Characterization of primar cell cultures derived from rat renal proximal tubules

Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E₁. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D₃-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na⁺-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron. This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC.     

The atokous-epitokous border is determined before the onset of heteronereid development in Platynereis dumerilii (Annelida,Polychaeta)

Summary In most nereids sexual maturation is accompanied by a dramatic reorganization of the body that enables swarming of the formerly benthic worms. However, a border exists between unchanged anterior (atokous) and metamorphosed posterior (epitokous) segments. The site of this atokous-epitokous border (a/e border) is different in sexually mature males and females of Platynereis dumerilii. There is no correlation between the total number of setigerous segments of a specimen and the location of the a/e border. The location of the a/e border and sexual development are affected neither by cutting off caudal segments of juveniles (including the prospective a/e border) nor by transecting the ventral nerve cord. When parapodia are transplanted from prospective epitokous regions to prospective atokous regions and vice versa, they maintain their original character during metamorphosis. The results presented here suggest that prospective atokous as well as epitokous characters are determined at or only very shortly after formation of the respective segments. Thus the a/e border is established well in advance of the onset of epitokous metamorphosis.     

Anti-idiotypic monoclonal antibody to a T-cell chronic lymphatic leukemia

Summary A murine anti-idiotypic monoclonal antibody (mAb), F1, (IgG_2a) was produced against the variable part of the T-cell receptor for antigen (T_i, /) on the tumor cells of a patient with T-cell chronic lymphatic leukemia (CD3⁺, 8⁺, 4^–). The molecular weight of the protein reactive with mAb F1, comodulation and coprecipitation with anti-CD3 antibody, and the restricted tumor-cell reactivity strongly support the anti-idiotypic nature of mAb F1. MAb F1 also stained 4% of peripheral blood lymphocytes of healthy donors. MAb F1 did not stimulate the tumor cells to DNA synthesis, but stimulated a fraction of the normal peripheral blood lymphocytes, mAb F1 did not mediate antibody-dependent cellular cytotoxicity or complement lysis to any significant degree in vitro. Three infusions of 1–10 mg anti-idiotypic mAb were given over a period of 4 weeks. The plasma half-life for mAb F1 was 3 h in the first 2 h after infusion and 44 h from 2 h to 120 h after infusion. After each treatment a rapid decrease of circulating tumor cells was seen. During the observation period an 80% reduction of the total circulating tumor cells was noted. After the second infusion, IgM and IgG antimouse antibodies were detected. Side-effects from therapy were fever, chills, nausea, vomiting, diarrhea, tachycardia, increase in systolic blood pressure and shortness of breath. Thus, in T-cell malignancies a major reduction of circulating tumor cells can be accomplished by low doses of anti-idiotypic mAb. Anti-idiotypic mAb might be a therapeutic agent of significant importance.     

Structure of the hirulog 3-thrombin complex and nature of the S' subsites of substrates and inhibitors.

The X-ray crystallographic structure of the human alpha-thrombin complex with hirulog 3 (a potent, noncleavable hirudin-based peptide of the "hirulog" class containing a beta-homoarginine at the scissile bond), which is isomorphous with that of the hirugen-thrombin crystal structure, was solved at 2.3-A resolution by starting with a model for thrombin derived from the hirugen-thrombin complex and was refined by restrained least squares methods (R = 0.132). Residues of hirulog 3 were well-defined in the electron density, which included most of the pentaglycine linker and the C-terminal helical turn that was disordered in a related structure of thrombin with hirulog 1. The interactions of D-Phe1'-Pro2'-beta-homoArg3' with the active site of thrombin were essentially identical to those of related structures of PPACK- (D-Phe-Pro-Arg chloromethyl ketone) and hirulog 1-thrombin, with the guanidinium function of the arginyl P1 residue forming a hydrogen-bonding ion pair with Asp189 of the S1 site. A noticeable shift in the CA atom of beta-homoArg3' due to the methylene insertion displaces the scissile bond from attack by Ser195, thus imparting proteolytic stability to the beta-homoArg hirulog derivative. Resolution of the pentaglycine spacer, linking N- and C-terminal functional domains into a single oligopeptide bivalent inhibitor, permitted delineation of corresponding S' subsites of thrombin. The position of Gly4' (P1') is stabilized by three hydrogen bonds with His57, Lys60F, and Ser195, while the conformational angles maintained in a strained, nonallowed configuration for non-glycyl amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the annexins as potential mediators of membrane fusion in exocytosis

Membrane fusion is a central event in the process of exocytosis. It occurs between secretory vesicle membranes and the plasma membrane and also among secretory vesicle membranes themselves during compound exocytosis. In many cells the fusion event is regulated by calcium. Since the relevant membranes do not undergo fusion in vitro when highly purified, much attention has been paid to possible protein mediators of these calcium-dependent fusion events. The annexins comprise a group of calcium-dependent membrane-aggregating proteins, of which synexin is the prototype, which can initiate contacts between secretory vesicle membranes which will then fuse if the membranes are further perturbed by the addition of exogenous free fatty acids. This review discusses the secretory pathway and the evidence obtained fromin vitro studies that suggests the annexins may be mediators or regulators of membrane fusion in exocytosis.     

Fine structure of external ‘genital’ papillae in the fresh water miteHydrovolzia placophora (Hydrovolziidae,Actinedida, Actinotrichida,Acari)

Numerous genital papillae are described for the first time in the freshwater miteHydrovoliza placophora. In contrast to other actinotrichid mites, these organs are located on the epimeral plates, thus well removed from the genital opening. Ultrastructurally, the papillae correspond to those of other freshwater mites and are thus likely involved in ion/osmoregulation.     

Nicotine Effects on Dopamine Clearance in Rat Nucleus Accumbens

Abstract: In vivo voltammetry was used to measure the clearance of exogenously applied dopamine (DA) in the nucleus accumbens following acute systemic nicotine administration in urethane-anesthetized rats. The IVEC-5 system was used for continuous in vivo electrochemical measurements. A finite amount of DA was pressure-ejected (25–100 nl, 200 µ M barrel concentration) at 5-min intervals from micropipettes (tip diameter, 10–15 µm) positioned 250 ± 50 µm from the recording electrode. The peak DA concentration after each DA ejection was significantly decreased in rats following nicotine, but not in rats given saline. In addition, when mecamylamine was administered 20 min before nicotine it clearly antagonized nicotine effects. These results suggest that nicotine may actually facilitate DA transporter systems within the nucleus accumbens.