specific complexes in solution

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

鲢、鳙在天然条件下的摄食强度(Ⅱ)武汉东湖鲢、鳙周年摄食强度的研究

武汉东湖的鲢(Hypophthalmichthy molitrix)鳙(Aristichthys nobilis)在天然条件下摄食强度具季节性变化。摄食强度高峰处于夏季,低谷处于冬季。在实验条件下,按周年采样期间水温变化范围,测定鱼的肠管排空率。食物通过鱼肠管时间(Y_p—h)与水温(X_t—℃)的关系为: 鲢Y_p=270.63 X_t~(0.6408) 鳙Y_p=280.46 X_t~(0.6642) 根据修正后Bajkov公式(D=C (24.A)/n),估算鱼的日粮。鱼日粮(Y_D)与水温(X_t)关系为: 鲢Y_D=0.2683e~(0.1503X_t) 鳙Y_D=0.0075X_t~(2.2715) 计算鱼在天然条件下周年月粮及年粮。鲢、鳙对天然饵料年消耗量分别为18.924公斤及17.39公斤,饵料系数分别为18.02及13.38。     

Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells

The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.     

Cytochemical properties of osteoblast cell membrane domains

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.     

Drugs of abuse--opiates.

Treating opiate-dependent patients can be difficult for many physicians because the patients'' life-styles, values, and beliefs differ from those of the physicians. Primary care physicians, however, are often involved in the treatment of the medical complications of opiate abuse, and physicians must often manage a patient''s opiate dependence until appropriate referral to a drug abuse treatment program can be arranged. Treatment is guided by an understanding of the patient''s addictive disease, for which there are specific diagnostic criteria, and an understanding of the pharmacology of opiates of abuse and the medications used in treating opiate dependence. The opiate agonist, methadone, is useful for both detoxification and maintenance. The opiate antagonist, naloxone, is the treatment of choice for opiate overdose, and naltrexone, also an opiate antagonist, is a useful adjunct in subgroups of opiate-dependent patients for preventing relapse. New medications for the treatment of opiate dependence are being developed.     

Kinetics of producing pro-urokinase in perfusion cultivations

Summary Better production of pro-urokinase from human cell line was observed with 5% serum containing medium than 10% or serum free medium on Cytodex II under perfusion chemostat operations, showing 0.8×10^–5 (IU/daycell) of maximum productivity at 0.020 (l/h) of dilution rate in 5% serum medium, which corresponds to 800 IU/mL at this dilution rate. Conversion of pro-urokinase was reduced in the serum-containing media.     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.