bHLH093/NFL and bHLH061 are required for apical meristem function in Arabidopsis thaliana.

The basic Helix-Loop-Helix (bHLH) transcription factors SCREAM/ICE1 and SCREAM2 have well-characterized roles in the terminal differentiation of stomatal guard cells in Arabidopsis thaliana. Here we report on the characterization of the functional roles of the remaining members of sub-group IIIB, bHLH093 and bHLH061. The bhlh093/bhlh061 double mutant failed to produce a primary inflorescence shoot and displayed greater phenotypic severity than bhlh093 and bhlh061 single mutants. An ultrastructural investigation revealed structural defects that develop in tissues surrounding the meristem prior to inflorescence emergence. The transition to flowering was restored in bhlh093/bhlh061 with the application of gibberellin to the apex. We also demonstrate that gibberellin application alleviates structural defects that develop in tissues surrounding the meristem and restore meristem activity. Furthermore, the bhlh093/bhlh061 double mutant was affected by delayed flowering, and the severity of the phenotype correlated with photoperiod and light intensity. Our results indicate that bHLH093 and bHLH061 function in the gibberellin-mediated establishment of functional apical meristems during the transition from vegetative to reproductive growth.

Generation and Functional Evaluation of Designer Monoterpene Synthases.

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.

Single centrosome manipulation reveals its electric charge and associated dynamic structure.

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.

Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy.

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 microm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50-300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of gamma-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.

Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.

Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes.

Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids, the most ancient and diverse class of natural products. Two distinct routes of IPP biosynthesis occur in nature: the mevalonate pathway and the recently discovered deoxyxylulose 5-phosphate (DXP) pathway. The evolutionary history of the enzymes involved in both routes and the phylogenetic distribution of their genes across genomes suggest that the mevalonate pathway is germane to archaebacteria, that the DXP pathway is germane to eubacteria, and that eukaryotes have inherited their genes for IPP biosynthesis from prokaryotes. The occurrence of genes specific to the DXP pathway is restricted to plastid-bearing eukaryotes, indicating that these genes were acquired from the cyanobacterial ancestor of plastids. However, the individual phylogenies of these genes, with only one exception, do not provide evidence for a specific affinity between the plant genes and their cyanobacterial homologues. The results suggest that lateral gene transfer between eubacteria subsequent to the origin of plastids has played a major role in the evolution of this pathway.

Management of patients needing antibiotic prophylaxis in a dental education setting.

The management of antibiotic prophylaxed (ABX) patients at an educational institution was evaluated to identify areas for improvement. Management criteria, reflecting guidelines to prevent oral-induced hematogenous microbial seeding, were pretested and applied to 1,225 record entries of eighty-five patients needing ABX for dental treatment between 1991 and 1996. Seven hundred twenty-two of the visits had 857 management or documentation problems, including no documentation indicating whether or not patients premedicated (n = 281); incomplete, insufficient, or repeated treatment (n = 214); and preventive concerns (n = 172), among others. The proportion of providers' patient visits with one or more management problems differed significantly (p < 0.001) by provider type, as did the distribution of problem categories (documentation, treatment, preventive, and scheduling concerns p < 0.001; compliance issues p < 0.005). Fifty-one percent of postgraduates' and 39 percent of faculty's record entries omitted patients' ABX status. Improved documentation, outcome measures, and patient, faculty and student education are indicated.

Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates.

To determine the molecular composition of the centrosome of a higher eukaryote, we carried out a systematic nano-electrospray tandem or MALDI mass spectrometry analysis of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. We have found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function.

Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes.

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.

Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: isopentenyl monophosphate kinase catalyzes the terminal enzymatic step.

In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including Escherichia coli, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (Mentha x piperita) and E. coli, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to approximately 308 aa with a mature size of approximately 33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and E. coli kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.

Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint.

Two distinct pathways are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids. The classical acetate/mevalonate pathway operates in the cytosol, whereas plastidial isoprenoids originate via a novel mevalonate-independent route that involves a transketolase-catalyzed condensation of pyruvate and D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate as the first intermediate. Based on in vivo feeding experiments, rearrangement and reduction of deoxyxylulose phosphate have been proposed to give rise to 2-C-methyl-D-erythritol-4-phosphate as the second intermediate of this pyruvate/glyceraldehyde-3-phosphate pathway (1-3). The cloning of an Escherichia coli gene encoding an enzyme capable of converting 1-deoxy-D-xylulose-5-phosphate to 2-C-erythritol-4-phosphate was recently reported (4). A cloning strategy was developed for isolating the gene encoding a plant homolog of this enzyme from peppermint (Mentha x piperita), and the identity of the resulting cDNA was confirmed by heterologous expression in E. coli. Unlike the microbial reductoisomerase, the plant ortholog encodes a preprotein bearing an N-terminal plastidial transit peptide that directs the enzyme to plastids where the mevalonate-independent pathway operates in plants. The peppermint gene comprises an open reading frame of 1425 nucleotides which, when the plastidial targeting sequence is excluded, encodes a deduced enzyme of approximately 400 amino acid residues with a mature size of about 43.5 kDa.

Genetic engineering of essential oil production in mint.

New approaches directed to unraveling monoterpene metabolism and secretion and recent progress in transformation protocols have set the stage for the systematic genetic engineering of essential oil production. This article focuses on specific strategies to improve the quality and quantity of mint essential oils.

Preferences for an influences on oral health prevention: perceptions of directors of nursing.

Directors of Nursing (DONs) from 196 of 206 Nebraska long-term-care (LTC) facilities were sent a pre-tested questionnaire. The aim was to assess available on-site dental services, existing oral health education and prevention programs as well as future needs/preferences, and the influence of 10 factors in assessing and maintaining residents' oral health. Of the 196 DONs contacted, 126 (64%) participated. Only 36% of DON responders reported having on-site dental services. DONs indicated a preference for nursing staff (NS) oral health inservice training over other educational and/or programmatic proposals. When asked to select the five most influential factors in assessing and maintaining residents' oral health in their respective facilities, DONs selected resident factors (mean, 2.8) more often than NS factors (mean, 2.2). DONs most often identified residents' (R') ability to perform oral hygiene (n = 99), R' cooperation with OH assistance (n = 98), R' interest in their oral health (n = 83), NS interest in R' dental health (n = 70), and NS time constraints (n = 69).

Monoaminergic effects of high-dose corticotropin in corticotropin-responsive pediatric opsoclonus-myoclonus.

Children with the opsoclonus-myoclonus syndrome (OMS) usually respond to corticotropin (adrenocorticotrophic hormone, ACTH) treatment but the mechanism of benefit is unknown. We previously showed that both cerebrospinal fluid (CSF) homovanillic acid (HVA) and 5-hydroxyindole-acetic acid (5-HIAA) concentrations are low in pediatric OMS. In this study, we measured levels of CSF Dopa, catecholamines, deaminated metabolites of catecholamines, as well as HVA and 5-HIAA in eight patients before and during treatment with ACTH. All the children were ACTH-responsive with 50-70% improvement in multiple clinical features of OMS. ACTH treatment reduced the HVA concentration in every child by a mean of 21% (p < 0.001). Treatment with ACTH was associated with significant correlations between dopaminergic markers such as HVA, dihydroxyphenylacetic acid (DOPAC), and Dopa. There were no significant changes in the CSF concentrations of the noradrenergic markers norepinephrine (NE) and dihydroxyphenylglycol (DHPG), or the serotonergic marker 5-HIAA. The only child with a marked inflammatory pattern in CSF, which was reversed by ACTH, was atypical for a large increase in NE and decrease in 5-HIAA during ACTH treatment. Beneficial effects of ACTH in OMS are not associated with normalization of HVA or 5-HIAA levels. The pattern of decreased HVA and unchanged DOPAC levels could reflect decreased extraneuronal uptake of catecholamines (which steroids inhibit) or decreased 0-methylation of catecholamines in nonneuronal cells.

A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.

Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.

Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase for shikonin biosynthesis in Lithospermum erythrorhizon cell suspension cultures.

The carbon skeleton of the naphthoquinone pigment shikonin, which is produced in Lithospermum erythrorhizon Sieb. et Zucc. cell-suspension cultures, is partly derived from the isoprenoid biosynthetic pathway. The requirement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34), a key enzyme of the mevalonate route to isoprenoids, for shikonin synthesis was investigated. Conserved regions of sequences from plant HMGR genes were used to design polymerase chain reaction (PCR) primers for the cloning of a cDNA fragment from L. erythrorhizon. The resulting 443-bp clone was used as a probe for Northern analyses and hybridized to an mRNA of approx. 2.5 kb. Under shikonin-producing conditions, microsomal HMGR enzyme activity as well as mRNA level closely correlated with the accumulation of shikonin derivatives. White light, which inhibits shikonin formation, was shown to strongly suppress HMGR gene expression. The results presented here indicate that HMGR plays a significant role in the regulation of shikonin biosynthesis and that the control appears to act at the transcriptional level.

A structural study of isolated mammalian centrioles using negative staining electron microscopy.

We have used a combination of centrifugation onto electron microscope grids and negative staining to study the structure of isolated mammalian centrioles. The technique relies on visualisation of structural detail by use of a goldthioglucose negative stain. The approach provides an easy structural definition of the mature and immature centriole and has revealed some novel proximal projections on the mature centriole. The rapid technique should prove of use in future analyses of centriolar structure and biochemistry.

Structure and function of the centriole in animal cells: progress and questions.

The centriole is a well-recognized, yet poorly understood, organelle present in many eukaryotic cells. Despite excellent electron-microscopic descriptions of its basic triplet microtubule structure, almost nothing is known of its specific molecular components. Here, Bodo Lange and Keith Gull survey centriole structure, duplication and maturation within the cell cycle and focus attention on the possible roles and function of centrioles as components of the centrosome in animal cells.