Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase

Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

Summary. In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively. Correspondence and reprints: Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik utca 2, 2462 Martonvásár, Hungary.     

Characterization of monoclonal B cell growth factor (BCGF) produced by a human T-T hybridoma

We previously demonstrated the development of a cloned human T cell hybridoma that secretes B cell growth factor (BCGF) in the absence of demonstrable interleukin 2 or B cell differentiation factor. Sephadex gel filtration chromatography demonstrated the m.w. of this factor to be 18 to 20K. The present studies were performed to further characterize the biochemical properties of the molecule and to determine its target cell specificity. Temperature stability studies showed the monoclonal BCGF to be stable at 37 degrees C for 12 hr and at 70 degrees C for 15 min; however, most (93%) of the activity was lost after incubation at 70 degrees C for 30 min. Aliquots of hybridoma supernatant were exposed to buffer solutions with variable pH with no diminution in activity over a pH range of 4.0 to 10.0 BCGF activity was not affected by 2-mercaptoethanol, neuraminidase, or nucleic acid denaturing enzymes. In contrast, all activity was destroyed by 10 M urea, trypsin, and chymotrypsin. Chromatofocusing demonstrated the isoelectric point of BCGF to be 6.3 to 6.6. Finally, absorption experiments demonstrated that BCGF activity was absorbed by large, activated B cells. Mitogen-stimulated T cell blasts, small resting B cells, and CESS cells failed to absorb BCGF activity from the hybridoma supernatant. These and future studies with purified monoclonal human BCGF should enhance our understanding of its immunochemical properties and of its role in the immunoregulation of human B cell responses.     

Total and semisynthesis and in vitro studies of both enantiomers of 20-fluorocamptothecin

Both enantiomers of 20-fluorocamptothecin and the racemate have been prepared by total synthesis. The (R)-enantiomer is essentially inactive in a topoisomerase-I/DNA assay, while the (S)-enantiomer is much less active than (20S)-camptothecin. The lactone ring of 20-fluorocamptothecin hydrolyzes more rapidly than that of camptothecin in PBS. The results provide insight into the role of the 20-hydroxy group in the binding of camptothecin to topoisomerase-I and DNA.     

Treatment of viral and neoplastic diseases with double-stranded RNA derivatives and other new agents

Many attempts have been made to inhibit viral and neoplastic diseases by targeting the RNA system. The pathophysiologic significance of the microRNA system and the therapeutic potential of its manipulation are discussed. Studies of double-stranded RNA derivatives are reviewed. The therapeutic potential of one of these compounds, polyI:MPC, is emphasized. Studies of other related antiviral and antineoplastic agents are discussed, including 2'-deoxyoligocytidilates and telomerase inhibitors.     

A role for lymphotoxin in primary Sjogren's disease

The etiology of salivary gland injury in primary Sj?gren's disease is not well understood. We have previously described a mouse model of Sj?gren's disease, IL-14α transgenic (IL14αTG) mice, which reproduces many of the features of the human disease. We now demonstrate a critical role for lymphotoxin α (LTA) in the pathogenesis of Sj?gren's disease in IL14αTG mice. IL14αTG mice express LTA mRNA in their salivary glands and spleen and produce soluble LTA protein in their salivary secretions. When IL14αTG mice were crossed with LTA(-/-) mice, the IL14αTG.LTA(-/-) mice retained normal salivary gland secretions and did not develop either lymphocytic infiltration of their salivary glands or secondary lymphomas. However, both IL14αTG and IL14αTG.LTA(-/-) mice produced similar amounts of IFN-α and had similar deposition of autoantibodies in their salivary glands. Both IL14α and IL14α/LTA(-/-) mice had similar B cell responses to T-dependent and T-independent Ags, L-selectin expression, and expression of RelA, RelB, and NF-κB2 in their spleens. These studies suggest that LTA plays a critical role in the local rather than systemic inflammatory process of Sj?gren's disease. Furthermore, local production of soluble LTA in the salivary glands of IL14αTG mice is necessary for the development of overt Sj?gren's disease. Autoantibody deposition alone is not sufficient to produce salivary gland dysfunction. We also demonstrate that LTA is increased in the salivary gland secretions and sera of patients with Sj?gren's disease, further strengthening the biological relevance of the IL14αTG model to understanding the pathogenesis of human disease.     

Effect of an interferon inhibitor on the antiproliferative signal of interferon-alpha.

We have described a nonantibody type inhibitor of interferons (IFN) in the blood of patients with AIDS, advanced neoplastic disorders, and lupus erythematosus in earlier reports (1,2). In the present study we show that the semipurified inhibitor blocks the antiproliferative signal of IFN-alpha in Daudi cells and the membrane potential shifting ability of IFN-alpha is modulated by the interferon inhibitor preparation (IFI).