Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation.

Subcellular fractions prepared from rat glial cells in culture (clonal line c6) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [3H] norepinephrine binding and enzyme activation were measured under identical experimental conditions. Binding sites for norepinephrine could be detected; their main characteristics were: apparant Km: 4 - 10-6 M, macimal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrennergic agonist) and the blocking agent propranolol were unable to compete with [3H] norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [3H] norepinephrine binding. As in several other systems so far studied, the presence on the glial cell's membrane of a large number of "catechol-binding sites" makes it difficult to characterize the beta-adrenergic receptor.     

Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.     

ARFGAP1 promotes the formation of COPI vesicles,suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.     

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.     

Synergistic effects in theα 1- andβ 1-adrenergic regulations of intracellular calcium levels in striatal astrocytes

1. Using indo-1 as a calcium fluorescent probe, we have observed the following in striatal astrocytes in primary culture. 2. The stimulation of alpha-adrenoceptors induces a rapid rise in cytosolic calcium resulting from an internal calcium mobilization followed by an external calcium influx (4-min duration). 3. The stimulation of beta 1-adrenoceptors evokes only a slight internal calcium mobilization (90-sec duration). 4. The simultaneous stimulation of beta 1- and alpha 1-adrenoceptors induces a more prolonged calcium influx (10 min). The latter phenomenon could explain the calcium-dependent synergistic effects of alpha 1 and beta stimulation on cAMP production already described in the brain.