Reduction in creatine metabolites in macrophages exposed to small molecule analogues of the anti-inflammatory parasitic worm product ES-62.

ES-62, a protein secreted by Acanthocheilonema viteae, is anti-inflammatory by virtue of covalently attached phosphorylcholine (PC) residues and thus a library of drug-like small molecule analogues (SMAs) based on its PC moieties has been designed for therapeutic purposes. Two members, SMAs 11a and 12b, were previously found to suppress production of pro-inflammatory cytokines by mouse bone marrow-derived macrophages (BMMs) exposed to cytosine-phosphate-guanosine oligodeoxynucleotides (CpG), agonists for Toll-like receptor 9. In order to explore the mechanism of action underlying such activities, an untargeted mass spectrometry-based metabolomics screen was undertaken. Stimulation of BMMs with CpG produced significant metabolic changes relating to glycolysis and the TCA cycle but the SMAs had little impact on this. Also, the SMAs did not promote alterations in metabolites known to be associated with macrophage M1/M2 polarization. Rather, BMMs exposed to SMAs 11a or 12b prior to CpG treatment, or even alone, revealed downregulation of metabolites of creatine, a molecule whose major role is in the transport of high energy phosphate from the mitochondria to the cytosol. These data therefore provide insight into a possible mechanism of action of molecules with significant therapeutic potential that has not previously been described for parasitic worm products.

Testing small molecule analogues of the Acanthocheilonema viteae immunomodulator ES-62 against clinically relevant allergens.

ES-62 is a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that protects against ovalbumin (OVA)-induced airway hyper-responsiveness in mice by virtue of covalently attached anti-inflammatory phosphorylcholine (PC) residues. We have recently generated a library of small molecule analogues (SMAs) of ES-62 based around its active PC moiety as a starting point in novel drug development for asthma and identified two compounds - termed 11a and 12b - that mirror ES-62's protective effects. In this study, we have moved away from OVA, a model allergen, to test the SMAs against two clinically relevant allergens - house dust mite (HDM) and cockroach allergen (CR) extract. We show that both SMAs offer some protection against development of lung allergic responses to CR, in particular reducing eosinophil infiltration, whereas only SMA 12b is effective in protecting against eosinophil-dependent HDM-induced allergy. These data therefore suggest that helminth molecule-induced protection against model allergens may not necessarily translate to clinically relevant allergens. Nevertheless, in this study, we have managed to demonstrate that it is possible to produce synthetic drug-like molecules based on a parasitic worm product that show therapeutic potential with respect to asthma resulting from known triggers in humans.

The intergenic transcribed spacer region 1 as a molecular marker for identification and discrimination of Enterobacteriaceae associated with acute oak decline.

AIMS: We assessed the veracity of intergenic spacer region 1 (ITS1) ribotyping for the rapid, inexpensive and accurate identification of Brenneria goodwinii and Gibbsiella quercinecans that are associated with acute oak decline (AOD) in the UK. METHODS AND RESULTS: Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) were applied for the typing of ITS1 PCR amplicons from strains of B. goodwinii, G. quercinecans and related species (n = 34). The number and length of ITS1 amplicons varied significantly between strains. ITS1 profiles generated via PAGE were used to differentiate species using a neighbour-joining phylogram. The ITS1 phylogram was compared against DNA gyrase B (gyrB) gene sequences from the same strains, demonstrating that ITS1 ribotyping is as effective as gyrB at resolving G. quercinecans and B. goodwinii to the species level. CONCLUSIONS: The ITS1 gene has been successfully employed as a novel marker to resolve newly described AOD-associated Enterobacteriaceae, B. goodwinii and G. quercinecans, to species level. SIGNIFICANCE AND IMPACT OF THE STUDY: ITS1 ribotyping of B. goodwinii and G. quercinecans provides equivalent sensitivity to the current standard method for strain identification (sequence analysis of the gyrB gene), but with reduced processing time and cost. Furthermore, the ITS1 gene is widely applicable as a rapid and inexpensive typing system for Enterobacteriaceae.

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.

Control of protein translation by phosphorylation of the mRNA 5'-cap-binding complex.

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5'-cap-binding complex in plants.

Dynamic distribution of BIMG(PP1) in living hyphae of Aspergillus indicates a novel role in septum formation.

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimG(PP1) in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.

P34(cdc2) kinase is associated with cortical microtubules from higher plant protoplasts.

The cell cycle regulatory enzyme p34(cdc2) kinase is known to be localized to the preprophase band, the spindle and the phragmoplast, but not to interphase cortical microtubules. This was investigated further by mechanically cleaving substrate-attached protoplasts to leave plasma membrane disks bearing microtubules freed of nuclear and cytosolic signal. Antibodies to PSTAIRE and to specific C-terminal peptides of cdc2a, were used in immunofluorescence, protein blotting and immunogold electron microscopy to demonstrate that antigen is located on the cortical microtubules of carrot, tobacco BY-2 and Arabidopsis cells.

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

Cellular basis of shoot apical meristem development.

Shoot apical meristems are composed of proliferating, embryonic type cells, that generate tissues and organs throughout the life of the plant. This review covers the cell biology of the higher plant shoot apical meristem (SAM). The first section describes the molecular basis of plant cell growth and division. The genetic mechanisms, that operate in meristem function and the identification of several key regulators of meristem behavior are described in the second section, and intercellular communication and coordination of cellular behavior in the third part. Finally, we discuss some recent results that indicate interaction between the cellular regulators, such as the cell cycle control genes and developmental regulators.

G2/M-phase-specific transcription during the plant cell cycle is mediated by c-Myb-like transcription factors.

Plant B-type cyclin genes are expressed specifically in late G2- and M-phases during the cell cycle. Their promoters contain a common cis-acting element, called the MSA (M-specific activator) element, that is necessary and sufficient for periodic promoter activation. This motif also is present in the tobacco kinesin-like protein gene NACK1, which is expressed with timing similar to that of B-type cyclin genes. In this study, we show that G2/M-phase-specific activation of the NACK1 promoter also is regulated by the MSA element, suggesting that a defined set of G2/M-phase-specific genes are coregulated by an MSA-mediated mechanism. In a search for MSA binding factors by yeast one-hybrid screening, we identified three different Myb-like proteins that interact specifically with the MSA sequence. Unlike the majority of plant Myb-like proteins, these Myb proteins, NtmybA1, NtmybA2, and NtmybB, have three imperfect repeats in the DNA binding domain, as in animal c-Myb proteins. During the cell cycle, the level of NtmybB mRNA did not change significantly, whereas the levels of NtmybA1 and A2 mRNAs fluctuated and peaked at M-phase, when B-type cyclin genes were maximally induced. In transient expression assays, NtmybA1 and A2 activated the MSA-containing promoters, whereas NtmybB repressed them. Furthermore, expression of NtmybB repressed the transcriptional activation mediated by NtmybA2. Our data show that a group of plant Myb proteins that are structurally similar to animal c-Myb proteins have unexpected roles in G2/M-phase by modulating the expression of B-type cyclin genes and may regulate a suite of coexpressed genes.

Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis.

A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.

Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar Bright Yellow-2 cells.

Plants possess two major classes of cyclin-dependent kinases (CDK) with cyclin-binding motifs PSTAIRE (CDK-a) and PPTA/TLRE (CDK-b). Tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells are the most highly synchronizable plant culture, but no detailed analysis of CDK activities has been reported in this system. Here we describe isolation of new PPTALRE CDKs (Nicta;CdkB1) from Bright Yellow-2 cells and present detailed analysis of the mRNA, protein and kinase activity levels of CdkB1, and the PSTAIRE CDKA during the growth and cell cycles. CdkA and CdkB1 transcripts are more abundant in exponential than in stationary phase cells, but the two genes show strikingly different regulation during the cell cycle. CdkA mRNA and protein accumulate during G1 in cells re-entering the cell cycle, and immunoprecipitated histone H1 kinase activity increases at the G1/S boundary. Aphidicolin synchronized cells show the highest CDKA-associated histone H1 kinase activity during S-G2 phases, although CdkA mRNA and protein levels are not significantly regulated. In contrast, CdkB1 transcripts are present at very low levels until S phase and CDKB1 protein and kinase activity is almost undetectable in G1. CdkB1 mRNA accumulates through S until M phase and its associated kinase activity peaks at the G2/M boundary, confirming that transcription of PPTALRE CDKs is cell cycle regulated. We suggest that CDKA kinase activity likely plays roles at the G1/S phase boundary, during S phase, and at the G2/M phase transition, and that CDKB1 kinase activity is present only at G2/M.

Microinjection reveals cell-to-cell movement of green fluorescent protein in cells of maize coleoptiles.

During the evaluation of dual-purpose plant/fungal expression systems, we found that green fluorescent protein (GFP) has the ability to move from cell to cell in the epidermis of Zea mays L. cv. Mutator coleoptiles as well as into underlying cortical cells. Movement of GFP was observed both when DNA encoding GFP and bacterially expressed GFP were microinjected into epidermal cells. This suggests that GFP is capable of cell-to-cell movement. From experiments using dextrans of known molecular weight linked to fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate, we estimate that the plasmodesmata of these cells have a size exclusion limit < 4.4 kDa. Cell-to-cell GFP movement did not occur when GFP was altered to include a nucleus- or endoplasmic reticulum-retention sequence. The fact that these transcripts differ from that of cytoplasmic GFP by a small number of nucleotides suggests that the transcripts are not capable of movement, but movement of nucleic acid cannot be excluded. Since GFP is widely used to study cell-to-cell movement and to localize the expression of transgenes, caution should be exercised when interpreting results where GFP expression is used for localization.

A type 2A protein phosphatase gene from Aspergillus nidulans is involved in hyphal morphogenesis.

A PCR-based approach, using degenerate oligonucleotide primers, was used to isolate fragments of two genes encoding type 2A protein phosphatases from the filamentous fungus, Aspergillus nidulans. The complete genomic sequence of one of these genes, pphA, was isolated and characterised. The pphA gene was predicted to encode a 329-residue protein which is about 85% identical to mammalian protein phosphatase 2A. Ectopic expression of the wild-type pphA+ gene slightly inhibited growth in some transformants; but a mutant form of pphA, in which R259 was mutated to Q, led to slow growth, delayed germ tube emergence and mitotic defects at low temperature. These results indicate that the pphA+ gene plays an important role in hyphal growth.

The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled.

D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.

Social controls on cell proliferation in plants.

Plant cell division occurs mainly in developing tissues and appears to be highly regulated in both space and time. Recently, genetic and molecular analyses have been able to dissect the function of cell proliferation in the processes of growth and development. Mutant studies have shown that plants have a compensatory mechanism whereby increased cell expansion can partially cover for defects in proliferation. Ectopic expression of developmental and cell-cycle regulators has indicated how growth rate is controlled at the molecular level in meristems and lateral organs.

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

Acute neurologic deterioration following lumbar puncture in an epidural abscess occurring 14 months after epidural catheter placement.

We report the case of a 19-year-old girl admitted to the hospital with a 2-month history of back pain and a 1-week history of severe weakness, who underwent a diagnostic lumbar puncture which was swiftly followed by acute neurologic deterioration requiring ventilation. She was subsequently shown to have an epidural abscess extending from the second cervical to the fifth lumbar vertebrae. She had received uneventful epidural analgesia for childbirth 14 months previously. The case is unusual in both the acute deterioration following lumbar puncture, and also in the length of time from epidural siting to abscess formation, if this were indeed the source of the infection.

Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2.

We have previously described the biochemical isolation of 65 kDa and 120 kDa microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAPs have subsequently been shown to be structural MAPs that reconstitute 30 nm cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devised a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the cold stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa MAPs are also associated with the cold-sensitive microtubules. On gradient gels, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC class known to move to the plus ends of microtubules. Antibodies raised against specific expressed sequences recognize the upper band, while the lower band is recognized by antibodies to the tobacco kinesin-related protein, TKRP125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homologous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cortical microtubules and the pre-prophase band, but anti-DcKRP120-2 does so only weakly. Both clearly stain the spindle and the phragmoplast, but in a proportion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-line where the plus ends of the microtubules overlap. We discuss the potential roles of these proteins during the microtubule cycle.