Mechanism of the enhancing effect of sorbitol on ileal Ca uptake in rat enterocytes

The effect of sorbitol on Ca uptake by isolated ileal epithelial cells was investigated. Intestinal cells were isolated from rat ileum by mechanical vibration.⁴⁵Ca uptake was approximately 2 times higher in cells exposed to 200 mM sorbitol ofd-alanine than in control cells. This enhancing effect of sorbitol on percentage Ca uptake decreased with increasing Ca concentrations in the incubation medium suggesting an effect on Ca entry velocity. The addition of 10 M nifedipine or 200 M verapamil to the incubation medium was devoid of any effect on Ca uptake in ileal cells, whereas 100 M trifluoperazine or chlorpromazine abolished the stimulatory effect of sorbitol. Finally, the effect of sorbitol on isolated cells was independent of a measurable change of cellular ATP content. In conclusion, the stimulatory effect of sorbitol on ileal Ca uptake is probably exerted through mechanisms other than an increase in intracellular ATP concentration. Sorbitol may enhance enterocyte Ca transport via a direct interaction with calmodulin and/or the Ca pump. It may also exert its effect through an inhibition of the basolateral Na Ca exchanger.     

Demonstration of non-idiotypic variable heavy chain (VH) antigens on human peripheral blood lymphocytes

In this study we have obtained evidence for the expression of Vh-like determinants on unstimulated human T lymphocytes as well as T lymphoblasts. These determinants were detected with antisera raised against isolated VH fragments of a human IgG3 cryoglobulin (KUP). The antisera detect idiotypic, VH subgroup and HV framework determinants and behave as anti-immunoglobulin antibodies when tested against peripheral blood mononuclear cells in immunofluorescence experiments. However, sensitive radiolabelling and immunoprecipitation techniques revealed a certain reactivity against highly purified T lymphocytes. The specificity of these T-cell-reactive antibodies has not been fully established, but the results suggest that the antisera contain antibodies directed at VH fragment-specific antigens or antigens not exposed on native immunoglobulins or isolated heavy chains.     

Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.     

Defects in neurofibromatosis 2 protein function can arise at multiple levels

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.      

Electrophysiological indices of processing aesthetics: Spontaneous or intentional processes?

Processes underlying aesthetic appreciation of formal graphic black and white patterns were investigated. In previous EEG studies on aesthetic and symmetry judgments, an early frontocentral negativity has been observed for not beautiful judgments, and interpreted as reflecting an impression formation. In addition, a lateralized late positivity, more pronounced in the aesthetic than in the symmetry judgment task, has been interpreted as reflecting evaluative categorization; and a sustained posterior negativity for not symmetric judgments, has been interpreted as reflecting a prolonged symmetry analysis. Here, we investigated whether these processes occur spontaneously, i.e. independent of an aesthetics task instruction or a judgment task demand. Participants were randomly assigned to two group conditions. In the Viewing condition, participants were instructed to view graphic patterns, and to detect a probe. In the Contemplation condition, participants were instructed to contemplate the beauty of the patterns, and to detect a probe. No aesthetics-related response was required in either condition. ERP results suggest that symmetry analysis occurred spontaneously. Evaluative categorization, reflected by a late positivity, required an aesthetics instruction and did not occur spontaneously. This process was elicited without an overt aesthetic judgment. An early frontocentral negativity for not beautiful patterns, interpreted as reflecting impression formation, did not occur (in contrast to previous studies that required an overt aesthetic judgment). Given the present data, aesthetic appreciation of graphic patterns requires different sub-processes. Aesthetic contemplation is reflected by a lateralized late positivity, whilst an aesthetic judgment is additionally reflected by an early frontocentral negativity. Aesthetic appreciation of beauty appears to require intention and is not spontaneous in character.     

Induction of B cell apoptosis by co-cross-linking CD23 and sIg involves aberrant regulation of c-myc and is inhibited by bcl-2

A novel system to study the effects of co-cross-linking CD23/FceRII and sIg on murine B lymphocytes utilizes a highly multivalent form of anti- Ig prepared by covalently linking anti-Ig antibodies to a DNP-dextran backbone. CD23-sIg co-cross-linking is accomplished by the addition of DNP-specific monoclonal IgE. Previous studies demonstrated that co- cross-linking CD23 and sIg significantly inhibited mouse B cell proliferation, especially at high doses of the multivalent anti-Ig. Interestingly, examination of early activation signals reveals no difference in B cells subjected to co-cross-linking conditions as compared to B cells activated with anti-Ig alone. Total cellular protein tyrosine phosphorylation levels are unchanged by co-cross- linking. Analysis of B cell mRNA reveals that co-cross-linking the receptors does not alter the expression levels of ornithine decarboxylase 8 h after stimulation as compared to the controls. In contrast, levels of the proto-oncogene c-myc were significantly elevated 1 h after inducing B cell activation under co-cross-linking conditions. However, it remains unclear whether this aberrant c-myc regulation plays any role in inducing apoptosis. In addition, on day 3 after stimulation, the co-cross-linking of CD23 and sIg resulted in the formation of apoptotic B cells, determined by both photomicroscopy of the B cell cultures and FACS analysis of B cell nuclei. B cells obtained from bcl-2 transgenic mice proliferated as well as controls, and failed to undergo apoptosis when CD23 and sIg were co-cross-linked on their surface. These studies indicate that co-cross-linking of CD23 with B cell sIg inhibits B cell proliferation by a mechanism that is distinct from that seen by co-cross-linking of the Fc gamma RII and sIg. In addition, these results suggest a means by which antigen- specific IgE can down-regulate additional B cell activation and IgE synthesis.      

The source and action of histamine in the isolated guinea-pig gallbladder

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H₁-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H₁-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H₂-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H₂-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H₃-receptor agonist, (R)--methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10^–10–10^–7 M) indicating little pre-synaptic H₃ activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H₁ and H₂ but not H₃ receptors.