Structural insights into the ene-reductase synthesis of profens.

Reduction of double bonds of α,ß-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.

Natural Product Biosynthesis in Escherichia coli: Mentha Monoterpenoids.

The era of synthetic biology heralds in a new, more "green" approach to fine chemical and pharmaceutical drug production. It takes the knowledge of natural metabolic pathways and builds new routes to chemicals, enables nonnatural chemical production, and/or allows the rapid production of chemicals in alternative, highly performing organisms. This route is particularly useful in the production of monoterpenoids in microorganisms, which are naturally sourced from plant essential oils. Successful pathways are constructed by taking into consideration factors such as gene selection, regulatory elements, host selection and optimization, and metabolic considerations of the host organism. Seamless pathway construction techniques enable a "plug-and-play" switching of genes and regulatory parts to optimize the metabolic functioning in vivo. Ultimately, synthetic biology approaches to microbial monoterpenoid production may revolutionize "natural" compound formation.

Episodic memory and autonoetic consciousness: a first-person approach.

Episodic memory is identified with autonoetic consciousness, which gives rise to remembering in the sense of self-recollection in the mental re-enactment of previous events at which one was present. Autonoetic consciousness is distinguished from noetic consciousness, which gives rise to awareness of the past that is limited to feelings of familiarity or knowing. Noetic consciousness is identified not with episodic but with semantic memory, which involves general knowledge. A recently developed approach to episodic memory makes use of 'first-person' reports of remembering and knowing. Studies using this approach have revealed many independent variables that selectively affect remembering and others that selectively affect knowing. These studies can also be interpreted in terms of distinctiveness and fluency of processing. Remembering and knowing do not correspond with degrees of confidence in memory. Nor does remembering always control the memory response. There is evidence that remembering is selectively impaired in various populations, including not only amnesic patients and older adults but also adults with Asperger's syndrome. This first-person approach to episodic memory represents one way in which that most elusive aspect of consciousness, its subjectivity, can be investigated scientifically. The two kinds of conscious experiences can be manipulated experimentally in ways that are systematic, replicable and intelligible theoretically.

Impact of encoding depth on awareness of perceptual effects in recognition memory.

Pictorial stimuli are more likely to be recognized if they are the same size, rather than a different size, at study and at test. This size congruency effect was replicated in two experiments in which the encoding variables were respectively undivided versus divided attention and level of processing. In terms of performance, these variables influenced recognition and did not influence size congruency effects. But in terms of awareness, measured by remember and know responses, these variables did influence size congruency effects. With undivided attention and with a deep level of processing, size congruency effects occurred only in remembering. With divided attention and with a shallow level of processing, size congruency effects occurred only in knowing. The results show that effects that occur in remembering may also occur independently in knowing. They support theories in which remembering and knowing reflect different memory processes or systems. They do not support the theory that remembering and knowing reflect differences in trace strength.

Episodic memory and remembering in adults with Asperger syndrome.

A group of adults with Asperger syndrome and an IQ-matched control group were compared in remember versus know recognition memory. Word frequency was also manipulated. Both groups showed superior recognition for low-frequency compared with high-frequency words, and in both groups this word frequency effect occurred in remembering, not in knowing. Nor did overall recognition differ between the two groups. However, recognition in the Asperger group was associated with less remembering, and more knowing, than in the control group. Since remembering reflects autonoetic consciousness, which is the hallmark of an episodic memory system, these results show that episodic memory is moderately impaired in individuals with Asperger syndrome even when overall recognition performance is not.

Memory illusions: false recall and recognition in adults with Asperger's syndrome.

As persons on the autistic spectrum are known not to use semantic features of word lists to aid recall, they might show diminished susceptibility to illusory memories that typically occur with lists of associated items. Alternatively, since such individuals also have poor source monitoring, they might show greater susceptibility. The authors found that adults with Asperger's syndrome (n = 10) recalled similar proportions of a nonpresented strong associate of the study list items, compared with controls (n = 15). In Experiment 2, rates of true and false recognition of study list associates did not differ significantly between Asperger (n = 10) and control (n = 10) participants. Moreover, the Asperger participants made fewer remember and more know judgments than controls for veridical but not for false recognitions. Thus, deficits found in some aspects of memory in people with Asperger's syndrome do not affect their susceptibility to memory illusions.

Response deadline and subjective awareness in recognition memory.

Level of processing and generation effects were replicated in separate experiments in which recognition memory was tested using either short (500 ms) or long (1500 ms) response deadlines. These effects were similar at each deadline. Moreover, at each deadline these effects were associated with subsequent reports of remembering, not of knowing. And reports of both knowing and remembering increased following the longer deadline. These results imply that knowing does not index an automatic familiarity process, as conceived in some dual-process models of recognition, and that both remembering and knowing increase with the slower, more controlled processing permitted by the longer response time.

Conjoint dissociations reveal involuntary "perceptual" priming from generating at study.

Incidental perceptual memory tests reveal priming when words are generated orally from a semantic cue at study, and this priming could reflect contamination by voluntary retrieval. We tested this hypothesis using a generate condition and two read conditions that differed in depth of processing (read-phonemic vs read-semantic). An intentional word-stem completion test showed an advantage for the read-semantic over the generate condition and an advantage for the generate over the read-phonemic condition, and completion times were longer than in a control test, prior to which there was no study phase. An incidental word-stem completion test showed equivalent priming for the read-semantic and read-phonemic study conditions, despite considerable power, and completion times were no longer than control, indicating that retrieval was involuntary, and insensitive to prior conceptual processing. The generate condition produced less priming than the read conditions, but significant priming nonetheless. The results show that priming from generating can be involuntary and suggest that lexical processes are responsible. They are also the first results conjointly showing a crossed double dissociation, a single dissociation, and a parallel effect across memory tests with identical physical retrieval cues.

Depth-of-processing effects on priming in stem completion: tests of the voluntary-contamination, conceptual-processing, and lexical-processing hypotheses.

Depth-of-processing effects on incidental perceptual memory tests could reflect (a) contamination by voluntary retrieval, (b) sensitivity of involuntary retrieval to prior conceptual processing, or (c) a deficit in lexical processing during graphemic study tasks that affects involuntary retrieval. The authors devised an extension of incidental test methodology--making conjunctive predictions about response times as well as response proportions--to discriminate among these alternatives. They used graphemic, phonemic, and semantic study tasks, and a word-stem completion test with incidental, intentional, and inclusion instructions. Semantic study processing was superior to phonemic study processing in the intentional and inclusion tests, but semantic and phonemic study processing produced equal priming in the incidental test, showing that priming was uncontaminated by voluntary retrieval--a conclusion reinforced by the response-time data--and that priming was insensitive to prior conceptual processing. The incidental test nevertheless showed a priming deficit following graphemic study processing, supporting the lexical-processing hypothesis. Adding a lexical decision to the 3 study tasks eliminated the priming deficit following graphemic study processing, but did not influence priming following phonemic and semantic processing. The results provide the first clear evidence that depth-of-processing effects on perceptual priming can reflect lexical processes, rather than voluntary contamination or conceptual processes.

Experiences of remembering, knowing, and guessing.

This article presents and discusses transcripts of some 270 explanations subjects provided subsequently for recognition memory decisions that had been associated with remember, know, or guess responses at the time the recognition decisions were made. Only transcripts for remember responses included reports of recollective experiences, which seemed mostly to reflect either effortful elaborative encoding or involuntary reminding at study, especially in relation to the self. Transcripts for know responses included claims of just knowing, and of feelings of familiarity. These transcripts indicated that subjects were often quite confident of the accuracy of their decisions, compared with those for guess responses. Transcripts for decisions associated with guess responses also expressed feelings of familiarity but additionally revealed various strategies and inferences that did not directly reflect memory for studied items. The article concludes with a historical and theoretical overview of some interpretations of the states of awareness measured by these responses.

The therapeutic potential of synthetic multivalent carbohydrates.

Recent progress has been made in constructing synthetic multivalent carbohydrate ligands targeted at known or probable multivalent carbohydrate ligand receptors. Multivalency can dramatically compensate for the weak affinities of individual carbohydrate ligand moieties. Multivalent branched ligand mimetics have been described bearing multiple mannoside, galactoside, lactoside and Sialyl Lewis X (SLe(x)) moieties. New methods for polymeric backbone construction have provided glyco-polymers of defined and controllable size. Promising examples of multivalent ligands giving significant increases in binding potency are being pursued. These types of agents have extensive therapeutic potential in treating infectious diseases or host abnormalities, and also offer exciting potential as drug-conjugate cell-targeting agents.

Molecular and genetic analysis of REC103, an early meiotic recombination gene in yeast.

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, ME14, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SK18, a gene that depresses the expression of yeast double-stranded ("killer") (ds)RNA viruses. REC103/SK18 is transcribed in mitotic cells and is induced approximately 15-fold in meiosis. REC103 has 26% amino acid identity to the Schizasaccharomyces pombe rec14+ gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.

Asperger's syndrome and memory: similarity to autism but not amnesia.

Two experiments are described in which the memory of adults with Asperger's syndrome is compared with that of verbal IQ controls. The results of the first experiment showed that the Asperger subjects resembled autistic adults and children in their failure to use category information to aid their free recall. In the second experiment, both groups of subjects showed similar priming effects in an implicit stem completion task and similar performance on an explicit cued recall task. Moreover, both groups also showed more priming for items that they had read at study and better recall for items that they had to generate at study, suggesting that the cued recall of the Asperger subjects did not result from contamination by automatic or involuntary processes.

Changes in memory awareness during learning: the acquisition of knowledge by psychology undergraduates.

First-year psychology students took multiple-choice examinations following each of 4 lecture courses and 3 laboratory research methods courses. One lecture course was later retested. Students indicated state of memory awareness accompanying each answer: recollective experience (remember), "just know" (know), feeling of familiarity (familiarity), or guess. On the lecture courses, higher performing students differed from other students because they had more remember responses. On research methods, higher performing students differed because they knew more, and in the delayed retest, higher performing students differed because they now knew rather than remembered more. These findings demonstrate a shift from remembering to knowing, dependent upon level attained, type of course, and retention interval, and suggest an underlying shift in knowledge representation from episodic to semantic memory. The authors discuss theoretical and educational implications of the findings.

Cloning maize telomeres by complementation in Saccharomyces cerevisiae.

Maize telomeric restriction fragments were cloned by virtue of their ability to function as telomeres on a linear plasmid in Saccharomyces cerevisiae. Nine maize telomeric YAC transformants (MTYs) were selected by hybridization to the Arabidopsis telomere repeat (CCCTAAA) from a pool of 1537 primary transformants. Bal31 digestion of MTY3 and MTY9 DNA indicated that the telomere hybridizing tracts are located at the terminus of the linear chromosome and therefore function as telomeres in yeast. Subclones generated for pMTY7 (pMTY7SC1) and pMTY9 (pMTY9ER) hybridized to Bal31 sensitive restriction fragments in maize DNA, indicating that maize telomeric restriction fragments had been cloned. Both pMTY7SC and pMTY9ER detected telomeric RFLPs, allowing the endpoints of seven chromosome arms to be determined. Additionally, pMTY7ER mapped to the centromeric regions of chromosomes 2 and 3, suggesting a relationship between centromeric and telomeric sequences. DNA sequencing of pMTY7SC and pMTY9ER revealed that both subclones contained CA-rich regions with sporadic occurrences of the telomere repeat and its degenerate repeats.

Physical and genetic mapping of chromosome 9S in maize using mutations with terminal deficiencies.

Deletion mapping was employed to determine the physical order of five morphological variants, pyd1, yg2, wd1, v28 and u31, with respect to restriction fragment length polymorphism (RFLP) markers located at the distal end of chromosome 9S in maize. The genetic materials used were a series of terminal-deficiency mutants, newly derived with McCLintock's original stocks developed in the 1940s, via breakage-fusion-bridge cycles. A combined physical map and genetic map has been constructed based on data gathered from both genetic complementation tests and RFLP analysis. The location of v31 in relation to RFLP markers was further determined by interval mapping. The physical distance between the healed telomeric end and the most distal RFLP marker in two terminal-deficiency lines was established by using pulsed field gel electrophoresis and verified by Bal31 digestion. The results from this study set a foundation for studies on the mechanism of healing of broken chromosome ends in higher plants.

The effect of acetylation and deacetylation on the disposition of dapsone and monoacetyl dapsone hydroxylamines in human erythrocytes in-vitro.

The fates of both dapsone and monoacetyl hydroxylamine have been studied in terms of acetylation and deacetylation within the human erythrocyte in-vitro. A comparison between the two metabolites showed equipotency in methaemoglobin generation at 15 min, although the monoacetyl derivative was the more rapid haemoglobin oxidizer. Within the erythrocytes, both dapsone and monoacetyl hydroxylamines were found to undergo acetylation, deacetylation and diacetylation. Of the inhibitors of acetylation studied, folate caused an increase in methaemoglobin formation associated with both metabolites, which led to a rise in both acetylated and non-acetylated amine formation. Amethopterin was associated with a rise in hydroxylamine mediated methaemoglobin formation which coincided with a fall in acetylated products. It is possible that the hydroxylamines undergo erythrocytic processes of acetylation and deacetylation before methaemoglobin-mediated reduction to their respective amines.

Acyclic nucleosides as antiviral compounds.

Acyclovir is an effective drug for the treatment of HSV and VZV infections, which after phosphorylation to the triphosphate, inhibits viral DNA polymerase. Acyclovir has low oral bioavailability, therefore prodrugs have been developed, and the L-valyl ester, valaciclovir, recently has been licensed for the treatment of shingles. Ganciclovir is used against CMV, and famciclovir, a lipophilic prodrug of penciclovir, is marketed for shingles. The acyclic nucleoside phosphonates are active against thymidine kinase-resistant viral strains. Promising analogs are PMEA (in clinical trial for the treatment of AIDS) and (S)-HPMPC (good in vivo activity against HSV, VZV, CMV, and EBV). Oligonucleotides incorporating acyclic nucleosides at the 3'-and 5'-ends, or constituted of amino acyclic nucleosides, are resistant to cleavage by nucleases and may be useful in antisense and/or antigene therapy. HEPT is active against HIV-1: It binds in a hydrophic pocket on reverse transcriptase, rather than in the polymerase active site. Some acyclic nucleosides are potent inhibitors of purine and pyrimidine nucleoside phosphorylase. These compounds may have a therapeutic niche in combination therapy with antiviral and anticancer nucleosides, and in the treatment of diseases involving the T-cell.

CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane.

The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats against 9,10-dimethyl-1,2-benz[a]anthracene tumorigenesis, thought to be due, at least in part, to induction of phase II detoxification. We investigated the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible for activation of several carcinogens, including dialkylnitrosamines. Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown to be a potent competitive inhibitor of CYP2E1 with a Ki of 37.0 +/- 4.5 microM. In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA). Sulforaphane at concentrations of > 0.8 microM inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone. Unscheduled DNA synthesis induced by NDMA (33.5 microM) in mouse hepatocytes was inhibited in a dose-dependent manner by sulforaphane at 0.064-20 microM. Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 micrograms/plate), a direct acting mutagen, in the Salmonella assay. It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of Salmonella employed and cytotoxic only at high concentrations (> or = 0.5 mM). These findings suggest that inhibition of CYP2E1 by sulforaphane may offer chemoprotection against carcinogenic substrates of this enzyme.