FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.

It is known that the phospholipids of the brain cells of fish are altered during cold adaptation. In particular, the 1-monounsaturated 2-polyunsaturated phosphatidylethanolamines (PEs) increase 2- to 3-fold upon adaptation to cold. One of the most striking changes is in the 18:1/22:6 species of PE. We determined how this lipid affected the bilayer-to-hexagonal-phase transition temperature of 16:1/16:1 PE. We found that it was more effective in lowering this transition temperature than were other, less unsaturated, PE species. In addition, it was not simply the presence of the 18:1/22:6 acyl chains which caused this effect, since the 18:1/22:6 species of phosphatidylcholine had the opposite effect on this transition temperature. Zwitterionic substances that lower the bilayer-to-hexagonal-phase transition temperature often cause an increase in the activity of protein kinase C (PKC). Indeed, the 18:1/22:6 PE caused an increase in the rate of histone phosphorylation by PKC which was greater than that caused by other, less unsaturated, PEs. The 18:1/22:6 phosphatidylcholine had no effect on this enzyme. The stimulation of the activity of PKC by the 18:1/22:6 PE is a consequence of this lipid's increasing the partitioning of PKC to the membrane.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity.

Overexpression of phytochrome B (phyB) in transgenic Arabidopsis results in enhanced deetiolation in red light. To define domains of phyB functionally important for its regulatory activity, we performed chemical mutagenesis of a phyB-overexpressing line and screened for phenotypic revertants in red light. Four phyB-transgene-linked revertants that retain parental levels of full-length, dimeric, and spectrally normal overexpressed phyB were identified among 101 red-light-specific revertants. All carry single amino acid substitutions in the transgene-encoded phyB that reduce activity by 40- to 1000-fold compared to the nonmutagenized parent. The data indicate that the mutant molecules are fully active in photosignal perception but defective in the regulatory activity responsible for signal transfer to downstream components. All four mutations fall within a 62-residue region in the COOH-terminal domain of phyB, with two independent mutations occurring in a single amino acid, Gly-767. Accumulating evidence indicates that the identified region is a critical determinant in the regulatory function of both phyB and phyA.

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.