Evaluation of butanone, carbon dioxide, and 1-octen-3-OL as attractants for mosquitoes associated with north central Florida bay and cypress swamps

In the sera of (NZB x NZW)F1 (B/W) mice that develop a lupus-like syndrome, increased levels of IgG antibodies (Ab) reacting with TNP have been detected before the appearance of IgG anti-DNA Ab and clinical symptoms. A single injection of trinitrophenyl-bovine serum albumin (TNP/BSA) in physiological saline into a young B/W mouse (3 months old), followed by fusion of its splenocytes 3 days later, gave rise to hybridomas simultaneously secreting IgM and IgG Ab with anti-TNP reactivity. Both mu and gamma chains were detected in culture supernatants by ELISA, and double isotype-producing cells were labeled by immunofluorescence. Molecular analysis of two of these double isotype-producing hybridomas showed the presence of mRNA coding for both mu and gamma chains of Ig, and this gamma mRNA could be translated in vitro into a gamma heavy (H) chain. Comparison of the H chain variable-region sequences of IgM and IgG revealed 100% homology between mu and gamma V(H) genes in one clone, while mu and gamma V(H) genes showed only 80% homology in the other clone. Both clones produced a single kappa light (L) chain. These two hybridomas, isolated from a B/W mouse, thus represent two different mechanisms of double isotype expression: the first one corresponds to an IgM to IgG switch, while the second one reflects a lack of allelic exclusion.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

The p65 subunit of NF-kappa B is redundant with p50 during B cell proliferative responses, and is required for germline CH transcription and class switching to IgG3

B cells lacking individual NF-kappa B/Rel family members exhibit defects in activation programs. We generated small resting B cells lacking p65 or p50 alone, or lacking both p50 and p65, then evaluated the ability of these cells to proliferate, secrete Ig, and undergo Ig class switching. B cells lacking p65 proliferated well in response to all stimuli tested. However, these cells demonstrated an isolated defect in switching to IgG3, which was associated with a decrease in gamma 3 germline CH gene expression. Whereas, previously reported, B cells lacking p50 alone had a severe proliferative defect in response to LPS, a moderate defect in response to CD40 ligand (CD40L), and normal proliferation to Ag receptor cross-linking using dextran-conjugated anti-IgD Abs (alpha delta-dex), B cells lacking both p50 and p65 exhibited severely impaired proliferation in response to LPS, alpha delta-dex, and CD40L. This defect could be overcome by simultaneous administration of alpha delta-dex and CD40L. In response to this latter combination of stimuli, B cells lacking both p50 and p65 secreted Ig and underwent isotype switching to IgG1 as efficiently as B cells lacking p50 alone. These data demonstrate a role for the p65 subunit of NF-kappa B in germline CH gene expression as well as functional redundancy between p50 and p65 during proliferative responses.     

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.     

Assembly of productive T cell receptor delta variable region genes exhibits allelic inclusion

The generation of a productive "in-frame" T cell receptor beta (TCR beta), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most alphabeta T cells express a single TCR beta chain and most B cells express single IgH and IgL chains. Assembly of TCR alpha and TCR gamma chain variable region genes exhibit allelic inclusion and alphabeta and gammadelta T cells can express two TCR alpha or TCR gamma chains, respectively. However, it was not known whether assembly of TCR delta variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR delta rearrangements in a panel of mouse splenic gammadelta T cell hybridomas. We find that, similar to TCR alpha and gamma variable region genes, assembly of TCR delta variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of gammadelta T cell development and regulation of rearrangement of TCR delta genes.     

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The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

Molecular characterization of IgA- and/or IgG-switched chronic lymphocytic leukemia B cells

The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a "subtype" of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/ IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH-D-JH gene sequence was joined with that of the C alpha gene in the B cells of the three IgA CLLs, and with that of the C gamma gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH-D-JH cDNA sequences were spliced to either C alpha or C gamma genes. In all five CLLs, the pattern of C mu DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the C mu exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.     

Expression of a CD3 epsilon transgene in CD3 epsilon(null) mice does not restore CD3 gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.     

Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells

Others have reported that the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator and down-regulator of most protein kinase C (PKC) isozymes, can induce apoptotic cell death of androgen-sensitive LNCaP but not androgen-insensitive PC-3 or DU 145 human prostate cancer cells. As a first step toward uncovering the mechanism by which TPA induces apoptosis of LNCaP cells, we quantified expression of PKC isozyme mRNAs in unmodified and TPA-resistant LNCaP cells and in naturally TPA-resistant PC-3, PC-3M, and DU 145 cells. All of the cell lines and normal prostate expressed RNAs for PKC alpha, delta, epsilon, eta, and mu; only DU 145 cells and normal prostate expressed PKC beta and theta RNAs, and none expressed PKC gamma. The amount of PKC alpha RNA and protein was 6- to 38-fold lower, and PKC mu RNA was 4.5- to 16.5-fold higher in unmodified and TPA-resistant LNCaP cells than in the androgen-independent cells. We examined the effects of TPA on PKC alpha and mu mRNA levels and on membrane translocation of PKC alpha. Incubation with TPA for 6 h or more induced 95% inhibition of cell growth, a transient 12-fold increase and 5-fold decrease in PKC alpha and mu mRNA levels, respectively, and prolonged translocation of PKC alpha to non-nuclear membranes in unmodified LNCaP cells and in TPA-resistant LNCaP cells from which TPA had been removed for 10 days. TPA-resistant LNCaP cells in the continuous presence of TPA, or 24 h after removal of TPA, had down-regulated PKC alpha and remained resistant to re-addition of TPA. These data demonstrate a strong correlation of the presence and absence of membrane PKC alpha with apoptosis and resistance to apoptosis, respectively.     

Monomeric IgG2 enhances Ig production and proliferation in human B cells

The effect of purified polyclonal human IgG subclasses on B-cell responses was studied using the human IgA-producing B-cell line GM-1056. IgG2 at concentrations of 0.01-1 microgram/mL enhanced both IgA production and proliferation, while IgG1, IgG3, and IgG4 each failed to do so at tested concentrations between 0.001 and 10 micrograms/mL. This enhancement was Fc gamma R mediated, since IgG2 Fc fragments enhanced IgA production and proliferation to the same extent as did the whole IgG2 molecule, whereas F(ab')2 fragments did not. However, in contrast to monomeric IgG2, aggregated IgG2, which was expected to bind Fc gamma RII on B cells, affected neither IgA production nor proliferation. Similarly, anti-CDw32 mAb (2E1, anti-Fc gamma RII), anti-CD 64 mAb (32.2 anti-Fc gamma RI), and anti-CD16 mAb (Leu 11a, anti-Fc gamma RIII) mAb each failed to stimulate GM-1056 cells, and more importantly did not block IgG2-induced stimulation. Of various cytokines tested, including IFN-alpha, IFN-gamma, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, IL-6 alone augmented IgG2-induced enhancement of IgA production and proliferation. Moreover, the IL-6 effect was lost following preabsorption with anti-IL-6 antibody but not following preabsorption with control antibody. IgG2 also enhanced Ig production and proliferation in tonsillar large activated B cells, while IgG1, IgG3 and IgG4 each failed to do so. In contrast, IgG2 had no effect on Ig production and proliferation in tonsillar small resting B cells or SAC-stimulated small B cells. IgG2-induced enhancement of Ig production and proliferation in large B cells was not blocked by 2E1, 32.2, or Leu 11a, while enhancement was augmented in a specific fashion by IL-6. These results indicate that monomeric IgG2 specifically enhances B cell responses via an Fc gamma R receptor distinct from Fc gamma RI, Fc gamma RII, and Fc gamma RIII, and that IL-6 may play a role in augmenting this response.     

Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses

PD-1, an Ig superfamily member, contains an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail. It is expressed in a minor fraction of CD4-CD8- normal thymocytes and induced in peripheral lymphocytes following activation. To assess the possible roles of PD-1 in the immune responses, PD-1-deficient (PD-1-/-) mice were generated by a gene-targeting strategy. PD-1-4- mice developed and grew normally. Although the thymus was apparently normal, PD-1-/- mice showed moderate but consistent splenomegaly, which reflected the increased cellularity of both lymphoid and myeloid cells. The proliferative response of B cells by anti-IgM antibodies, but not of T cells by an anti-CD3 (145-2C11) mAb in vitro, was augmented in PD-1-/- mice as compared with control littermates. PD-1-/- mice showed increased serum levels of IgG2b, IgA and most strikingly IgG3, while those of IgM and IgG1 were comparable with control mice. Furthermore, PD-1-/- mice exhibited significantly augmented IgG3 anti-DNP antibody response to a type 2 T-independent antigen, DNP-Ficoll, with comparable IgM and IgG1 antibody responses with littermate controls. In the peritoneal cavity, the B-1 cell population in PD-1-/- mice exhibited significantly reduced expression of CD5, a negative regulator of B-1 cell activation, despite a marginal increase in the number of B-1 cells. Thus, PD-1 was suggested to be involved in the negative regulation for particular aspects of B cell proliferation and differentiation including class switching.     

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During B cell development, the surface expression of CD79 alpha/CD79 beta heterodimers had been thought to begin in the pre-B cell stage where the heterodimers constitute pre-B cell receptors together with mu heavy and surrogate light chains. Thereafter, in mature B cells, CD79 alpha/CD79 beta associates with surface Ig to form B cell antigen receptors. In this study, we revealed by using newly established mAb that CD79 beta was expressed on the surface of pro-B cells which had not undergone the productive Ig gene rearrangement. Biochemical analysis showed that CD79 beta on pro-B cells existed either as monomers or as disulfide-linked heterodimers with CD79 alpha, non-covalently associated with four unidentified membrane molecules. Our finding that CD79 beta is expressed on earlier B-lineage cells than previously expected coincides with the recent study in which CD79 beta-deficient mice exhibit a blockade of B cell differentiation at the pro-B cell stage. Thus, it is speculated that the CD79 beta-containing complexes on pro-B cell surfaces may function to induce early B cell differentiation.     

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.     

Expression of genes for human globin in the cell line K-562: an experimental model for the study of fetal erythropoiesis

Human leukemia K-562(S) cells are a useful model system to study the relationship between cell proliferation and induced erythroid differentiation. In these studies K-562(S) cells were cultured in alpha -medium, 10% fetal calf serum and induced to express erythroid genes by 75 microM hemin, 1.2 mM butyric acid or 1.5 ng/ml actinomycin D. Cell number was determined using a ZF Coulter Counter and the increase in the proportion of hemoglobin-containing cells was detected by a specific colorimetric reaction with benzidine. The characterization of the synthesized hemoglobins was performed by cellulose-acetate gel electrophoresis of post-mitochondrial supernatants. By cloning K-562(S) cells in semi-solid medium (O,33% agar) containing 75 microM hemin a variant cell line, denominated K-562(S6), have been isolated which does not undergo terminal cell division but does express human globin genes and accumulates on the average 12 pg of Hb/cell. K-562(S6) cells accumulate, upon exposure to 75 microM hemin, mostly Hb Gower 1 (zeta 2 epsilon 2) and low amounts of Hb X (epsilon 2 gamma 2) and Hb Portland (zeta 2 gamma 2), being suitable for studies focused on the expression of embryonic globin genes and on the molecular mechanisms controlling the switching from embryonic-type to fetal-type hemoglobin accumulation, when in the human embryo zeta and epsilon globin genes become less active, being sharply increased accumulation of alpha and gamma globin chains.