Selectivity of recognition of variable (V) regions of autoantibodies by intravenous immunoglobulin (IVIg)

In the present study, we demonstrate that intravenous immunoglobulin (IVIg) is capable of binding to variable (V) regions of anti-endothelial cell antibodies (AECA) of healthy donors and patients with systemic lupus erythematosus (SLE). Among V regions of AECAs, IVIg selectively recognized certain idiotypes expressed by the autoantibodies of a given individual, in the case of both natural and SLE-associated AECAs. These observations provide new and direct evidence that IVIg interacts idiotypically with V regions of autoantibodies and that the efficacy of such interaction depends on individual autoantibody specificity. Our findings may be relevant for the understanding of the mechanisms that control expression of natural autoantibody activity in serum and for that of the differences in response to IVIg therapy that are seen between patients with autoimmune disease.     

The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C

A clone encoding the guinea pig (gp) min K potassium channel was isolated and expressed in Xenopus oocytes. The currents, gpIsK, exhibit many of the electrophysiological and pharmacological properties characteristic of gpIKs, the slow component of the delayed rectifier potassium conductance in guinea pig cardiac myocytes. Depolarizing commands evoke outward potassium currents that activate slowly, with time constants on the order of seconds. The currents are blocked by the class III antiarrhythmic compound clofilium but not by the sotalol derivative E4031 or low concentrations of lanthanum. Like IKs in guinea pig myocytes, gpIsK is modulated by stimulation of protein kinase A and protein kinase C (PKC). In contrast to rat and mouse IsK, which are decreased upon stimulation of PKC, myocyte IK and gpIsK in oocytes are increased after PKC stimulation. Substitution of an asparagine residue at position 102 by serine (N102S), the residue found in the analogous position of the mouse and rat min K proteins, results in decreased gpIsK in response to PKC stimulation. These results support the hypothesis that the min K protein underlies the slow component of the delayed rectifier potassium current in ventricular myocytes and account for the species-specific responses to stimulation of PKC.     

Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties

Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date. Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems. Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule. To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain. Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting. Both isoforms were expressed in all tissues tested but at different levels. Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized. CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties. More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates. These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics. These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles.     

Immunoreactivity of neural crest-derived cells in thymic tissue developing under the rat kidney capsule

In order to study the functional development of a thymus in an experimental model, small pieces of adult rat thymic tissue were cultured for 9 days and implanted under the kidney capsule of littermates. The tissues were examined with a panel of antibodies raised against thymic and neural factors and neural crest cells at intervals from 5 to 13 days. At 5 days post-implantation, there were groups of L1+ cells within the implants that reacted with antibodies raised against neural and neural crest cell markers. L1+ cells were highly mitotic, rounded cells measuring 8.7 +/- 0.6 micrometer in diameter. Double immunostaining with different combinations of antibodies showed that 94% of the L1+ cells were also TH+, and many were HNK-1/NCAM+, PGP 9.5+, NGF+, chromogranin A+, VIP+, S100+, CGRP+, GAD+, and A2B5+. A few were also pan-cytokeratin+. These results indicate that these cells are derived from neural crest derived cells and belong to the neuroepithelial line of development. The L1+ cells were most numerous before nerves appeared (about Day 9) and reduced in number and extent as the thymus differentiated. The neural crest cells occasionally had long cytoplasmic extensions, but it was not possible to decide if they formed the nerves that appeared in the implants. Adult thymuses also contained a population of L1+ and HNK-1/NCAM+ cells, mainly in the subcapsular cortex, the septa, and the medulla. These cells could be a source of neural crest cells able to repopulate the implant. The adult thymus may always contain a reservoir of cells potentially capable of producing neuropeptides and transmitter factors required for thymic growth and regeneration.     

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.     

Histopathology of preneoplastic and neoplastic lesions of the esophagus in BUF rats ingesting diethylnitrosamine

Carcinomas developed in the mucosa of the esophagus in BUF strain rats ingesting diethylnitrosamine in the diet. The stages of hyperplasia, hyperplastic nodules, small carcinomas, and large, well-developed carcinomas were observed. Carcinomas, which were well differentiated, poorly differentiated, or undifferentiated, invaded the adjacent tissue but did not metastasize.