B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Cutting edge: rho activation and actin polarization are dependent on plexin-A1 in dendritic cells

We recently identified expression of the semaphorin receptor, plexin-A1, in dendritic cells (DCs); however, its function in these cells remains to be elucidated. To investigate function and maximize physiological relevance, we devised a retroviral approach to ablate plexin-A1 gene expression using small hairpin RNA (shRNA) in primary bone marrow-derived DCs. We show that plexin-A1 localizes within the cytoplasm of immature DCs, becomes membrane-associated, and is enriched at the immune synapse in mature DCs. Reducing plexin-A1 expression with shRNA greatly reduced actin polarization as well as Rho activation without affecting Rac or Cdc42 activation. A Rho inhibitor, C3, also reduced actin polarization. These changes were accompanied by the near-ablation of T cell activation. We propose a mechanism of adaptive immune regulation in which plexin-A1 controls Rho activation and actin cytoskeletal rearrangements in DCs that is associated with enhanced DC-T cell interactions.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Buried Alive: The Behavioural Response of the Mussels,Modiolus modiolus and Mytilus edulis to Sudden Burial by Sediment

Sedimentation in the sea occurs through natural processes, such as wave and tidal action, which can be exacerbated during storms and floods. Changes in terrestrial land use, marine aggregate extraction, dredging, drilling and mining are known to result in substantial sediment deposition. Research suggests that deposition will also occur due to the modern development of marine renewable energy. The response to individual burial under three depths of sediment, three sediment fractions and five burial durations was investigated in two mussel species, Modiolus modiolus and Mytilus edulis in specialist mesocosms. Both mussel species showed substantial mortality, which increased with duration of burial and burial by finer sediment fractions. M. modiolus was better able to survive short periods of burial than M. edulis, but at longer durations mortality was more pronounced. No mortality was observed in M. modiolus in burial durations of eight days or less but by 16 days of burial, over 50% cumulative mortality occurred. Under variable temperature regimes, M. edulis mortality increased from 20% at 8°C to over 60% at 14.5 and 20°C. Only M. edulis was able to emerge from burial, facilitated by increased byssus production, laid mostly on vertical surfaces but also on sediment particles. Emergence was higher from coarse sediment and shallow burials. Byssus production in M. edulis was not related to the condition index of the mussels. Results suggest that even marginal burial would result in mortality and be more pronounced in warm summer periods. Our results suggest that in the event of burial, adult M. modiolus would not be able to emerge from burial unless local hydrodynamics assist, whereas a small proportion of M. edulis may regain contact with the sediment water interface. The physiological stress resulting in mortality, contribution of local hydrodynamics to survival and other ecological pressures such as mussels existing in aggregations, are discussed.     

Antigenic variants of the nonspecific cross-reacting antigen (NCA)

Monoclonal antibodies (Mab) were prepared against nonspecific cross-reacting antigen (NCA) and were selected on the basis of their absence of reactivity with carcinoembryonic antigen (CEA). Four Mab were found which allowed the characterization on CEA of three epitopes, defined A, B, and C. These epitopes were all located on the peptidic moiety of this highly glycosylated antigen and were present on NCA molecules of heterogeneous m.w. (greater than 100,000, 80,000, and 48,000 m.w., the latter being the most abundant). The amount of NCA was estimated in 251 human sera both by a conventional RIA, using a rabbit antiserum, and by EIA, using different Mab: Mab 4, 18, and 33, which reacted, respectively, with epitopes A, B, and C. Each assay gave a different value of the absolute concentration of NCA in the serum. On the whole, Mab 4 gave lower values, whereas Mab 18 and 33 gave higher values as compared to RIA. Furthermore, whereas all of the human sera contained NCA which was measurable by RIA, 67 sera typed negative in EIA when using Mab 4 or 18. Eight additional sera were negative in more than one EIA. Negativity when using Mab 33 was observed in only one serum, which was also negative with Mab 4 and 18. Twenty-five of 30 sera which were negative with Mab 4 came from cancer patients, and 32 of 37 sera negative with Mab 18 came from normal subjects and noncancer patients, giving a statistically highly significant difference between the two groups of sera (p less than 0.001). Analysis of tissue perchloric extracts and NCA samples purified from these extracts gave similar results. Three extracts (one from lung, two from cancer tissue) and the corresponding NCA samples were negative with Mab 18. The discrepancies observed in these assays are best explained by assuming the existence of antigenic variants of NCA which have not been described previously. These variants appear to exist in various proportions in the different sera. The variants may represent antigenically complete and incomplete molecules. Alternatively, most of the NCA molecules may be incomplete, lacking one or another of the several NCA-specific epitopes. Sequential immunoprecipitation experiments were in favor of the second hypothesis, showing that most of the NCA molecules were incomplete, lacking either epitope A or B.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.     

Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors

In the striatum, stimulation of dopamine D2 receptors results in attenuation of glutamate responses. This effect is exerted in large part via negative regulation of AMPA glutamate receptors. Phosphorylation of the GluR1 subunit of the AMPA receptor has been proposed to play a critical role in the modulation of glutamate transmission, in striatal medium spiny neurons. Here, we have examined the effects of blockade of dopamine D2-like receptors on the phosphorylation of GluR1 at the cAMP-dependent protein kinase (PKA) site, Ser845, and at the protein kinase C and calcium/calmodulin-dependent protein kinase II site, Ser831. Administration of haloperidol, an antipsychotic drug with dopamine D2 receptor antagonistic properties, increases the phosphorylation of GluR1 at Ser845, without affecting phosphorylation at Ser831. The same effect is observed using eticlopride, a selective dopamine D2 receptor antagonist. In contrast, administration of the dopamine D2-like agonist, quinpirole, decreases GluR1 phosphorylation at Ser845. The increase in Ser845 phosphorylation produced by haloperidol is abolished in dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) knockout mice, or in mice in which the PKA phosphorylation site on DARPP-32 (i.e. Thr34) has been mutated (Thr34-->Ala mutant mice), and requires tonic activation of adenosine A2A receptors. These results demonstrate that dopamine D2 antagonists increase GluR1 phosphorylation at Ser845 by removing the inhibitory tone exerted by dopamine D2 receptors on the PKA/DARPP-32 cascade.