Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5'-diphosphate bound conformation

Li(+) is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li(+) competes with Mg2+ for the Mg2+ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7Li T1 relaxation measurements and fluorescence spectroscopy with the Mg2+ fluorophore furaptra, we detected Li(+)/Mg(2+) competition in three preparations: the purified G-protein transducin (Gt), stripped rod outer segment membranes (SROS), and SROS with purified Gt reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li+ in the presence of fixed amounts of Mg(2+), the apparent Li(+) binding constant decreased due to Li(+)/Mg(2+) competition. Whereas for SROS the competition mechanism was monophasic, for G(t), the competition was biphasic, suggesting that in G(t), Li(+)/Mg(2+) competition occurred with different affinities for Mg(2+) in two types of Mg(2+) binding sites. Moreover, as [Li(+)] increased, the fluorescence excitation spectra of both ROS-T and G(t) were blue shifted, indicating an increase in free [Mg(2+)] compatible with Li(+) displacement of Mg(2+) from two low affinity Mg(2+) binding sites of G(t). G(t) release from ROS-T membrane was also inhibited by Li(+) addition. In summary, we found evidence of Li(+)/Mg(2+) competition in G(t)-containing preparations.     

Sympatric natural Saccharomyces cerevisiae and S. paradoxus populations have different thermal growth profiles

Saccharomyces cerevisiae and its close congener S. paradoxus are typically indistinguishable by the phenotypic criteria of classical yeast taxonomy, but they are evolutionarily distinct as indicated by hybrid spore inviability and genomic sequence divergence. Previous work has shown that these two species coexist in oak-associated microhabitats at natural woodland sites in North America. Here, we show that sympatric populations of S. cerevisiae and S. paradoxus from a single natural site are phenotypically differentiated in their growth rate responses to temperature. Our main finding is that the S. cerevisiae population exhibits a markedly higher growth rate at 37 degrees C than the S. paradoxus population; we also find possible differences in growth rate between these populations at two lower temperatures. We discuss the implications of our results for the coexistence of these yeasts in natural environments, and we suggest that thermal growth response may be an evolutionarily labile feature of these organisms that could be analyzed using genomic approaches.     

A method for rapidly screening functionality of actin mutants and tagged actins

Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three β-actin mutants that have been associated with diseases. Published: October 25, 2004.     

Arsenobetaine in Atlantic salmon (Salmo salar L.): influence of seawater adaptation

Glycine betaine has been suggested to improve the maintenance of ionic and osmotic homeostasis during seawater adaptation in teleost fish. Arsenobetaine may also behave as an osmolyte, due to its structural similarity to glycine betaine. The influence of seawater adaptation on intestinal uptake and muscle accumulation of arsenobetaine in the teleost Atlantic salmon (Salmo salar L.) was investigated. Atlantic salmon (freshwater and seawater adapted) were given a single oral dose of arsenobetaine, which was absorbed over the intestine within 6 h after exposure. Seawater adapted Atlantic salmon had significantly higher levels of accumulated arsenobetaine in blood compared to the freshwater adapted salmon. However, seawater adaptation had no effect on the levels of accumulated arsenobetaine in muscle tissue. Similar retention of the administered dose was found in muscle tissue in both freshwater and seawater adapted salmon, with 49+/-6% and 50+/-10% retention after 144 h, respectively. Results indicate that muscle retention was not influenced by salinity in seawater adapting teleosts.     

Vertebrate organogenesis: getting the heart into shape

Recent mutant analysis in zebrafish points to an important role for oriented cell division in cardiac chamber formation and reveals its molecular control by a novel signal from the heart's interior.     

Motor physiology: a brain of two halves

Brain changes after stroke suggest that undamaged areas may 'take over' the function of damaged regions. Recent studies using magnetic stimulation to disrupt the healthy human brain shed new light on the potential for dynamic compensation across the motor system.     

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.     

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.