Dopamine system genes associated with parenting in the context of daily hassles

The current study examined the molecular genetic foundations of sensitive parenting in humans and is the first to test the interaction between genes and environment in modulating parental sensitive responses to children. In a community sample of 176 Caucasian, middle class mothers with their 23-month-old toddlers at risk for externalizing behavior problems, the association between daily hassles and sensitive parenting was investigated. We tested whether two dopamine-related genes, dopamine D4 receptor ( DRD4 ) and catechol-O-methyltransferase ( COMT ) gene polymorphisms, modulate parents' vulnerability to the negative influence of daily hassles on sensitive parenting behavior to their offspring. Sensitive parenting was observed in structured settings, and parents reported on their daily hassles through a standard questionnaire. In parents with the combination of genes leading to the least efficient dopaminergic system functioning ( COMT val/val or val/met, DRD4 -7Repeat), more daily hassles were associated with less sensitive parenting, and lower levels of daily hassles were associated with more sensitive parenting d  = 1.12. The other combinations of COMT and DRD4 polymorphisms did not show significant associations between daily hassles and maternal sensitivity, suggesting differential susceptibility to hassles depending on parents' dopaminergic system genes. It is concluded that the study of (multiple) gene–environment interactions (in the current case: gene by gene by environment interaction, G × G × E) may explain why some parents are more and others less impacted by daily stresses in responding sensitively to their offspring's signals.     

The effect of daily hassles on salivary IgA: experimental evidence.

The study aimed at evidencing the suppressive effect of daily hassles on salivary immunoglobulin A (S-IgA); in fact, previous research did not detect a clear relationship between the two variables. Twenty-four subjects were tested as to the hassles that had occurred in their recent life and to S-IgA; the tests were repeated three times, with an interval of four weeks between each. The results show that the variation of S-IgA values between times 2 and 3 is associated with the variation of hassles between times 1 and 2 (r = -.46, p < .01).     

The "second stalk" of Escherichia coli ATP synthase: structure of the isolated dimerization domain

The b subunit of E. coli F(0)F(1)-ATPase links the peripheral F(1) subunits to the membrane-integral F(0) portion and functions as a "stator", preventing rotation of F(1). The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b(62-122). Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b(62-122) dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 A, and a radius of gyration of 27 A, values that are consistent with an alpha-helical coiled-coil structure. The crystal structure of b(62-122) has been solved and refined to 1.55 A. The protein crystallized as an isolated, monomeric alpha helix with a length of 90 A. Combining the crystal structure of monomeric b(62-122) with SAXS data from the dimer in solution, we have constructed a model for the b(62-122) dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.     

Role of Mood in Outcome of Biofeedback Assisted Relaxation Therapy in Insulin Dependent Diabetes Mellitus

Stressful life events and negative mood have been associated with elevated blood glucose and poor self-care in individuals with diabetes. The purpose of this controlled study was to determine the effect of mood state, specifically depression, anxiety, and daily hassles on the outcome of biofeedback assisted relaxation in insulin dependent diabetes mellitus. Eighteen subjects completed the study, nine in biofeedback assisted relaxation and nine in the control group. There were no significant group differences in blood glucose between those receiving biofeedback assisted relaxation and the subjects continuing usual care. Five of the nine experimental subjects and one of the nine control subjects were identified as succeeders according to an arbitrary criterion. Treatment failures were more depressed, more anxious, and took longer to complete the protocol than succeeders. Statistically significant correlations were found between high scores on inventories measuring depression, anxiety, and hassles intensity and higher blood glucose levels and smaller changes in blood glucose as a result of treatment. It is suggested that mood has an important impact on the response to biofeedback assisted relaxation. Further research is necessary to determine whether assessment of anxiety and depression followed by appropriate treatment where necessary should precede biofeedback assisted relaxation in insulin dependent diabetes.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

Population genetic structure,diversity and stocking effect of the oriental weatherloach (<Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in an isolated island

Genetic endemism of island organisms and the threat to such organisms provided by artificially introduced genes are aspects of major interest in evolutionary and conservation studies of fishes. In this paper the genetic population structure of the oriental weatherloach, Misgurnus anguillicaudatus, in Sado Island of Japan was elucidated by phylogeographic analysis based on partial mitochondrial control region sequences. The specimens were sampled at 62 sites in Sado Island and 14 sites on the mainland close to the island. We found various haplotypes of different origins, most of which had already been reported from the mainland and other places of Japan. This suggests that the loach has been historically introduced to the island from various regions of Japan. Of the 62 sites on the island, cultured/nonnative individuals were confirmed to have been stocked at eight specific sites for feeding of re-introduced Japanese crested ibis (Nipponia nippon). By a Mantel test, geographical and genetic distances were not significantly correlated among the local populations in Sado Island. However a significant correlation was found when the eight stocked local populations were excluded from the analysis. This implied that the genetic distribution pattern of the loach on the island has been disturbed by the stocking. In addition, the nucleotide diversity values of stocked local populations were significantly higher than those of other local populations, also a likely outcome of the stocking. In conclusion, the loach on the island likely had their origins in multiple historical introductions and colonizations, where more recent stocking for the ibis has caused further genetic disturbance to their local populations.     

Voltammetry of a "protein on a rope"

A periplasmic electron-transfer protein, cytochrome c(555)(m) from Aquifex aeolicus contains a 62-residue N-terminal extension by which it is anchored to the membrane--most probably via a thioester bond to its N-terminal cysteine. This linker can act as a "rope" to tether the protein close to its reaction partners. Mimicking this principle, a recombinant cytochrome c(555)(m), expressed in Escherichia coli, has been attached covalently to a gold electrode modified with 6-mercaptohexan-1-ol. The "tethered" cytochrome c(555)(m) displays remarkably fast electron-transfer kinetics, with an electrochemical exchange rate constant k(0) of 1.4 x 10(4) s(-1). The results show that fast electron transfer is associated with weak interactions: importantly, the tethered cytochrome can explore many different orientations without escaping into solution.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Current "corrected" calcium concept challenged.

There is wide individual variation in the number of millimoles of calcium bound per gram of albumin in the serum. Individual regression coefficients for serum calcium concentration on serum albumin concentration have been determined in 62 people (25 of our own patients and 37 reported by others). The 95 percentile range was 0-007-0-053 mmol/g, with a median value of 0-025 mmol/g. Accordingly, it is not valid to "correct" a person''s measured serum total calcium concentration for variations in serum albumin concentration using an average regression coefficient. Rather, the individual''s own regression coefficient must be used. A tourniquet test seems to be the simplest technique for determining this value. Even then, precise interpretation of an individual''s corrected serum calcium concentration is possible only when an appropriate reference range for corrected serum calcium concentration has been established. Such an appropriate reference range must be determined from an adequate number of normal people using the individual''s own correction factor in each case.     

Genealogy of the "Green Revolution" gene in rice

During the "Green Revolution" of rice, high-yielding varieties (HYVs) were developed using a semi-dwarf gene (sd1 or OsGA20ox2). The presence or absence of the two mutant alleles (DGWG type in Dee-geo-woo-gen and JKK type in Jikkoku) were surveyed by PCR using 256 accessions of eight wild and two cultivate rice species. The DGWG allele was detected in a landrace (Oryza sativa) and two accessions of wild rice (O. rufipogon), all of which are from China, showing their limited distribution. Genealogical studies of the OsGA20ox2 gene showed that the 62 sequences of O. sativa and O. rufipogon included 20 distinct haplotypes, indicating that the species complex contained OsGA20ox2 genes from two different lineages. The silent site nucleotide diversities (pi and theta(w)) were extremely low in Japonica rice, suggesting a genetic bottleneck. The haplotype network showed that the DGWG and JKK alleles were derived in different lineages. The DGWG carrier (W1944) had unique polymorphisms in the surrounding region of the locus, suggesting that the DGWG allele has been preserved in the wild progenitor, rather than that the DGWG allele has been introgressed from HYVs to W1944. Although a semi-dwarfing plant is a weak competitor under saturated fields, the crossing experiment revealed that the DGWG variant might have been preserved as a hidden variation in the genetic background of wild rice, without expressing a short-stature.     

Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C(delta) and atom Lys61-C(beta) (2.92 A) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.     

Fourier-transform infrared studies of CaATPase partitioning in phospholipid mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 with 1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-stearoyl-2-oleoylphosphatidylcholine

CaATPase from rabbit sarcoplasmic reticulum has been reconstituted into binary lipid mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1,2-dipalmitoylphosphatidylcholine-d62 (DPPC-d62) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC)/DPPC-d62. Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor temperature-induced structural alterations in the individual lipid components in the presence and absence of protein. A simple two-state model is used to construct a phase diagram that is in good agreement with one constructed from differential scanning calorimetry data, for the POPE/DPPC-d62 (protein-free) system. Although these two lipids are miscible over at least most of the composition range, substantial deviations from ideal behavior are observed. An estimate of the nonideality of mixing in both the gel and liquid-crystalline phases is obtained from regular solution theory. The phase diagram for SOPC/DPPC-d62 shows gel-phase immiscibility. FT-IR studies of ternary (POPE/DPPC-d62/CaATPase) complexes indicate that both lipid components are disordered by protein at all temperatures studied. In addition, their melting events are broadened and shifted to lower temperatures compared with the appropriate binary lipid mixture. Semiquantitative estimates for the fraction of each lipid melted are obtained from the model. The effect of protein on SOPC/DPPC-d62 mixtures depends on that total lipid to protein ratio. At low protein levels, SOPC is preferentially selected by CaATPase, so that bulk lipid is enriched in DPPC-d62. At high levels of protein, both lipid components are selected. The applicability of vibrational spectroscopy for determination of the partitioning preferences of membrane proteins into regions of particular chemical structure or physical order in a complex lipid environment is demonstrated.     

Axoneme specialization embedded in a "generalist" beta-tubulin

The relationship between the primary structure of the beta-tubulin C-terminal tail (CTT) and axoneme structure and function is explored using the spermatogenesis-specific beta2-tubulin of Drosophila. We previously showed that all beta-tubulins used for motile 9 + 2 axonemes contain a conserved sequence motif in the proximal part of the CTT, the beta-tubulin axoneme motif. The differential ability of tubulin isoforms and abilities of beta2-tubulin C-terminal truncations to form axonemes led us to hypothesize that the axoneme motif is essential for axoneme formation and the distal half of the CTT was less important. The studies we report here indicate that it is not that simple. Unexpectedly, some changes in the core sequence of the axoneme motif did not disrupt formation of motile axonemes. And, while deletion of the distal CTT did not disrupt the ability to produce functional sperm [Popodi et al., Cell Motil Cytoskeleton 2005;62:48-64], changing the amino acid sequence in this region can. Thus both regions are important. The deep conservation of the axoneme motif in all eukaryotic groups implies that the presence of the sequence motif confers a functional advantage. The central pair is the axoneme structure most sensitive to perturbations in tubulin molecules; we hypothesize central pair assembly is facilitated by the presence of this motif. Our data reveal that beta2-tubulin has robust properties for axoneme assembly, and that axonemal specializations are embedded in both the CTT and the body of the beta2 molecule.     

A "glyconutrient sham"

The discipline of glycobiology contributes to our understanding of human health and disease through research, most of which is published in peer-reviewed scientific journals. Recently, legitimate discoveries in glycobiology have been used as marketing tools to help sell plant extracts termed "glyconutrients." The glyconutrient industry has a worldwide sales force of over half a million people and sells nearly half a billion dollars (USD) of products annually. Here we address the relationship between glyconutrients and glycobiology, and how glyconutrient claims may impact the public and our discipline.     

"Wide-open" 1.3 A structure of a multidrug-resistant HIV-1 protease as a drug target

This report examines structural changes in a highly mutated, clinical multidrug-resistant HIV-1 protease, and the crystal structure has been solved to 1.3 A resolution in the absence of any inhibitor. This protease variant contains codon mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90 that confer resistance to protease inhibitors. Major differences between the wild-type and the variant include a structural change initiated by the M36V mutation and amplified by additional mutations in the flaps of the protease, resulting in a "wide-open" structure that represents an opening that is 8 A wider than the "open" structure of the wild-type protease. A second structural change is triggered by the L90M mutation that results in reshaping the 23-32 segment. A third key structural change of the protease is due to the mutations from longer to shorter amino acid side chains at positions 82 and 84.     

Signal integration and diversification through the p62 scaffold protein

Signal specificity of multifunctional enzymes is achieved through protein-protein interactions involving specific domains on scaffold proteins. p62 (also known as sequestosome 1) is such a scaffold protein that possesses PB1 and UBA domains, and the TRAF6 binding sequence. Proteins recruited to these domains enable p62 to integrate kinase-activated and ubiquitin-mediated signaling pathways. The biological function of p62 has been studied in diverse systems and processes such as osteoclastogenesis, inflammation, differentiation, neurotrophin biology and obesity. The availability of mice in which p62 has been genetically inactivated is providing new insight into the mechanism and function of p62 at a whole-organism level.     

HLA-A29 sub-types and "Birdshot" choroido-retinopathy susceptibility: a possible "resistance motif" in the HLA-A29.1 molecule]

The Birdshot choroidoretinopathy (BSCR) is an ocular disease strongly associated with HLA-A29. The HLA-A29 specificity can be split using immunoelectrofocusing in two subtypes A29.1 and A29.2. BSCR susceptibility is exclusively linked to the HLA-A29.2 molecule. The sequence of HLA-A29.2 was established (EMBL X60108 and found to be identical between patients and healthy individuals. A single difference was found (H----D) 102) in the extra cellular domains between HLA-A29.2 and HLA-A29.1. The HLA-A29 sub-types shares the consensus HLA class I sequence (D102). The mutation exhibited by HLA-A29.1 (H102) is unique to that molecule. The ancestral type is thus HLA-A29.2 that confers the susceptibility to BSCR whereas HLA-A29.1 has arisen from a more recent mutation conferring resistance to BSCR. Another single amino-acid difference between HLA-A29.1 and HLA-A29.2 was found in the intracytoplasmic part of the molecule, HLA-A29.2 exhibiting the HLA-A consensus sequence whereas A29.1 shares with AW33.1 the mutation S----F321. In addition, the A29 specificity was assigned to L and Q amino-acids at position 62-63, which can interact with peptides into the binding groove. No specific T or B epitope of susceptibility could be considered involving the region of the mutation discriminating HLA-A29.2 from HLA-A29.1. The HLA-A29.1 mutation is unable to interact with the T cell receptor and did not seem to induce significant structural changes in the peptide-binding groove. Conversely, its position suggests that the A29.1 mutation might interfere with the binding of an accessory molecule, the CD8 molecule being the most likely candidate for that role.     

Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight

Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU(gf.w.)(-1), which in turn decreased progressively to 4-5 log CFU(gf.w.)(-1) after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU(gf.w.)(-1), thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.     

Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement

HeLa cell p34, homolog of the yeast cdc2+/CDC28 protein kinase, has been investigated. p34 was phosphorylated at two or more sites and existed in a complex with p13, the previously identified homolog of the suc1+ gene product of S. pombe. A fraction of the most highly phosphorylated form of p34 was also associated with p62, a newly identified protein that became phosphorylated in vitro. The phosphorylation state of p34, its association with p62, and the protein kinase activity of the complex were each subject to cell cycle regulation. In newly born cells early in G1, p34 was unphosphorylated, not associated with p62, and inactive as a protein kinase. Each of these conditions was reversed in G2 and the p34/p62 complex was maximally active as a protein kinase, with respect to both endogenous and exogenous substrates, during mitotic metaphase. p34 may act to regulate the G2/M transition in HeLa cells.