Membrane-bound and secreted IgA contain structurally different alpha-chains

Three different forms of alpha-chains are synthesized by BF0.3 and 615.2, two cloned cell lines derived from the murine B lymphoma 1.29. The three forms of alpha-chains differ in size, pI, cellular location, and rate of turnover. They were identified by means of lactoperoxidase-catalyzed radioiodination, internal 14C or 35S labeling, and immunofluorescence techniques as membrane-bound(alpha m), secreted (alpha s), and intracellular (alpha ic) proteins. Comparison of immunoglobulin products of the two lymphoma lines with those of a hybridoma cell line, Id 150, which secretes IgA of the 1.29 idiotype but lacks membrane IgA, confirmed the assignments of alpha m, alpha s, and alpha ic. Results of biosynthetic labeling of BF0.3, 615.2, and Id 150 in the presence and absence of tunicamycin suggest that the difference in m.w. and charge observed between alpha m and alpha s can be attributed to differences in primary amino acid structure rather than different degrees of glycosylation.     

Enhanced immune responses in transgenic mice expressing a truncated form of the lymphocyte semaphorin CD100

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5(+) B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.     

Involvement of Sema4A in the progression of experimental autoimmune myocarditis

Dilated cardiomyopathy often results from autoimmunity triggered by microbial infections during myocarditis. However, it remains unclear how immunological disorders are implicated in pathogenesis of autoimmune myocarditis. Here, we demonstrated that Sema4A, a class IV semaphorin, plays key roles in experimental autoimmune myocarditis (EAM). Dendritic cells pulsed with myosin heavy chain-α peptides induced severe myocarditis in wild-type mice, but not in Sema4A-deficient mice. In adoptive transfer experiments, CD4⁺ T-cells from wild-type mice induced severe myocarditis, while CD4⁺ T-cells from Sema4A-deficient mice exhibited considerably attenuated myocarditis. Our results indicated that Sema4A is critically involved in EAM by regulating differentiation of T-cells.     

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO₂ in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.     

Metabolic effects of the major component of bovine growth hormone

Bovine growth hormone, subjected to DEAE-cellulose chromatography, yielded one major and several minor components. The various chromatographic fractions of bovine growth hormone were compared with the parent material for their ability to promote hormone effects in vivo and in vitro. The major component of bovine growth hormone was homogeneous by acrylamide-gel electrophoresis, rechromatography and sedimentation equilibrium. Its amino acid composition was similar to that of the parent hormone. The major component possessed all the qualitative activities present in the original heterogeneous material, including promotion of acute hypoglycaemia and hypolipaemia. In studies in vitro in adipose-tissue segments the major component of the hormone increased entry of glucose and its oxidation to CO₂, conversion of glucose into glyceride glycerol, release of glycerol and incorporation of histidine into adiposetissue protein. Other chromatographic fractions of bovine growth hormone were not homogeneous and possessed some but not all of the metabolic activities attributed to the hormone preparations or its major component. Thus, the metabolic effects obtained with bovine growth-hormone preparations in vivo and in vitro can be obtained with the major homogeneous component of the hormone. This observation precludes the possibility that the metabolic effects obtained with bovine growth-hormone preparations are due to the combined actions of a number of components found therein.     

Flavone-catalyzed apoptosis in Scutellaria baicalensis

In response to mechanical damage, roots of Scutellaria baicalensis undergo cell death within 24 h. The flavone baicalein was identified as the factor regulating apoptosis in the damaged roots of S. baicalensis. Plant apoptosis is known to be triggered by oxidative damage of DNA through oxidative bursts, whereas baicalein causes apoptosis in Scutellaria cells by a copper-dependent oxidation of nuclear DNA without inducing an oxidative burst. S. baicalensis possesses an interesting system for quickly producing this apoptosis-inducing flavone in its cells. Intact Scutellaria cells contain little baicalein but store a large amount of baicalin (baicalein 7-O-β-D-glucuronide). Stress treatment of Scutellaria cells immediately initiates hydrolysis of baicalin by endogenous β-glucuronidase, and the resulting baicalein is immediately translocated to the nucleus, leading to apoptosis. Thus, S. baicalensis possesses a unique apoptosis-inducing system that is linked with metabolism of baicalin.     

Expression and function of an early activation marker restricted to human B cells

A B cell-specific monoclonal antibody (anti-Ba) was prepared. In two-color FACS analysis the anti-Ba reacted with a subpopulation of Ig+ or B1+ cells obtained from tonsils, but did not react with most B1+ cells derived from PBL. Activation of B cells from PBL with TPA or anti-mu induced Ba expression and the addition of PHA-conditioned supernatant with anti-mu-enhanced Ba expression. Other B cell activators, such as Staphylococcus aureus Cowan I (Staph-A) or PWM plus T cells, could induce Ba expression. Ba expression was observed 6 hr after stimulation and reached a peak level at 72 hr. Ba expression was strictly restricted to B cells. H-7, a specific inhibitor of protein kinase C (C-kinase), displayed a dose-dependent inhibitory effect on Ba expression, showing dependency on C-kinase for Ba expression. Anti-Ba inhibited B cell proliferation induced by anti-mu and B-BCGF distinct from BSF-1. The results presented in this study suggest that the Ba antigen on B cells may be comparable to the Tac antigen on T cells.     

Isotype switching in murine pre-B cell lines

Isotype switching at the pre-B cell stage was studied by employing A-MuLV-transformed cell lines. Two gamma 2b-producing cell lines that did not have other cytoplasmic heavy chains or light chains were established from A-MuLV-transformed cell lines. One clone (SL2-1-52) arose spontaneously from a non-Ig-producing cell line (SL2-1) during in vitro culture. Another clone (AT11-2-24-6-99) underwent isotype switching from a mu-producing cell line (AT11-2-24-6), which had been derived from another non-Ig-producing line (AT11-2). Southern blot analysis of two gamma 2b-producing clones was performed in comparison to that of their respective parent clones. The results showed that isotype switching can operate at the stage of pre-B cells by a CH gene deletion mechanism without utilizing the switch region. In addition, the possibility is presented that deletion of the intervening CH genes could occur prior to the formation of the functional V region-coding DNA segment. This indicates that the prior expression of mu chains is not obligatory for the expression of other isotypes and that isotype commitment could occur in pre-B cells.