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Aims: Definition of sense and antisense microRNA matches in 3’utr. Background: Matches of mature microRNAs (m-miRs) in human 3’utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5’utr and cds should be by far fewer, but could follow similar patterns). Objective: To ascertain if the sense and antisense m-miR fragments in 3’utr occur at similar or different levels. Methods: Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. Results: The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3’utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3’utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. Conclusion: The m-mir matches of various sizes in 3’utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3’utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

Background: Same-nucleotide repeats (iterons) are strongly expressed in many DNA regions and RNA classes. These repeats serve importantly in association of polynucleotides and proteins, but have not been characterized in miRNAs. Methods: Iterons and nucleotide strings were quantified in currently known human miRNAs, including some comparisons with miRNAs of other species. Results: Human 5p miRNAs have significantly more G iterons than other miRNA groups. The 3p miRNAs have an inverse excess of C iterons. The miRNAs lacking functional counter-stems (which we differentiate as 5n or 3n by position in pre-miRNAs) also have a large excess of G iterons. In 5p miRNAs G and C iterons have much higher density in the seed compared to the post-seed region. This difference is lower in 5n and 3n sequences, and much lower in 3p sequences. In all groups the contiguous GC strings constitute a larger part of sequences than the AU strings. A surplus of G or C iterons and of GC strings should enable a more stable association with the target mRNAs. Conclusion: From the available evidence, the G iteron- and GC-rich miRNAs should also interact more readily with miRNA-processing and similar proteins.

Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25–28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (

The kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex were examined by stopped-flow fluorescence spectroscopy, using a modified version of the RecA protein that contains a fluorescent reporter group in the ssDNA binding site. The results indicated that: i) an active RecA-ssDNA complex was formed more rapidly on dT200 than on dT50 when either ATP or dATP was provided as the nucleotide cofactor, and ii) active complex formation occurred more rapidly with dATP than with ATP on either dT50 or dT200. The dependence on both the identity of the nucleotide cofactor and the length of the ssDNA effector indicated that active complex formation occurs by a cooperative mechanism and that dATP is more effective than ATP in mediating the interactions between RecA monomers that drive this process. Interestingly, the time courses of dATP-mediated active complex formation were closely similar to those that were obtained with ATPγS, an effectively non-hydrolyzable ATP analog that strongly stabilizes the active conformation of the RecA-ssDNA complex. These results provide mechanistic insight into the enhanced ssDNA binding and DNA strand exchange activities that are observed when dATP is provided in place of ATP in RecA biochemical assays.

Sulfur is vital for primary and secondary metabolism in plant roots. To understand the molecular and morphogenetic changes associated with loss of this key macronutrient, we grew Arabidopsis (Arabidopsis thaliana) seedlings in low-sulfur conditions. These conditions induced a cascade of cellular events that converged to produce a profound intracellular phenotype defined by large cytoplasmic inclusions. The inclusions, termed low-sulfur Pox, show cell type- and developmental zone-specific localization. Transcriptome analysis suggested that low sulfur causes dysfunction of the glutathione/ascorbate cycle, which reduces flavonoids. Genetic and biochemical evidence indicated that low-sulfur Pox are the result of peroxidase-catalyzed oxidation of quercetin in roots grown under sulfur-depleted conditions.

Highlights • Homoiterons like GGGGGGG stabilize ribosomal RNAs of thermophile prokaryotes. • In eukaryotes, homoiterons are much more abundant in RNA of the larger subunit (LSU). • The LSU repeats increase with phylogenetic rank to 28% entire RNA sequence in hominids. • In mammal LSU RNAs, these repeats constitute 45% of the massive expansion segments. • These repeats may help in anchoring of ribosomes and export of secretory proteins., Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

BACKGROUND Heptahelical G protein coupled receptors (GPCRs) support numerous sensory and metabolic functions and differ considerably in levels of expression. GPCR protein levels should link to regulation of GPCR mRNAs by microRNAs (miRs), which might significantly depend on numbers, size and GC content of the canonical antisense matches in mRNAs. These parameters of GPCR mRNAs have not been studied in detail. METHODS Canonical matching profiles of human GPCR mRNAs and miRs were examined using segments of 7-15 nucleotides in windows shifted by one position over the entire microRNA sequence. RESULTS Human GPCRs mRNAs within larger function-related groups have a quite homogenous matching with miRs. Both the GC content and the melting temperature (and hence also the binding energy) are appreciably higher in 5'utr compared to 3'utr matches of the same length. Increase in the GC content correlates significantly with length in the ubiquitous matches of 7-12 nucleotides. However, several GPCR groups strongly differ in overall match numbers and density. The untranslated regions of sensory receptor mRNAs, especially the olfactory and Taste-2 mRNAs, have the lowest match numbers and density and the fewest miR partners. The glucagon and frizzled families show the highest canonical matching. CONCLUSION Partnership of GPCR mRNAs and miRs could significantly relate to the type of function of the receptor proteins, with mRNAs of the sensory receptors having the lowest and those of metabotropic GPCRs the highest targeting. This could be of interest regarding GPCR regulation by exogenous miRs.

Background: Canonical hydrogen-bonding multi-nucleotide matches of microRNAs (miRs) with mRNAs are considered as important in mRNA regulation. MiR "seed" positions 2-8 are frequently viewed as mRNA partners, but there is ample evidence for use of other (and even non-contiguous) miR parts. No detailed information is available about canonical matching, and the GC content of the matches is rarely considered, although it should have a major regulatory potential. Methods: Sequences of 2586 human miRs and of 5'utr, cds and 3'utr in 18810 human mRNAs were examined for number and GC content of contiguous Watson-Crick antisense matches of six or more nucleotides (nt) in successive windows shifted by 1 nt. Results: Frequency of the antisense matches is within all sectors similar for segments of up to 10 nt starting at positions 1-10 of miR sequences, with decrease of 3.5 to 4-fold for each 1-nt increment. Adenine and uracil rich elements (ARE-like) are very frequent in cds and 3'utr. All mRNAs have matches of up to 10 nt, and most also those of 11-15 nt. The match density is largest in 5'utr, and the match number in cds. The 5'utr and cds matches average much higher GC content than those of 3'utr. The GC content of matches is above that for the whole sector in 5'utr and cds, but lower in 3'utr. Conclusion: Human mRNA matches across miR sequences constitute a positionally similar matrix of canonical hydrogen-bonding reactivity. This presents ample opportunities for contiguous binding independent of miR position. The ubiquitous 10 to 15-nt matches could serve as binding foci. Interaction of miRs with the abundant GC-rich 5'utr and cds counterparts could be important in the regulation of mRNA-ribosome interaction as well as in mRNA disposal. The lower density and GC content of a majority of 3'utr matches could mainly support a dynamic regulation by miRs.

Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that serine and asparagine are functionally interchangeable at position 240. The S240K mutation, in contrast, essentially eliminated the ability of the RecA protein to utilize ATP as a nucleotide cofactor. The [S240K]RecA protein was able to catalyze the hydrolysis of dATP, however, suggesting that the absence of the 2′-hydroxyl group reduced an inhibitory interaction with the Lys240 side chain. Interestingly, the [S240K]RecA protein was able to promote an efficient LexA cleavage reaction but exhibited no strand exchange activity when dATP was provided as the nucleotide cofactor. This apparent separation of function may be attributable to the elevated S0.5 value for dATP for the [S240K]RecA protein (490 μM, compared to 20–30 μM for the wild type and [S240N]RecA proteins), and may reflect a differential dependence of the LexA co-protease and DNA strand exchange activities on the nucleotide cofactor-mediated stabilization of the functionally-active state of the RecA–ssDNA complex.

The T-cell death-associated gene 8 (TDAG8) is a pH-sensing GPCR with a reported immune-specific expression profile. Here, we demonstrate pH-induced activation of TDAG8 receptor cloned from rodent brain (rTDAG8). Cloned rTDAG8 transcript showed 88–95% homology with human and mouse transcripts of lymphoid origin. RT-PCR revealed high expression of TDAG8 in forebrain limbic regions. Extracellular acidification induced significantly elevated intracellular cyclic AMP, and phosphorylated CREB in TDAG8 expressing cells. Acidification-induced LDH release was significantly attenuated in cells expressing TDAG8, suggesting neuroprotective potential against acidosis-related cell injury. Our results open up new areas of investigation into the relevance of TDAG8 in pH homeostasis and pathological states associated with acid-base dys-regulation in the brain such as ischemia and panic disorder.

The heptahelical G protein coupling receptors oligomerize extensively via transmembrane domains, in association with heterotrimeric G proteins. This provides higher affinity for agonists, conformational stability necessary for signal transduction, and protection from intracellular proteinases. The oligomerization is relevant to organismic pathophysiology and could be targeted by natural or modified agonists. For Supplement material, please see the online version of the article.

With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.

The neuropeptide Y (NPY) Y2 receptors and the pancreatic polypeptide Y4 receptors from rabbit kidney cortex are isolated largely as approximately 180 kDa complexes constituted of one receptor dimer and one G-protein heterotrimer, similar to NPY receptors expressed in the Chinese hamster ovary (CHO) cells. As expected, kidney and CHO cell Y2 dimers are converted into monomers by increasing concentrations of a selective agonist. Prevalence of dimeric Y2 receptors in the kidney could be related to low plasma levels of Y2 agonists, and possibly also to a relatively low concentration of Gi alpha subunits.

Treatment with pertussis toxin in addition to a stable inhibition of G(i)alpha subunits of G-proteins also strongly reduced human neuropeptide Y Y(1) receptors expressed in Chinese hamster ovary (CHO) cells. This was reflected in abolition of the inhibition by Y(1) agonists of forskolin-stimulated adenylyl cyclase in intact cells, and of Y(1) agonist stimulation of GTPgammaS binding to particulates from disrupted cells. The loss of both receptor and G(i)alpha subunit function was attenuated by ammonium chloride, an inhibitor of acid proteinases, pointing to a chaperoning co-protection of active pertussis toxin-sensitive Galpha subunits and Y(1) receptors. The surface complement of the Y(1) receptor was changed a little in conditions of approximately 85% decrease of the Y(1) population, but the rate of the Y(1) receptor-linked internalization of agonist peptides was reduced about 70%. The preserved receptor fraction consisted of monomers significantly coupled to G(q)alpha subunits. The persistent pertussis toxin-insensitive internalization of agonists with the Y(1) receptor may reflect a rescue or alternative switching that could be important for cell functioning in neuropeptide Y-rich environments. The results are compatible with a loss, due to G(i)alpha subunit inactivation by the toxin, of a large Y(1) receptor reserve constituted of oligomers associating with heterotrimeric G-proteins.

The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)-Y(2) receptor (38-40%). The pharmacological profile of rGIR was investigated using (125)I-PYY(3-36), a Y(2)-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y(2) receptor, in that the Y(2)-selective C terminus fragments of NPY and PYY (NPY(3-36) and PYY(3-36)) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY(3-36)>PYY(3-36)=NPY>NPY(13-36)>Ac, Leu NPY(24-36)>[D-Trp(32)]-NPY>Leu(31), Pro(34)-NPY=hPP. NPY and Y(2)-selective agonists NPY(3-36) and PYY(3-36) led to significant activation of (35)S-GTPgammaS binding to rGIR transfected cells. BIIE0246, a specific Y(2) antagonist, displaced (125)I-PYY(3-36) binding to rGIR with high affinity (95nM). Activation of (35)S-GTPgammaS binding by Y(2)-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY-Y(2) receptor.

Attention deficit hyperactivity disorder (ADHD) is a complex, multifactorial disorder characterized by physical hyperactivity and behavioural disinhibition. Short interval cortical inhibition (SICI), measured in motor cortex with transcranial magnetic stimulation, is reduced in ADHD and correlates with symptom severity. However, ADHD medication-induced changes in SICI vary widely among normal individuals and have not been well studied in children with ADHD. Therefore, we undertook this study to measure and compare effects of two ADHD medications, methylphenidate (MPH), a psychostimulant, and atomoxetine (ATX), a selective norepinephrine reuptake inhibitor, on SICI in children with ADHD. In addition, we wished to determine whether a genetic variation in the dopamine transporter (DAT1), a site of action of MPH, could influence the effects of MPH or ATX on SICI. We performed a randomized, double-blind, single-dose, crossover study comparing 0.5 mg/kg MPH with 1.0 mg/kg ATX in 16 children with ADHD, aged 8-17. Seven were homozygotes and 9 heterozygotes for the DAT1 variable number of tandem repeats 10-repeat allele. Medication and genotype effects on SICI were estimated with repeated measures, mixed model regression. We found that MPH and ATX had similar effects on SICI. However, medication effects differed significantly by DAT1 genotype [ F (2,13) = 13.04, P = 0.0008]. Both MPH and ATX increased SICI in heterozygotes but not in 10-repeat homozygotes. In conclusion, MPH and ATX have similar effects on SICI in children with ADHD. A genetic variation in DAT1, previously linked to ADHD risk and MPH behavioural responses, influences the neurophysiological effects of both MPH and ATX.

The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dT(n) oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of Mg2+, the results indicated that the SsbEc protein was able to bind to the dT(n) oligomers in the SSB(35) mode, with only two of the four subunits of the tetramer interacting with the dT(n) oligomers. In the presence of Mg2+, however, the results indicated that the SsbEc protein was bound to the dT(n) oligomers in the SSB(65) mode, with all four subunits of the tetramer interacting with the dT(n) oligomers. The SsbA protein behaved similarly to the SsbEc protein under all conditions, indicating that it undergoes Mg2+ -dependent changes in its DNA binding modes that are analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind to the dT(n) oligomers in an SSB(65)-like mode in either the presence or the absence of Mg2+, suggesting that it may not exhibit the pronounced negative intrasubunit cooperativity in the absence of Mg2+ that is required for the formation of the SSB(35) mode. Additional experiments with a chimeric SsbA/B protein indicated that the structural determinants that govern the transitions between the different DNA binding modes may be contained within the N-terminal domains of the SSB proteins.

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC 50 ∼ 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu 31 Pro 34 ) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu 31 Pro 34 ) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.

The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nucleoside triphosphate, ITP, as the nucleotide cofactor. However, although both reactions are stimulated by single-stranded DNA-binding (SSB) protein, the ITP-dependent reaction differs from the ATP-dependent reaction in that it is observed only at low SSB protein concentrations, whereas the ATP-dependent reaction proceeds efficiently even at high SSB protein concentrations. Moreover, the circular ssDNA-dependent ITP hydrolysis activity of the RecA protein is strongly inhibited by SSB protein (suggesting that SSB protein displaces RecA protein from ssDNA when ITP is present), whereas the ATP hydrolysis activity is uninhibited even at high SSB protein concentrations (because RecA protein is resistant to displacement by SSB protein when ATP is present). These results suggest that SSB protein does not stimulate the ITP-dependent strand exchange reaction presynaptically (by facilitating the binding of RecA protein to the circular ssDNA substrate) but may act postsynaptically (by binding to the displaced strand that is generated when the circular ssDNA invades the linear dsDNA substrate). Interestingly, the mechanistic characteristics of the ITP-dependent strand exchange reaction of the E. coli RecA protein are similar to those of the ATP-dependent strand exchange reaction of the RecA protein from Streptococcus pneumoniae. These findings are discussed in terms of the relationship between the dynamic state of the RecA-ssDNA filament and the mechanism of the SSB protein-stimulated three-strand exchange reaction.

Streptococcus pneumoniae is a naturally transformable bacterium that is able to incorporate DNA from its environment into its own chromosome. This process, known as transformational recombination, is dependent in part on themmsA gene, which encodes a protein having a sequence that is 40% identical to that of the Escherichia coli RecG protein, a junction-specific DNA helicase believed to be involved in the branch migration of recombinational intermediates. We have developed an expression system for the MmsA protein and have purified the MmsA protein to more than 99% homogeneity. The MmsA protein has DNA-dependent ATP hydrolysis and DNA junction-helicase activities that are similar to those of the E. coli RecG protein. The effect of the MmsA protein on the S. pneumoniae RecA protein-promoted three-strand exchange reaction was also investigated. In the standard direction (circular single-stranded (ss) DNA + linear double-stranded (ds) DNA → linear ssDNA + nicked circular dsDNA), the MmsA protein appears to promote the branch migration of partially exchanged intermediates in a direction opposite of the RecA protein, resulting in a nearly complete inhibition of the overall strand exchange reaction. In the reverse direction (linear ssDNA + nicked circular dsDNA → circular ssDNA + linear dsDNA), however, the MmsA protein appears to facilitate the conversion of partially exchanged intermediates into fully exchanged products, leading to a pronounced stimulation of the overall reaction. These results are discussed in terms of the molecular mechanism of transformational recombination.

The ATP-dependent three-strand exchange activity of the Streptococcus pneumoniae RecA protein (RecA(Sp)), like that of the Escherichia coli RecA protein (RecA(Ec)), is strongly stimulated by the single-stranded DNA-binding protein (SSB) from either E. coli (SSB(Ec)) or S. pneumoniae (SSB(Sp)). The RecA(Sp) protein differs from the RecA(Ec) protein, however, in that its ssDNA-dependent ATP hydrolysis activity is completely inhibited by SSB(Ec) or SSB(Sp) protein, apparently because these proteins displace RecA(Sp) protein from ssDNA. These results indicate that in contrast to the mechanism that has been established for the RecA(Ec) protein, SSB protein does not stimulate the RecA(Sp) protein-promoted strand exchange reaction by facilitating the formation of a presynaptic complex between the RecA(Sp) protein and the ssDNA substrate. In addition to acting presynaptically, however, it has been proposed that SSB(Ec) protein also stimulates the RecA(Ec) protein strand exchange reaction postsynaptically, by binding to the displaced single strand that is generated when the ssDNA substrate invades the homologous linear dsDNA. In the RecA(Sp) protein-promoted reaction, the stimulatory effect of SSB protein may be due entirely to this postsynaptic mechanism. The competing displacement of RecA(Sp) protein from the ssDNA substrate by SSB protein, however, appears to limit the efficiency of the strand exchange reaction (especially at high SSB protein concentrations or when SSB protein is added to the ssDNA before RecA(Sp) protein) relative to that observed under the same conditions with the RecA(Ec) protein.

Hair analysis for the major metabolite of cocaine, benzoylecgonine (BZE) was performed in a cohort of 251 predominantly African-American newborns utilizing a modified radioimmunoassay (RIA) (Abuscreen Roche Laboratories, Nutley, N.J., USA). Maternal drug intake was confirmed by positive urine drug screen for cocaine metabolites at the time of delivery. The BZE concentrations reported here are the composites of both the parent compound cocaine (hydrolyzed to BZE) and the native BZE metabolized by the patient and deposited in the hair follicle. Hair analysis as an indicator for gestational cocaine exposure had a sensitivity of 88%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 85%. The range of BZE was 99–23,300 ng/g of hair, with 79% having hair BZE levels

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

Background Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. Results We report the analysis of changes in the transcription of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes may represent metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of the main virulence associated genes or known human colonization factors. Here, we documented regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vWbp, encoded on SaPIbov5). Colonization with isogenic-deletion strains (Δvwbp and ΔscpA) did not alter the ex vivo nasal S. aureus colonization compared to wild type. Conclusions Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-915) contains supplementary material, which is available to authorized users.

Behavioral sensitization to psychostimulants involves neuroadaptation of stress-responsive systems. We have identified and sequenced a glucocorticoid-induced receptor (GIR) cDNA from rat prefrontal cortex. The full-length GIR cDNA encodes a 422 amino acid protein belonging to G-protein-coupled receptor superfamily. Although the ligand for GIR is still unknown, the dendrogram construction indicates that GIR may belong to peptide receptor subfamily (e.g., substance P receptor), with more distant relationship to subfamilies of glycoprotein hormone receptors (e.g., thyrotropin receptor) and biogenic amine receptors (e.g., dopamine receptor). GIR shares 31–34% amino acid identity to the tachykinin receptors (substance P receptor, neurokinin A receptor, and neurokinin B receptor). GIR mRNA is expressed preferentially in brain, and its neuronal expression is relegated to limbic brain regions, particularly in forebrain. GIR transcript levels are increased significantly and persistently in prefrontal cortex for 7 d after discontinuation of chronic amphetamine exposure. The induction of GIR expression by amphetamine is associated with augmented behavioral activation. These findings suggest that modulation of GIR expression may be involved in behavioral sensitization, and GIR may play a role at the interface between stress and neuroadaptation to psychostimulants.

The Escherichia coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for the RecA protein-promoted three-strand exchange reaction. An open reading frame encoding a protein similar in size and sequence to the E. coli SSB protein has been identified in the Streptococcus pneumoniae genome. The open reading frame has been cloned, an overexpression system has been developed, and the protein has been purified to greater than 99% homogeneity. The purified protein binds to ssDNA in a manner similar to that of the E. coli SSB protein. The protein also stimulates the S. pneumoniae RecA protein and E. coli RecA protein-promoted strand exchange reactions to an extent similar to that observed with the E. coli SSB protein. These results indicate that the protein is the S. pneumoniae analog of the E. coli SSB protein. The availability of highly-purified S. pneumoniae SSB protein will facilitate the study of the molecular mechanisms of RecA protein-mediated transformational recombination in S. pneumoniae.

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a circular bacteriophage phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution. These results indicate that the strand exchange activity of the [P67G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted strand exchange reaction that is dependent on ADP. It is likely that the [P67G/E68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type RecA protein.

The x-ray crystal structure of theEscherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence (66GPESSGKT73) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which serine 69 was replaced by a glycine residue. We have found that the [S69G]RecA mutation has a differential effect on the hydrolysis of various nucleoside triphosphates. The [S69G]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of rATP, ddATP, and dATP with turnover numbers of 10, 20, and 36 min−1, respectively. The wild type RecA protein, in contrast, hydrolyzes each of these nucleoside triphosphates with similar turnover numbers of 20–24 min−1. Significantly, the [S69G]RecA protein promotes strand exchange with all three nucleoside triphosphates, and the rate of strand exchange is directly proportional to the rate of hydrolysis of each of the nucleotide cofactors. These findings with the [S69G]RecA protein provide support for the existence of a mechanistic coupling between NTP hydrolysis and DNA strand exchange.

Objective To evaluate serotonin transporter protein (5HTPR) binding in platelets from children and adolescents with major depression (MDD) compared to normal controls using the selective ligand 3 H-paroxetine. Method Children and adolescents with MDD ( n = 24) defined by DSM-III-R criteria and normal controls ( n = 22) were compared by platelet 5HTPR kinetic analysis with the hypothesis that 5HTPR is reduced in MDD. A subset of MDD subjects ( n = 18) continued to participate in a fixed-dose, open-label sertraline trial for 6 weeks followed by drug-free washout and repeated 5HTPR analysis. Results Sex, prepubertal status, and age had no effect on 5HTPR. Medication-free MDD subjects differed from controls in reduced binding capacity (B max ) ( p p max elevation from baseline was associated with nonresponse and suicide attempt history. Conclusion Earlier literature in this population is replicated with regard to reduced platelet 5HTPR B max in MDD. Findings support a continuum of 5HTPR involvement in MDD across the developmental spectrum. J. Am. Acad. Child Adolesc. Psychiatry. 1998, 37(7):777–784.

Sindbis virus expression has been used for in vitro investigations of antigen processing, presentation and epitope mapping. The recent development of a replication-deficient recombinant Sindbis virus expression vector has made in vivo expression possible with minimal pathogenic risk. Advantages of Sindbis virus over other available viral systems include a comparatively smaller genome size making it possible to clone larger inserts, the ability to infect a wide range of host cell types with reduced pathogenicity for humans. These features suggest the possible utility of Sindbis virus for the in vivo delivery of genes to neural cells. We used the recombinant Sindbis viral expression system to target delivery of the lacZ gene to neuronal cells of mice by stereotactic surgery. Sindbis viral mRNA obtained by in vitro transcription was used to transfect baby hamster kidney (BHK) cells. As shown by histochemistry, beta-galactosidase (beta-gal) was expressed in approximately 50% of transfected cells. Cells were then cotransfected with DH-BB helper sequences that enabled the recombinant Sindbis virus RNA packaging. Nonreplicative Sindbis viral stock was collected 24 h after transfection. BHK cells were then infected with viral stock and histochemistry analysis was performed. Again, approximately 50% of the cells expressed beta-gal. The same viral stock was infused into the nucleus caudatus/putamen and nucleus accumbens septi and histochemical analysis of frozen sections from the relevant brain areas confirmed that beta-gal was expressed in neurons in a time-dependent manner. beta-Gal was detected at 24 h after inoculation and was present for at least 14 days, with maximum expression at 48 h. These results suggest that a nonreplicative Sindbis virus expression system may be useful for delivery of foreign genes into the central nervous system (CNS).

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways., The murine G protein-coupled receptor 83 (Gpr83) is expressed widely in the brain, but its physiological role is largely unknown. Here Müller et al. show that Gpr83 regulates systemic energy metabolism in part by modulating ghrelin signalling in the arcuate nucleus of the hypothalamus.

Previous studies of serotonin transporter protein (5HTPR) indexed in platelets by 3H-imipramine demonstrate reduction in children with comorbid obsessive-compulsive disorder (OCD) and Tourette's syndrome (TS).To use the 5HTPR selective ligand 3H-paroxetine and homogeneous diagnostic groups to reevaluate these findings.Platelet Kinetic binding parameters were evaluated using standard techniques from medication-free child and adolescent patients with OCD (n = 18), with TS (n = 10), and normal controls (n = 19).Baseline binding capacity (Bmax) was significantly reduced in patients with OCD (1,342 +/- 952 fmol/mg; protein p.01) compared with normal controls (2,486 +/- 1309 fmol/mg) and TS patients (2,420 +/- 1,069 fmol/mg; p.05). Among OCD patients who were subsequently treated on an open-label basis with selective serotonin reuptake inhibitor (SSRI), Bmax values at baseline differentiated between responders (1,718 +/- 1,041 fmol/mg) and nonresponders (802 +/- 713 fmol/mg protein; p.05). Response to SSRI was greatest in patients with a positive family history of OCD. Among responders (n = 10), baseline Yale-Brown Obsessive Compulsive Scale and Bmax were positively correlated (r = .76, p = .01), as was Clinical Global Impression (r = .67, p = .03).Platelet 5HTPR capacity (Bmax) is reduced in children and adolescents with OCD, but not in those with TS. 5HTPR may be an indirect measure of basal serotonergic tone.

Metabolic labeling experiments were performed using eukaryotic cells transfected with the human dopamine (DA) transporter cDNA. Autophosphorylation in the presence and absence of the transporter substrate DA, was analyzed. Dopamine transporter (DAT) was phosphorylated in the absence of DA and dephosphorylated in the presence of the substrate. The functional significance of this phenomenon was checked by incubating cells with phosphorylation promoting agents, all of which reduced substrate uptake and ligand binding significantly. It is shown that at least one site of phosphorylation on DAT is a serine residue. These experiments suggest that the state of phosphorylation of the DAT may play an important role in its biological function.

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.

Site-directed mutagenesis was used to replace His-163 in the Loop 1 region of the recA protein with a tryptophan residue. The [H163W]recA protein binds single-stranded DNA (ssDNA), catalyzes ssDNA-dependent ATP hydrolysis, and is fully active in the three-strand exchange reaction. In addition, the fluorescence properties of the Trp-163 reporter group are very sensitive to the binding of nucleotide cofactors to the H163W]recA-ssDNA complex. The fluorescence of Trp-163 is modestly quenched by the binding of ADP (21%) and strongly quenched by the nonhydrolyzable ATP analog, ATP gamma S (70%); since ADP and ATP gamma S stabilize the closed and open conformations of the recA-ssDNA complex, respectively, the quenched states observed with these nucleotides likely reflect differences in the fluorescence properties of tryptophan 163 in these two states. ATP has a more complex time-dependent effect on Trp-163 fluorescence. When ATP is added to [H163W]recA-ssDNA complexes, there is an immediate quenching of Trp-163 fluorescence (44%) which is intermediate in intensity between that observed with ADP and ATP gamma S. The ATP-induced quenching gradually decreases with time as the pool of ATP is converted to ADP by the ATP hydrolysis activity of the [H163W]recA protein. These results are discussed with regard to the nucleotide cofactor-dependent conformational transitions of the recA-ssDNA complex.

Background Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated. Methodology/principal findings The complexes that were formed by the SsbA and SsbB proteins on dT(n) oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dT(n) oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dT(n) oligomers via a "shared-strand binding" mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins. Conclusion/significance These results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.

The cerebral vasculature of five anaesthetised rabbits was perfused with a perfluorocarbon emulsion via the internal carotid arteries, and the effluent from the jugular veins analysed for ATP, substance P (SP), endothelin (ET) and arginine vasopressin (AVP). Viability of the preparation was monitored periodically by the electrocorticogram, oxygen uptake, carbon dioxide release and perfusion pressure. The basal rate of infusion of 7.8±1.26ml·min−1 resulted in an infusion pressure of 114.0±22.1 mmHg and when increased first to 10.5±1.53ml·min−1 and then to 15.0±1.87ml·min−1, rose to 163.0±33.1mmHg and to 170.0±33.2mmHg, respectively. Between each 3-min period of increased flow the rate was returned to the basal rate for 6 min. of the four vasoactive substances, ET was released at the largest rate during the initial period of basal flow, 65.3±10.7pmol·min−1. This increased further when the infusion rate rose to 10.5 ml·min−1, but was significant only when the infusion rate was increased to 15.0 ml·min−1. ATP was released at 41.5±11.5pmol·min−1 during the initial period of basal flow. Its release significantly increased with flow and peaked at 15.0 ml·min −1. SP was released at a rate of 13.3±8.2pmol·min−1 during the initial period of basal flow. Its rate of release was increased significantly the second time the flow was increased to 10.5 ml·min−1 and increased even further when the flow was increased to 15.0 ml·min−1. AVP was detected in one third of the samples collected during the initial period of basal flow, being released at a rate of 0.65±047pmol·min−1. Only in the period following the 15.0 ml·min−1 rate of flow was there a sustained increased release of AVP. These results are consistent with the view that ATP, SP, ET and AVP are released from vascular endothelial cells during changes in flow, and may contribute to the adjustments in local cerebrovascular tone.

We recently described two mutant recA proteins, (G160N)recA and (H163A)recA, which have full single-stranded DNA-dependent ATP hydrolysis activity but which are unable to promote the ATP-dependent strand exchange reaction under standard reaction conditions (pH 7.5). These mutant proteins, however, are able to promote strand exchange at pH 6.0 to 6.8. Here we show that this activation correlates with a pH-dependent decrease in the S0.5 value for ATP, with the (H163A)recA protein becoming active in strand exchange at pH values where the S0.5(ATP) decreases below 100 microM. We also show that the (H163A)recA protein is active in strand exchange over the range of pH 6.0-8.2 if dATP (or ddATP) is used in place of ATP as a cofactor; dATP is hydrolyzed by (H163A)recA protein at the same rate as ATP but has an S0.5 value lower than 100 microM across this pH range. These results are discussed with regard to the general significance of the S0.5 value in determining whether a nucleoside triphosphate will be able to stabilize the recA-single-stranded DNA filament in the strand exchange active conformational state.

The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted. In the presence of dsDNA, in contrast, there was a kinetic lag before a linear phase of ATP hydrolysis was achieved. The nonlinear kinetics of the dsDNA-dependent ATP hydrolysis reaction could be modeled by a kinetic scheme in which helicase II undergoes a time-dependent transition from an ATPase-inactive to an ATPase-active form. Order of addition experiments indicated that this transition was not due to a rate-limiting association event between helicase II and any other component of the reaction. Instead, agarose gel assays showed that progressive unwinding of the dsDNA occurs during the same time period as the lag phase of the ATP hydrolysis reaction. No significant ATP hydrolysis was observed when the linear dsDNA was replaced with closed circular dsDNA, suggesting that the ATP hydrolysis reaction requires a dsDNA substrate that can be unwound to the complementary single strands. These results are consistent with a model in which the lag phase of the dsDNA-dependent ATP hydrolysis reaction corresponds to progressive unwinding of the dsDNA, with the ATP hydrolysis reaction arising from helicase II molecules that are bound to the separated single strands.

We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine. Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties. Both the [H97A]recA protein and the [K248A]recA protein bind poorly to single-stranded DNA, have no single-stranded DNA-dependent ATP hydrolysis activity, and do not promote renaturation of complementary single-stranded DNA molecules or the ATP-dependent three-strand exchange reaction. Furthermore, both mutant proteins are defective in Mg(2+)-induced helical filament formation. To account for these results, we propose that the mutation of either histidine 97 or lysine 248 alters subunit interactions between recA monomers and that this leads to the loss of cooperative single-stranded DNA binding and DNA pairing activities. This proposal is consistent with the recently determined x-ray structure of the recA protein, which shows that although histidine 97 and lysine 248 are distant from one another in the monomer structure, these two residues are on the opposing complementary faces of the recA subunit and pack against each other at the interface between adjacent recA monomers in the helical filament (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

For many G-protein coupling receptors (GPCRs), the upkeep of receptor dimers could depend on association with functional Gi α subunits. This is known for Y1, Y2 and Y4 neuropeptide Y receptors [presented in the companion paper (Estes et al., Amino Acids, doi: 10.1007/s00726-010-0642-z , 2010)]. Interactions with transducers use mainly intracellular domains of the receptors. Intracellular loops 1 and 2 in GPCRs are short and lack extensive helicity that could support transducer anchoring. Interaction with G-proteins is known to use the juxtamembrane Helix 8 in the fourth intracellular domain, for which we document a helix-stabilizing n/(n + 4) pattern of large hydrophobic sidechains. Another intracellular helix located in the C-terminal portion of the third intracellular loop does not display a strong stabilizing pattern, and is found in many studies to serve dynamically in association and activation of transducers and effectors. We show that these tracts share features across metazoan phyla not only in opsins and opsin-like receptors (including the Y receptors), but also in Taste-2 and Frizzled receptors. Similarities of these helices across GPCR groups could have both phylogenetic and functional roots.

A concentration-dependent elevation of intracellular calcium ([Ca2+]i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (or = 100 microM in 1 ml with 1 x 10(6) cells). Oxidation of NAD(P)H preceded a rise in [Ca2+]i. The elevation of [Ca2+]i was reversible at50 microM tBOOH exposure and the return to the steady state [Ca2+]i correlated temporally with repletion of NAD(P)H. At50 microM tBOOH, the changes in NAD(P)H and [Ca2+]i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at50 microM tBOOH, oxidation of NADPH predominated, while at50 microM tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca2+]i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca2+]i at50 microM tBOOH; (ii) the sustained elevation of [Ca2+]i at50 microM tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca2+]i did not depend on influx of extracellular Ca2+. We speculate that at low tBOOH, Ca2+ was released from the NADPH/NADP(+)-sensitive mitochondrial Ca2+ pool while higher tBOOH caused additional Ca2+ release from GSH/GSSG-sensitive nonmitochondrial Ca2+ pools with sustained elevation of [Ca2+]i due to decreased mitochondrial Ca2+ reuptake.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and carcinogenic studies, and considered to be nutritionally adequate, were compared with respect to neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling B6C3F1 mice were fed either AIN-76A (a purified diet) or NIH-07 (a natural ingredient diet), with or without 2-AAF, for up to 2 years. Dosages of 2-AAF were administered to males at 0, 40, 60, or 80 ppm in each diet and to females at 0, 150, 200, or 250 ppm. Each group consisted of 96 mice, except the groups of females dosed at 0 and 150 ppm, which consisted of 120 and 72 mice, respectively. The incidence of malignant liver tumors was significantly greater in all AIN-fed groups compared to corresponding NIH-fed groups, as was the total incidence of tumors (malignant + benign). Similarly, the incidence of malignant and total bladder tumors was greater in AIN-fed groups of mice administered the two high doses of 2-AAF for each sex compared to NIH-fed groups. No malignant bladder tumors were observed among any of the groups of mice receiving control diets. This was also true for the males on low dosages (40 ppm) of 2-AAF. The AIN-fed group of low dose females (150 ppm) developed 4% incidence of malignant bladder tumors. This study dramatically showed the importance of diet selection in chronic carcinogenic studies and suggests that results obtained with the use of a purified diet may differ both qualitatively and quantitatively from results obtained with a natural ingredient diet.

We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue. The new [Ala-163]recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein. Despite being proficient in ssDNA-dependent ATP hydrolysis, the [Ala-163]recA protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions, pH 7.5. The [Ala-163]recA protein does exhibit three-strand exchange activity at pH 6.0-7.0, however, and the induction of strand exchange activity at low pH correlates directly with the activation of an ATP-dependent isomerization of the mutant protein. Thus, the [Ala-163]recA protein is functionally similar to our previously described mutant [Asn-160]recA protein (Bryant, F.R. (1988) J. Biol. Chem. 263, 8716-8723; Muench, K.A., and Bryant, F. R. (1990) J. Biol. Chem. 265, 11560-11566). Trypsin proteolysis studies indicate that the [Ala-163]recA and [Asn-160]recA proteins, like the wild-type recA protein, are organized into carboxyl-terminal and amino-terminal domains of nearly equal size. According to this structural model, the [Ala-163]recA and [Asn-160]recA mutations may lie in a linker region joining these two domains. We speculate that the [Ala-163]recA and [Asn-160]recA mutations interfere with an ATP-dependent conformational change of the recA protein that perhaps involves a change in the relative orientation of the carboxyl-terminal and amino-terminal domains.

We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F. R. (1988) J. Biol. Chem. 263, 8716-8723). Although the [Asn-160]recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP). We have found that the [Asn-160]recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH. The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the [Asn-160]recA protein. This ATP-dependent isomerization is characterized by the conversion of the [Asn-160]recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein. In contrast to the pronounced pH sensitivity of the [Asn-160]recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4. These results show that the [Asn-160] mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the [Asn-160]recA protein (or the [Asn-160]recA-ssDNA complex) relieves this mechanistic defect. Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the [Asn-160]recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism.

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.