Sterile H2O Research Articles

Antimicrobial activity of Angocin® Anti-Infekt N, a combination of nasturtium (Tropaeoli majoris herba) and horseradish (Armoraciae rusticanae radix)

Aims: Angocin® Anti-Infekt N (Repha, Langenhagen, Germany) is a native preparation comprised of the haulm of nasturtium (N) and the root of horseradish (H). The active ingredients are isothiocyanates that are cleaved from precursors (glucosinolates) after oral intake. Clinical trials have shown that this drug is effective for treatment of urinary and upper respiratory tract infections [1]. Objective: Antimicrobial susceptibility testing of the preparation. Methods: A gas-test was used to investigate the antimicrobial activity of the volatile mustard oils that are released by the preparation. Gently dried and ground N and H were applied to the lids of Mueller Hinton-agar plates (ratio of 2.5:1) and mixed with sterile H2O. Amounts of 3.31mg N/1.25mg H to 400mg/160mg were tested in 65mm, amounts of 200mg/80mg up to 1200mg/480mg in 95mm plates. Various clinically relevant bacterial species were tested (20 isolates each). The organisms were plated onto the agar plates and placed above the test substances. The plates were sealed with tape and incubated at 37° C for 92h. Colony forming units were counted after 24h and 92h, and the MIC90 was determined for each bacterial species. Susceptibilities were checked by use of synthetic mustard oils and with agar- and broth dilution methods. Results: H. influenzae: MIC90 50mg N/20mg H, M. catarrhalis: 100mg/40mg, E. coli: 400mg/160mg, P. aeruginosa: 400mg/160mg, P. vulgaris 400mg/160mg, MSSA and MRSA 400mg/160mg, S. pyogenes 400mg/160mg, S. pneumoniae 400mg/160mg, K. pneumoniae 1000mg/400mg. Conclusion: A broad in vitro antibacterial activity was found against both, gram-negative and gram-positive bacteria, including MRSA and P. aeruginosa. Given that one capsule of the preparation contains 200mg N/80mg H and that the advised daily intake averages out at 15–25 capsules, clinical efficacy can be explained.

Human Ikaros Function in Activated T Cells Is Regulated by Coordinated Expression of Its Largest Isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.

Caffeine Targets TOR Complex I and Provides Evidence for a Regulatory Link between the FRB and Kinase Domains of Tor1p

Caffeine Targets TOR Complex I and Provides Evidence for a Regulatory Link between the FRB and Kinase Domains of Tor1p

Gel Retardation Assays for DNA-binding Proteins

MATERIALS 10x Tris-glycine buffer TBE buffer may be used in place of Tris-glycine. 6x Gel-loading buffer I 32P-labeled control DNA 32P-labeled target DNA of >20 bp (specific activity 2 x 107 cpm/μg [ Labeling of the DNA fragment can be accomplished by phosphorylation (Dephosphorylation of DNA Fragments with Alkaline Phosphataseor Phosphorylating the 5' Termini of Oligonucleotides), filling in of a 3'-recessed end using a DNA polymerase (Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase Ior Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I), or by using PCR to incorporate radiolabeled nucleotides into the body of the probe (The Basic Polymerase Chain Reaction). Control nuclear extract or protein fraction Ficoll 400 (20% w/v) Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-μl aliquots at -20°C. Nuclear extract or Protein fraction(s) Prepare the extract by one of the methods described in Mapping Protein-binding Sites on DNA by DNase I Footprinting. Poly(dI-dC) (1 mg/ml) Gel Retardation Assays for DNA-binding Proteins -Sambrook and Rus... file:///C:/Users/MILI%20JOON/Documents/protocol/Electrophoresis,...

Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

Mutational Analysis of the Ras Converting Enzyme Reveals a Requirement for Glutamate and Histidine Residues

The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.

Mutator Phenotypes Caused by Substitution at a Conserved Motif A Residue in Eukaryotic DNA Polymerase δ

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.

Identification of proteins induced or upregulated by Fusarium head blight infection in the spikes of hexaploid wheat (Triticum aestivum)

Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley. It causes economic losses due to reduction in both yield and quality. Although FHB resistance has been well documented and resistant cultivars have been developed to reduce incidence and severity of FHB, there is a limited understanding of the molecular mechanisms involved in plant resistance against the infection and spread of F. graminearum. In the current study, 2-dimensional displays of proteins extracted from wheat spikelets infected with F. graminearum were compared with those from spikelets inoculated with sterile H2O. Fifteen protein spots were detected that were either induced (qualitatively different) or upregulated (quantitatively increased) following F. graminearum infection of spikelets of 'Ning7840', a resistant cultivar. These proteins were identified by LC-MS/MS analysis. Proteins with an antioxidant function such as superoxide dismutase, dehydroascorbate reductase, and glutathione S-transferases (GSTs) were upregulated or induced 5 d after inoculation with F. graminearum, indicating an oxidative burst of H2O2 inside the tissues infected by FHB. An ascorbate-glutathione cycle is likely involved in reduction of H2O2. Expression of proteins with highest similarity to dehydroascorbate reductase and TaGSTF5 (a glutathione S-transferase) differed following FHB infection in susceptible and resistant cultivars. A 14-3-3 protein homolog was also upregulated in FHB-infected spikelets. In addition, a PR-2 protein (beta-1, 3 glucanase) was upregulated in FHB-infected spikes, which is in accord with a previous study that analyzed transcript accumulation.

First report of fusarium wilt of eggplant caused by Fusarium oxysporum f. sp. melongenae in Turkey

In 2002 eggplant production in several glasshouses, polytunnels and fields was surveyed in the provinces of Adana and Mersin, southern Turkey. Some plants exhibited leaf chlorosis and slight vein clearing on outer leaflets, followed by yellowing and dropping of leaves, then vascular discoloration of the stem and finally death of the above-ground parts. Isolations were made from the discoloured stem vascular tissue. Necrotic tissue fragments were surface sterilized (2% NaOCl) and plated on potato dextrose agar (PDA). A Fusarium species was the only fungus isolated, and appeared from most fragments. Single-spore isolates were obtained, and the culture characteristics and micromorphology were investigated using PDA and carnation leaf agar (Nelson et al., 1983). All isolates obtained were identified as Fusarium oxysporum due to production of characteristic three- to five-septate, sickle-shaped macroconidia, with a foot-shaped basal cell, ellipsoid microconidia borne in false heads on short monophialides, and chlamydospores in culture. A typical cream-coloured colony developed on PDA, with purple pigmentation on the reverse side (Booth, 1971; Nelson et al., 1983). Fusarium wilt of eggplant is caused by Fusarium oxysporum f. sp. melongenae. Pathogenicity tests were performed twice for each isolate using a root-dip inoculation method modified from Biles & Martyn (1989). In total, 74 Fusarium isolates were tested on seedlings of Solanum melongena (eggplant cv. Pala) at the six-leaf stage. Wounded roots were submerged for 10 min in a conidial suspension (1 × 106 conidia mL−1 in sterile H2O), while control plants were dipped in sterile tap water. Seedlings were transplanted into pots and maintained in a growth chamber. After 3 weeks, severity of wilt symptoms was assessed on the leaves using a wilt index. All the isolates tested were pathogenic to eggplant. Symptoms observed on inoculated plants were similar to those in commercial glasshouses, including leaf chlorosis and necrosis; all inoculated plants died. Control plants showed no symptoms, and two other members of the Solanaceae (tomato and pepper) tested with the same method displayed no symptoms. Fusarium oxysporum f. sp. melongenae was successfully reisolated from the stems of disease-affected inoculated plants, thereby completing Koch's postulates. This is the first report of F. oxysporum f. sp. melongenae in Turkey. This study is dedicated to the memory of Assoc. Prof. Dr Yeter Canıhoş who passed away in 2002.

Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

First Report of Pyricularia grisea Causing Gray Leaf Spot on Kikuyugrass (Pennisetum clandestinum) in the United States.

Kikuyugrass (Pennisetum clandestinum) is a warm-season turfgrass that has been adopted for use in fairways and roughs in a number of subtropical areas including southern California, Mexico, Australia, and South Africa. During August 2003, a foliar disease of Kikuyugrass was reported from a number of golf courses in southern California. Examination of diseased plants showed the presence of dark, olive green-to-brown lesions on the foliage. Incubation of these plants in a moist chamber for 12 h led to the production of numerous pyriform conidia from these lesions that were characteristic of Pyricularia grisea. Single-spore isolates of the fungus were obtained from infected kikuyugrass samples by transferring conidia to acidified 1.5% water agar and then transferring single, germinated conidia to one-quarter-strength potato dextrose agar. Colony morphology and conidia production were consistent with that described for P. grisea (1). Koch's postulates were performed separately for two single-spore isolates (OSGC-1 and CCCC-1) obtained from infected kikuyugrass. For each isolate, 2-week-old, glasshouse-grown seedlings of kikuyugrass (cv. 'AZ-1') and perennial ryegrass (Lolium perenne) grown in 75-mm pots in soilless media were inoculated with conidia from either OSGC-1 or CCCC-1. For each test, six pots of both kikuyugrass and ryegrass were inoculated, and the tests were conducted three times for each isolate. Conidia were obtained from isolates grown on clarified V8 agar in 100-mm petri plates for 14 days at 25°C. Suspensions were made by adding 10 ml of sterile distilled H2O (sdH2O) to the plates, scraping the surface of the media to dislodge the conidia, filtering the suspension through cheesecloth, and then adjusting the final concentration to 1 × 106 conidia/ml with sdH2O. Seedlings were inoculated with the conidial suspensions with an aerosol applicator, placed in plastic boxes lined with wet paper towels, and sealed to provide adequate moisture for infection. Boxes were incubated at 28°C for 48 h after which time the covers were removed and the plants maintained in ambient glasshouse conditions at approximately 28°C. In all three replicated experiments, kikuyugrass seedlings inoculated with OSGC-1 or CCCC-1 developed symptoms of disease approximately 5 days after inoculation, while inoculated perennial ryegrass did not, even 14 days after inoculation. Symptomatic kikuyugrass leaves were taken randomly from plants from each of the three replicated tests, surface disinfested in 0.3% sodium hypochlorite for 30 s, rinsed with sdH2O, blotted dry, and placed onto acidified water agar in petri plates. Twenty-four hours later, abundant sporulation was observed from symptomatic tissue with conidiophores bearing conidia typical of P. grisea. To our knowledge, this is the first report of gray leaf spot being caused by P. grisea on Pennisetum clandestinum in North America. Reference: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971.

Curcumin Inhibits Formation of Amyloid β Oligomers and Fibrils, Binds Plaques, and Reduces Amyloid in Vivo

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.

Casein Kinase Iϵ Plays a Functional Role in the Transforming Growth Factor-β Signaling Pathway

The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

Olfactory response in the temporal cortex of the elderly measured with near-infrared spectroscopy: a preliminary feasibility study.

The pathway for the olfactory response may be affected at an early stage of Alzheimer's disease. Measurement of the olfactory response in the elderly is therefore of particular interest. In this feasibility study, near-infrared spectroscopy was used to measure the olfactory response in 21 patients aged 56 to 79 years. Eight subjects had no memory complaints whereas 13 had subjective memory complaints, mild cognitive impairment, or very mild Alzheimer's disease. The optodes were placed over the temporal lobe, with the emitting optode over the pole and the receiving optode over the superior gyrus. The response to vanilla (1% in sterile H2O) in a test tube held immediately beneath the nostrils was compared to the response to sterile H2O only. Four control subjects had a clearly definable response with increased oxyhemoglobin and decreased deoxyhemoglobin bilaterally. The response was measured as the sum of the deviation of oxygenated and deoxygenated hemoglobin from baseline mean. With a cut-off determined after examination of responses to vanilla and sham stimulus, group difference was significant for response to vanilla (chi2 test, P = 0.03). Response amplitudes to vanilla in the patient group were within the range of those to sham stimuli.

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p. Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking. We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189. In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex. The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A. This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

First report of Fusarium proliferatum causing rot of garlic bulbs in North America

In September 2001, bulbs of garlic (Allium sativum cv. Italian Red) rotted in a drying shed at the Plant Introduction farm in Pullman, Washington. Softened cloves displayed water-soaked, tan lesions, often with white mycelium near the bulb axis. Similar symptoms were noted in the same cultivar commercially grown in Idaho, 10 km from Pullman, and purchased at a retail outlet in July 2002. In each case, surface-disinfested tissue plated on agar produced a Fusarium sp. with catenate microconidia borne on polyphialides. Pathogenicity tests were conducted using eight garlic plants (W6-672) grown in the glasshouse. Three plants and a control were used for each of two replications. For each treated plant, six injections of 25 µL of isolate AsatF1 (106 conidia mL−1 in nutrient yeast extract broth) were delivered to developing cloves. Controls received sterile broth only. At 55 days, tissue was dissected from cloves in each replicate, disinfested (15 s in 70% ethanol, 3 min in 0.5% v/v NaOCl) and plated on antibiotic malt extract agar for recovery of fungi. The experiment was repeated once. Subsequent experiments employed cured garlic (cv. Italian Red grown in California) with five garlic bulbs inoculated with AsatF1 and five serving as controls. Treatments were replicated and the inoculations repeated, but substituting isolate AsatF8 for AsatF1 Conidia (106 mL−1 in sterile H2O) were injected into each bulb at three sites, while sterile H2O was injected into controls. After 11 weeks bulb tissue was disinfested as above, and portions of tissue incubated on nutrient agars. Every bulb injected with AsatF1 in greenhouse experiments displayed rot corresponding to material rotted in the drying shed. No controls displayed symptoms. Tissue excised from garlic cloves with symptoms and plated on agar produced Fusarium isolates indistinguishable from AsatF1. The fungus did not grow from controls. All cured garlic bulbs injected with isolates AsatF1 or AsatF8 developed symptoms. Internal tan rot progressed from injection sites toward the clove apex, with occasional white mycelium in rot cavities. Fusarium isolates indistinguishable from isolates AsatF1 or AsatF8 were isolated from all treatment bulbs. No controls developed symptoms, or produced Fusarium when tissue was incubated on agar. Isolates were identified as Fusarium proliferatum (Nelson et al., 1983; Nirenberg & O’Donnell, 1998). Previously, F. proliferatum has been reported on onion in the north-western USA (Mohan et al., 1997), and isolates AsatF1 or AsatF8 were also pathogenic to onion (data not shown). This is the first report of a Fusarium in section Liseola attacking garlic in North America. Seefelder et al. (2002) have reported F. proliferatum and fumonisins in garlic bulbs in Germany.

Distinct Protein Kinase C Isoforms Mediate Regulation of Vascular Endothelial Growth Factor Expression by A2A Adenosine Receptor Activation and Phorbol Esters in Pheochromocytoma PC12 Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A(2A) adenosine receptor (A(2A)AR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A(2A)AR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha, PKCepsilon, and PKCzeta, and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A(2A)AR occurs following deactivation of PKCzeta.

In Vitro Antimicrobial Activity of Various Medication Preparations on E. faecalis in Root Canal Dentin

The purpose of this study was to evaluate the antimicrobial activity of several medication preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens, 5 mm in height. The smear layer was removed and the samples were autoclaved and then incubated at 37 degrees C/5% CO2 for 24 h in brain-heart infusion (BHI) broth containing 7.0 x 10(4) colony forming units per ml of E. faecalis. The samples were washed in phosphate buffered saline and mounted to individual culture wells with sticky wax. Test medications were applied to fill the canal lumina; medication groups were: (a) sterile H2O (positive control); (b) a 10% mixture of 1.0 g Ca(OH)2 USP in 10 ml sterile H2O; (c) 10% Ca(OH)2 in 0.12% chlorhexidine gluconate (Peridex); (d) Peridex; and (e) uninoculated BHI (negative control). The samples were incubated at 37 degrees C/5% CO2 for 24 h. Dentin samples for quantitative microbiology were then obtained with consecutive sterile burs (ISO 029, 035, 042). All three experimental groups demonstrated significantly greater antimicrobial activity than the positive control (p < 0.001). Group 2 demonstrated significantly greater antimicrobial activity than Group 3 or Group 4 at all dentin depths (p < 0.05). These results suggest that 10% Ca(OH)2 may be more effective than Peridex or 10% Ca(OH)2 in Peridex for the elimination of E. faecalis from dentin tubules.

Antimicrobial Activity of Several Calcium Hydroxide Preparations in Root Canal Dentin

The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)2) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degrees C in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)2 USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)2 USP (0.1 g/ml H2O); group 3, Pulpdent TempCanal paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)2 can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)2 may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.