Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

How many sphygmomanometric cuff inflations are necessary to obtain a hemodynamic baseline?

The purpose of this study was to determine the minimum number of consecutive blood pressure cuff inflations required to obtain seated stable resting baseline measurements of heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP). Sixty male college students aged 18 to 31 years volunteered as study subjects. Thirteen observations of HR, SBP, DBP, and MAP were recorded at 90-second intervals for each subject using a Critikon-Dinamap monitor. Stable readings for SBP and MAP were obtained in 6.5 minutes or 3 to 5 cuff inflations in the population tested. Using this procedure, additional age- and gender-specific norms could be established for normal and hypertensive subjects. Knowing the approximate quantity and frequency of blood pressure cuff inflations needed to generate baseline minimum measurements of HR, SBP, DBP, and MAP will be helpful in studies of cardiovascular reactivity, as well as for clinical and psychophysiologic treatment of hypertension.     

Enhancement of antidepressant potency by a potentiator of AMPA receptors

1. AMPA receptor potentiators (ARPs) exhibit antidepressant-like activity in preclinical tests (for example, the forced swim test) that are highly predictive of efficacy in humans. Unlike most currently used antidepressants, ARPs do not elevate extracellular levels of biogenic amines (e.g., 5HT, NE) in prefrontal cortex at doses that are active in the forced swim test.2. The present series of experiments examined the effects of combining the ARP, LY 392098, with biogenic amine-based antidepressants in the forced swim test. Male, NIH Swiss mice were placed in a cylinder of water and observed for attempted escape behaviors and immobility.3. LY 392098 dose-dependently decreased immobility as did a range of classical antidepressants. At doses of LY 392098 below those that decreased immobility, this compound significantly increased the potency with which fluoxetine and citalopram (SSRI antidepressants), imipramine (tricyclic antidepressant), duoxetine (norepinephrine/serotonin uptake blocker), nisoxetine (norepinephrine uptake inhibitor), and rolipram (PDE4 inhibitor) decreased immobility in the forced swim test with potency shifts upward of 5-fold (fluoxetine, imipramine, and rolipram). Likewise, ineffective doses of the traditional antidepressants potentiated the effects LY 392098 with shifts in the dose-effect functions that were 10-fold or more for citalopram, fluoxetine, imipramine, and duloxetine.4. Combined with other evidence for a role of AMPA receptors in the efficacy of antidepressants, the current data suggest that the addition of an ARP may augment the activity and perhaps the onset of the therapeutic effects of biogenic amine and second messenger-based antidepressants.     

Association of neuregulin 1 with schizophrenia confirmed in a Scottish population

Recently, we identified neuregulin 1 (NRG1) as a susceptibility gene for schizophrenia in the Icelandic population, by a combined linkage and association approach. Here, we report the first study evaluating the relevance of NRG1 to schizophrenia in a population outside Iceland. Markers representing a core at-risk haplotype found in Icelanders at the 5' end of the NRG1 gene were genotyped in 609 unrelated Scottish patients and 618 unrelated Scottish control individuals. This haplotype consisted of five SNP markers and two microsatellites, which all appear to be in strong linkage disequilibrium. For the Scottish patients and control subjects, haplotype frequencies were estimated by maximum likelihood, using the expectation-maximization algorithm. The frequency of the seven-marker haplotype among the Scottish patients was significantly greater than that among the control subjects (10.2% vs. 5.9%, P=.00031). The estimated risk ratio was 1.8, which is in keeping with our report of unrelated Icelandic patients (2.1). Three of the seven markers in the haplotype gave single-point P values ranging from .000064 to .0021 for the allele contributing to the at-risk haplotype. This direct replication of haplotype association in a second population further implicates NRG1 as a factor that contributes to the etiology of schizophrenia.     

Competition between MutY and mismatch repair at A x C mispairs In vivo

We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A. C mispairs in vivo, converting them to G. C pairs. In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A. T-->G. C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type.     

Anthocyanin pigments and leaf flavonoids in the family araceae

Anthocyanins, variously identified in inflorescence, fruit, leaf or petiole of 59 representative species of the Araccae, are of a simple type. The most common pigment is cyanidin 3-rutinoside, while pelargonidin 3-rutinoside and cyanidin 3-glucoside are regularly present. Two rare pigments are: cyanidin 3-gentiobioside in Anchomanes and Rhektophyllum, both in the subfamily Lasioideae; and delphinidin 3-rutinoside in Schismatoglottis concinna. In a leaf survey of 144 species from 58 genera, flavone C-glycosides (in 82%) and proanthocyanidins (in 35–45%) were found as the major flavonoids. In the subfamily Calloideae, subtribe Symplocarpeae, flavonols replace glycoflavones as the major leaf components but otherwise flavonols are uncommon in the family (in 27% of the sample) and more usually co-occur with flavone C-glycosides. Two new flavonol glycosides were characterized from Lysichiton camtschatcense: kaempferol 3-(6-arabinosylgalactoside)and kaempferol 3-xylosylgalactoside. Simple flavones, luteolin and chrysoeriol (in 6%) were found only in the subtribes Arinae and Cryptocoryninae, subfamily Aroideae. Flavonoid sulphates were identified in only four taxa: glycoflavone sulphates in two Culcasia species and Philodendron ornatum and a mixture of flavone and flavonol sulphates in Scindapsus pictus. Caffeic ester sulphates were more common and their presence in Anthurium hookeri was confirmed. These results show that the Araceae are unusual amongst the monocots in their simple and relatively uniform flavonoid profile; no one subfamily is clearly distinguished, although at tribal level some significant taxonomic patterns are observed. The best defined groups are the subfamilies Lasioideae and Monsteroideae, and the tribes Symplocarpeae and Arophyteae, and the subtribe Arinae. The greatest chemical diversity occurs in Anthurium and Philodendron, but this may only reflect the fact that these are the two largest genera in the family. The origin and relationship of the Araccae to other monocot groups are discussed in the light of the flavonoid evidence.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.