Experimental genetic approaches to addiction

Drugs of abuse are able to elicit compulsive drug-seeking behaviors upon repeated administration, which ultimately leads to the phenomenon of addiction. Evidence indicates that the susceptibility to develop addiction is influenced by sources of reinforcement, variable neuroadaptive mechanisms, and neurochemical changes that together lead to altered homeostasis of the brain reward system. Addiction is hypothesized to be a cycle of progressive dysregulation of the brain reward system that results in the compulsive use and loss of control over drug taking and the initiation of behaviors associated with drug seeking. The view that addiction represents a pathological state of reward provides an approach to identifying the factors that contribute to vulnerability, addiction, and relapse in genetic animal models.     

Continual production of phosphatidic acid by phospholipase D is essential for antigen-stimulated membrane ruffling in cultured mast cells

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.     

Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems

The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5 flanking sequences were fused to -glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (–358 to –211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (–211 to –80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.     

Proliferative response of peripheral blood lymphocytes to mitogens in the owl monkey Aotus nancymae

The new world primate Aotus sp. has been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and P. vivax. The present study examined the in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) isolated from Aotus monkeys, utilizing a wide range of mitogens. Results presented herein demonstrate that the in vitro proliferative response of PBMCs from the Aotus sp. is quite variable from monkey to monkey for each of the mitogens assessed. PBMCs from the Aotus monkey exhibited a delayed kinetic proliferative response and, particularly, a different sensitivity to proliferation in response to various concentrations of Phytohemagglutinin-P and favin lectins, the phorbol ester Phorbol myristate acetate and the calcium ionophore ionomycin. Altogether, our findings are consistent with the conclusion that the in vitro proliferative response of PBMCs from the Aotus differ in their activation requirements compared with PBMCs from humans.     

A marine dinoflagellate, Amphidinium eilatiensis n. sp., from the benthos of a mariculture sedimentation pond in Eilat, Israel

A species of Amphidinium bloomed in a mariculture sedimentation pond that was used to grow bivalves near the Gulf of Eilat, Israel. Its overall length averaged 13 microm, the hypocone was 11 microm, and its width was 8 microm. It has a ventral ridge. The sulcus begins at the longitudinal flagellar pore and does not project forward in the apex toward the transverse flagellar pore and left margin of the cingulum. The sulcus is a very shallow groove that projects variably about a third of the body length toward the antapex. The cingulum is a deep groove as it circles the cell from the left ventral side to the dorsal side and then becomes very shallow on the right ventral side as it arches posterior toward the longitudinal flagellar pore. Using a modified method for studying dinoflagellate chromosomes in the SEM, we observed 31 chromosomes. The plastid is dorsal and peripheral with 6 ventrally projecting peripheral digital lobes that wrap around the sides of the ventral and posterior nucleus. Amphidinium eilatiensis n. sp. is morphologically closest to Amphidinium carterae and Amphidinium rhynchocephalum, but it does not have the obvious thecal plates or polygonal units described for the former species. Instead, it has a series of spicules, bumps, and ridges on its surface. It differs from A. rhynchocephalum by two morphological characters: surface morphology and gross plastid architecture. The amplified fragments of the rDNA from A. eilatiensis n. sp. isolated from 2 separate sedimentation ponds in Eilat include the 3'- end of the SSU rDNA (about 100 nt), the whole ITS region (ITS1 + 5.8S + ITS2) and the 5'-end of the LSU rDNA (about 900 nts). The total length of the sequences ranged from 1,460 nt. (A. eilatiensis isolate #1) to 1,461 nts. (A. eilatiensis isolate #2). The latter sequences are identical, the difference in length being due to three insertions. Amphidinium eilatiensis is genetically more closely related to A. carterae than to A. klebsii, with respectively 2.36% and 6.93% of sequence divergence.     

Allometric cascade: a model for resolving body mass effects on metabolism

Expanding upon a preliminary communication (Nature 417 (2002) 166), we here further develop a "multiple-causes model" of allometry, where the exponent b is the sum of the influences of multiple contributors to control. The relative strength of each contributor, with its own characteristic value of b(i), is determined by c(i), the control contribution or control coefficient. A more realistic equation for the scaling of metabolism with body size thus can be written as BMR=MR(0)Sigmac(i)(M/M(0))(bi), where MR(0) is the "characteristic metabolic rate" of an animal with a "characteristic body mass", M(0). With M(0) of 1 unit mass (usually kg), MR(0) takes the place of the value a, found in the standard scaling equation, b(i) is the scaling exponent of the process i, and c(i) is its control contribution to overall flux, or the control coefficient of the process i. One can think of this as an allometric cascade, with the b exponent for overall energy metabolism being determined by the b(i) and c(i) values for key steps in the complex pathways of energy demand and energy supply. Key intrinsic factors (such as neural and endocrine processes) or ecological extrinsic factors are considered to act through this system in affecting allometric scaling of energy turnover. Applying this model to maximum vs. BMR data for the first time explains the differing scaling behaviour of these two biological states in mammals, both in the absence and presence of intrinsic regulators such as thyroid hormones (for BMR) and catecholamines (for maximum metabolic rate).     

Sequence and diversity of MHC <Emphasis Type="Italic">DQA</Emphasis> and<Emphasis Type="Italic"> DQB</Emphasis> genes of the owl monkey<Emphasis Type="Italic"> Aotus nancymaae</Emphasis>

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.