The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells

Although Fas ligand (FasL) is well characterized for its capacity to deliver a death signal through its receptor Fas, recent work demonstrates that FasL also can receive signals facilitating antigen (Ag)-specific proliferation of CD8+ T cells. The fact that the gld mutation differentially influences the proliferative capacity of CD8+ and CD4+ T cells presented the intriguing possibility that a single molecule may play opposing roles in these two subpopulations. The present study focuses on how these positive and negative regulatory roles are balanced. We show that naive CD4+ T cells are responsive to FasL-mediated costimulation on encounter with Ag when Fas-mediated death is prevented. Thus, the machinery responsible for transducing the FasL positive reverse signal operates in both CD4+ and CD8+ T cells. Instead, differential control of FasL expression distinguishes the role of FasL in these two T cell subpopulations. FasL costimulation occurs immediately on T cell receptor ligation and correlates with the up-regulation of FasL expression on CD8+ and naive CD4+ T cells, both of which are sensitive to the FasL costimulatory signal. Conversely, FasL-initiated death occurs late in an immune response when high levels of FasL expression are maintained on CD4+ T cells that are sensitive to Fas-mediated death, but not on CD8+ T cells that are relatively insensitive to this signal. This careful orchestration of FasL expression during times of susceptibility to costimulation and conversely, to death, endows FasL with the capacity to both positively and negatively regulate the peripheral T cell compartment.

Parsing executive processes: Strategic vs. evaluative functions of the anterior cingulate cortex

Event-related functional MRI and a version of the Stroop color naming task were used to test two conflicting theories of anterior cingulate cortex (ACC) function during executive processes of cognition. A response-related increase in ACC activity was present when strategic processes were less engaged, and conflict high, but not when strategic processes were engaged and conflict reduced. This is inconsistent with the widely held view that the ACC implements strategic processes to reduce cognitive conflicts, such as response competition. Instead, it suggests that the ACC serves an evaluative function, detecting cognitive states such as response competition, which may lead to poor performance, and representing the knowledge that strategic processes need to be engaged.

Epistatic and independent functions of Caspase-3 and Bcl-XL in developmental programmed cell death

The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-XL directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-XL in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR)

The target of rapamycin protein (TOR) is a highly conserved ataxia telangiectasia-related protein kinase essential for cell growth. Emerging evidence indicates that TOR signaling is highly complex and is involved in a variety of cellular processes. To understand its general functions, we took a chemical genomics approach to explore the genetic interaction between TOR and other yeast genes on a genomic scale. In this study, the rapamycin sensitivity of individual deletion mutants generated by the Saccharomyces Genome Deletion Project was systematically measured. Our results provide a global view of the rapamycin-sensitive functions of TOR. In contrast to conventional genetic analysis, this approach offers a simple and thorough analysis of genetic interaction on a genomic scale and measures genetic interaction at different possible levels. It can be used to study the functions of other drug targets and to identify novel protein components of a conserved core biological process such as DNA damage checkpoint/repair that is interfered with by a cell-permeable chemical compound.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice.

Interleukin 1 receptor antagonist (IL-1ra) is a cytokine whose only known action is competitive inhibition of the binding of interleukin 1 (IL-1) to its receptor. To investigate the physiological roles of endogenously produced IL-1ra, we generated mice that either lack IL-1ra or overproduce it under control of the endogenous promoter. Mice lacking IL-1ra have decreased body mass compared with wild-type controls. They are more susceptible than controls to lethal endotoxemia but are less susceptible to infection with Listeria monocytogenes. Conversely, IL-1ra overproducers are protected from the lethal effects of endotoxin but are more susceptible to listeriosis. Serum levels of IL-1 following an endotoxin challenge are decreased in IL-1ra nulls and increased in IL-1ra overproducers in comparison to controls. These data demonstrate critical roles for endogenously produced IL-1ra in growth, responses to infection and inflammation, and regulation of cytokine expression.

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.

Differential functions of the two Src homology 2 domains in protein tyrosine phosphatase SH-PTP1.

SH-PTP1 (also known as PTP1C, HCP, and SHP) is a non-transmembrane protein tyrosine phosphatase (PTPase) containing two tandem Src homology 2 (SH2) domains. We show here that the two SH2 (N-SH2 and C-SH2) domains in SH-PTP1 have different functions in regulation of the PTPase domain and thereby signal transduction. While the N-terminal SH2 domain is both necessary and sufficient for autoinhibition through an intramolecular association with the PTPase domain, truncation of the C-SH2 domain [SH-PTP1 (delta CSH2) construct] has little effect on SH-PTP1 activity. A synthetic phosphotyrosine residue (pY) peptide derived from the erythropoietin receptor (EpoR pY429) binds to the N-SH2 domain and activates both wild-type SH-PTP1 and SH-PTP1 (delta CSH2) 60- to 80-fold. Another pY peptide corresponding to a phosphorylation site on the IgG Fc receptor (Fc gamma RIIB1 pY309) associates with both the C-SH2 domain (Kd = 2.8 microM and the N-SH2 domain (Kd = 15.0 microM) and also activates SH-PTP1 12-fold. By analysis of the effect of the Fc gamma RIIB1 pY309 peptide on SH-PTP1 (delta CSH2), SH-PTP1 (R30K/R33E), SH-PTP1 (R30K/R136K), and SH-PTP1 (R136K) mutants in which the function of either the N- or C-SH2 domain has been impaired, we have determined that both synthetic pY peptides stimulate SH-PTP1 by binding to its N-SH2 domain; binding of pY ligand to the C-SH2 domain has no effect on SH-PTP1 activity. We propose that the N-terminal SH2 domain serves both as a regulatory domain and as a recruiting unit, whereas the C-terminal SH2 domain acts merely as a recruiting unit.

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.

Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is an important mechanism for the regulation of gene expression. The members of the SR protein family of pre-mRNA splicing factors have distinct functions in promoting alternative splice site usage. Here we show that SR proteins are required for the first step of spliceosome assembly, interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Further, we find that individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA.

The Future Direction of Delaware Law (Including a Brief Exegesis on Fee Shifting Bylaws)

Delaware sets the governance standards for most public companies. The ability to attract corporations could not be explained solely by the existence of a favorable statutory regime. Delaware was not invariably the first or the only state to implement management friendly provisions. Given the interpretive gaps in the statute and the critical importance of the common law in the governance process, courts played an outsized role in setting legal standards. The management friendly nature of the Delaware courts contributed significantly to the state’s attraction to public corporations. A current example of a management friendly trend in the case law had seen the recent decisions setting out the board’s authority to adopt bylaws under Section 109 of the Delaware General Corporation Law (DGCL), particularly those involving the shifting of fees in litigation against the corporation or its directors. The DGCL allows bylaws that address “the business of the corporation, the conduct of its affairs, and its rights or powers or the rights or powers of its stockholders, directors, officers or employees.” The broad parameters are, however, subject to limits. Bylaws cannot be inconsistent with the certificate of incorporation or “the law.” Law includes the common law. The Delaware courts have used the limitations imposed by “the law” to severely restrict the reach of shareholder inspired bylaws. The courts have not used the same principles to impose similar restraints on bylaws adopted by the board of directors. This can be seen with respect to bylaws that restrict or even eliminate the right of shareholders to bring actions against management and the corporation. In ATP Tour, Inc. v. Deutscher Tennis Bund the court approved a fee shifting bylaw that had littl relationship to the internal affairs of the corporation. The decision upheld the bylaw as facially valid.The decision ignored a number of obvious legal infirmities. Among other things, the decision did not adequately address the requirement in Section 109(b) that bylaws be consistent with “the law.” The decision obliquely acknowledged that the provisions would “by their nature, deter litigation” but otherwise made no effort to assess the impact of this deterrence on shareholders causes of action. The provision in fact had the practical effect of restricting, if not eliminating, litigation rights granted by the DGCL and the common law. Perhaps most significantly, however, the bylaws significantly limited common law rights of shareholders to bring actions against the corporation and the board. Given the high dismissal rates for these actions, fee shifting bylaws imposed a meaningful risk of liability on plaintiffs. Moreover, because judgments in derivative suits were paid to the corporation, shareholders serving as plaintiffs confronted the risk of liability without any offsetting direct benefit. By preventing suits in this area, the bylaw effectively insulated the behavior of boards from legal challenge. The ATP decision was poorly reasoned and overstepped acceptable boundaries. The management friendly decision threatened the preeminent role of Delaware in the development of corporate law. The decision raised the specter of federal intervention and the potential for meaningful competition from the states. Because the opinion examined the bylaw in the context of non-stock companies, the reasoning may remain applicable only to those entities and never make the leap to for-profit stock corporations. Nonetheless, the analysis reflects a management friendly approach that does not adequately take into account the impact of the provision on the rights of shareholders.