First Report of "Candidatus Liberibacter solanacearum" on Field Tomatoes in the United States.

In August 2008, 30% of tomato (Solanum lycopersicum) plants in plots in Lubbock County, Texas showed yellowing, lateral stem dieback, upward leaf curling, enlargement of stems, adventitious roots, and swollen nodes. Yellowing in leaves was similar to that seen with zebra chip disease (ZC) of potato that was confirmed in a potato field 112 km away in July 2008 and was associated with a 'Candidatus Liberibacter' species (1), similar to findings earlier in 2008 in New Zealand and California (2,3). Tissue from four symptomatic plants of cv. Spitfire and two of cv. Celebrity were collected and DNA was extracted from midribs and petioles with a FastDNA Spin Kit (Qbiogene, Inc., Carlsbad, CA,). PCR amplification was done with 16S rRNA gene primers OA2 and OI2c, which are specific for "Ca. Liberibacter solanacearum" from potato and tomato and amplify a 1.1-kb fragment of the 16S rRNA gene of this new species (1,3). Amplicons of 1.1 kb were obtained from all samples and these were sequenced in both orientations (McLab, San Francisco, CA). Sequences of the 16S rRNA gene were identical for both Spitfire and Celebrity and were submitted to the NCBI as GenBank Accession Nos. FJ939136 and FJ939137, respectively. On the basis of a BLAST search, sequence alignments revealed 99.9% identity with a new species of 'Ca. Liberibacter' from potato (EU884128 and EU884129) in Texas (1); 99.7% identity with the new species "Ca. Liberibacter solanacearum" described from potato and tomato (3) in New Zealand (EU849020 and EU834130, respectively) and from the potato psyllid Bactericera cockerelli in California (2) (EU812559, EU812556); 97% identity with 'Ca L. asiaticus' from citrus in Malaysia (EU224393) and 94% identity with both 'Ca. L. africanus' and 'Ca. L. americanus' from citrus (EU921620 and AY742824, respectively). A neighbor-joining cladogram constructed using the 16S rRNA gene fragments delineated four clusters corresponding to each species, and these sequences clustered with "Ca. L. solanacearum". A second PCR analysis was conducted with the CL514F/CL514R primer pair, which amplifies a sequence from the rplJ and rplL ribosomal protein genes of "Ca. L. solanacearum". The resulting 669-bp products were 100% identical to a sequence reported from tomato in Mexico (FJ498807). This sequence was submitted to NCBI (GU169328). ZC, a disease causing losses to the potato industry, is associated with a 'Candidatus Liberibacter' species (1-3) and was reported in Central America and Mexico in the 1990s, in Texas in 2000, and more recently in other states in the United States (4). In 2008, a "Ca. Liberibacter solanacearum" was detected on Capsicum annuum, S. betaceum, and Physalis peruviana in New Zealand (3). Several studies have shown that the potato psyllid, B. cockerelli, is a potential vector for this pathogen (2,4). To our knowledge, this is the first report of "Ca. Liberibacter solanacearum" in field tomatoes showing ZC-like foliar disease symptoms in the United States. References: (1). J. A. Abad et al. Plant Dis. 93:108, 2009 (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) G. A. Secor et al. Plant Dis. 93:574, 2009.

The role of transcription factors cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER) in epileptogenesis.

Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing approximately threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.

Darwin's Down house.

Six years after his return from the voyage of the Beagle, Darwin moved his family to the country, where he spent the rest of his life conducting experiments, writing, and raising a family. English Heritage recently purchased Down House and is restoring the house and grounds to look as they did during Darwin's life.

Attenuation of premature estrous behavior in postpartum beef cows synchronized to estrus using GnRH and PGF2alpha.

The efficacy of GnRH and PGF2alpha (7-day injection interval) for estrus synchronization is diminished by estrous expression before PGF2alpha (premature estrus; PE). Effects of modifications to GnRH-PGF2alpha protocols on the incidence of PE and other indicators of reproductive performance were evaluated. In Experiment 1, Angus-based crossbred cows (n=51) received 25 mg of PGF2alpha i.m. on Day 0. Animals were randomly assigned by parity and interval postpartum to receive GnRH 100 microg i.m. on either Day -7 or Day -6. Estrous detection and AI were conducted from Day -3 to Day 5. Treatment had no effect on the incidence of PE, estrous response, conception rate per AI or synchronized pregnancy rate (6- vs. 7-day interval; 8 vs. 15%; 92 vs. 93%; 77 vs. 76%; 71 vs. 70%, respectively). In Experiment 2, Angus cows (n=150) received GnRH 100 microg i.m. on Day -7 and 25 mg PGF2alpha i.m. on Day 0. Animals were randomly assigned by parity, interval postpartum, and body condition score to receive either no further treatment (Control) or 0.5 mg melengestrol acetate/hd/d from Day -7 to Day -1 (MGA). Estrous detection and AI were conducted from Day -2 to Day 7. Fewer (P < 0.05) MGA-treated cows were detected in PE (0%) compared to controls (7%). Treatment had no effect on estrous response or synchronized pregnancy rates (Control vs. MGA; 78 vs. 84%; 52 vs. 60%, respectively). Conception rate per AI of cows > or = 60 days postpartum were not affected by treatment (Control vs. MGA; 79 vs. 73%) however, control cows < 60 days postpartum tended (P < 0.10) to have lower conception rates per AI (39%) than did their MGA-treated counterparts (69%). In summary, 6- and 7-day GnRH-PGF2alpha injection intervals resulted in similar synchronized reproductive performance. Inclusion of MGA feeding between GnRH and PGF2alpha injections eliminated the occurrence of premature estrus and improved conception rate per AI of late-calving cows.

A comparison of the bioequivalence of 0.5% fenbendazole top dress pellets or 10% fenbendazole oral suspension against a spectrum of equine parasites.

A controlled test was conducted to assess the efficacy bioequivalence of a single dose of 0.5% fenbendazole (FBZ) top dress pellets to a 10% FBZ suspension formulation (Panacur suspension 10%, Hoechst Roussel Vet). Thirty horses with naturally-acquired parasite infections, in replicates of three, were used. Strongyle egg per gram counts were not significantly different (P>0.1) between groups pretreatment, but FBZ treated groups were significantly different from the control group post-treatment. At necropsy, which occurred seven to nine days post-treatment, two methods of nematode recovery were compared to assess whether a small aliquot can be used in a control test to determine efficacy against large as well as small strongyles. Both post mortem worm recovery techniques revealed similar efficacies of both formulations (>95%) against small and large strongyles, but large differences in the number of worms recovered. Six species of small strongyles comprised 96% of all the small strongyles recovered: Coronocyclus coronatus, Cylicocyclus insigne, Cylicostephanus longibursatus, Cylicocyclus brevicapsulatus, Cylicocyclus nassatus, and Cyathostomum catinatum. The results of this study demonstrated therapeutic bioequivalence between FBZ formulations and also the need to sample at least a 10% aliquot to accurately estimate number of large strongyles. No adverse reactions to treatment were detected.

The Human Genome Diversity Project: medical benefits versus ethical concerns.

By the year 2005 the entire human genome should have been sequenced and the genes identified. But the resulting genomic sequence, although a marvelous accomplishment, will be a composite of just a handful of individuals selected at random. The Human Genome Diversity Project was proposed as a means to overcome these limitations by obtaining genetic information from many diverse populations of the world. This would give medical geneticists a handle on the variations in susceptibility to disease among different populations, as well as being of anthropological value. But would such a project risk exploiting the indigenous populations involved?

2.5-W, continuous-wave, 629-nm solid-state laser source.

We report an efficient, high-power, cw, 629-nm laser source based on a diode-pumped Nd:YAG laser and a periodically poled lithium niobate (PPLN) frequency converter. This device integrates two separate frequency-conversion steps in a single crystal, taking advantage of the ability to fabricate PPLN with nearly arbitrary grating periods and phase-matching temperatures. This device uses a single PPLN crystal that has two grating regions in series. The first region quasi-phase matches a standard optical parametric oscillator process (1064nm?1540nm +3450nm), and the second region quasi-phase matches a sum-frequency process whereby the pump and the signal light make red light (1064nm+1540nm ?629nm). Using a four-mirror ring cavity, we were able to convert 21% of the 1064-nm pump to 629-nm output, yielding 2.5W of red output with 11.8W of input.

DNA on a chip: serving up the genome for diagnostics and research.

It is expected that by the year 2003 the entire human genome will be sequenced, providing us with new insight into human disease. The amount of human sequence information that is already available (only a few percent of the total) is, however, already much greater than can be routinely evaluated with existing, commonly used diagnostic technology. New technology based on attaching DNA to a chip for parallel hybridization analysis is generating much interest in both the basic research and the clinical diagnostic community. It will increase, by orders of magnitude, our ability to evaluate an individual's genetic heritage and to conduct basic genetic research. The new technology will be a major advance in screening for genetic diseases, but the ability to treat these newly understood genetic defects will lag significantly behind the new-found diagnostic capabilities.

Drugs from the sea: harvesting the results of aeons of chemical evolution.

Despite the recent development of combinatorial chemistry for the rapid generation of thousands of new chemicals, the pharmaceutical industry still looks to the natural environment to uncover novel compounds that extend the boundaries of our chemical imagination. The terrestrial environment has been mined for such compounds for many years with great success. Now the world's oceans are beginning to yield hidden treasures. Many of these chemicals are structurally complex, stretching the prowess of modern organic chemists to their limits as they attempt to mimic the synthetic versatility of nature. In the future, these marine compounds are likely to yield entirely new classes of drugs that will be a valuable contribution to our ability to treat human disease.

Purification and properties of a human platelet inositol 1,4,5-trisphosphate 3-kinase.

An inositol 1,4,5-trisphosphate 3-kinase (Ins(1,4,5)P3 3-kinase) has been purified 943-fold from a 30,000g human platelet extract and has a specific activity of 283 nmol/min/mg protein and an apparent Km for inositol 1,4,5-trisphosphate of 0.76 microM; the optimal pH for the enzymatic activity was 7.2. Under both denaturing and nondenaturing conditions, the kinase preparation contained two polypeptides, both of which exhibited Ca2+/calmodulin-dependent Ins(1,4,5)P3 3-kinase activity. In the presence and absence of calmodulin, Ins(1,4,5)P3 3-kinase exhibited a biphasic response to Ca2+, being stimulated between 10(-7) and 10(-6) M Ca2+ and inhibited when the Ca2+ level was further increased. Ins(1,4,5)P3 3-kinase was stimulated by calmodulin approximately 10-fold, requiring 55 nM calmodulin for a half-maximal effect. Calmodulin stimulation was immediately reversed upon chelation of Ca2+ by ethylene glycol bis (beta-amino-ethyl ether) N,N'-tetraacetic acid consistent with a mechanism of activation involving a direct interaction of calmodulin with Ins(1,4,5)P3 3-kinase. Since we have previously shown that Ins(1,4,5)P3 3-kinase can also be phosphorylated and consequently inactivated by protein kinase C in vitro (Lin, A. N., Barnes, S., and Wallace, R. W., 1990, Biochem. Biophys. Res. Commun. 170, 1369-1376), Ins(1,4,5)P3 3-kinase appears to be a key enzyme in the inositol phosphate signaling pathway and as such may play an important role in human platelet function.

Induction of ICAM-1 by TNF-alpha, IL-1 beta, and LPS in human endothelial cells after downregulation of PKC.

The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.

Intercellular adhesion molecule-1 gene expression in human endothelial cells. Differential regulation by tumor necrosis factor-alpha and phorbol myristate acetate.

Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein expressed on the surface of inflamed endothelium which mediates in part the extravasation of granulocytes into sites of infection or injury. ICAM-1 mRNA is not detected in unstimulated human umbilical vein endothelial cells (HUVECs), but accumulates transiently following tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA) treatment with maximal steady state levels occurring at 2 or 4 h, respectively. Pretreating HUVECs with PMA for 72 h down-regulates protein kinase C and inhibits the subsequent induction of ICAM-1 mRNA by PMA, but does not affect TNF-alpha-induced message accumulation. Nuclear run-on assays showed that the ICAM-1 gene is transcribed under basal conditions in HUVECs, and that TNF-alpha stimulates transcriptional activity 3- to 4-fold within 30 min of treatment. In contrast, PMA has little effect on ICAM-1 gene transcription up to 4 h following stimulation. Message stability studies established that ICAM-1 mRNA induced by PMA has a longer half-life than the TNF-alpha-induced message. These results suggest that PMA acts through protein kinase C to up-regulate ICAM-1 expression primarily at a post-transcriptional level by stabilizing ICAM-1 mRNA, whereas TNF-alpha transcriptionally regulates ICAM-1 gene expression through an undefined, protein kinase C-independent pathway.

Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction.

Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAM-1 expression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAM-1 expression of human umbilical vein endothelial cells activated alternatively by TNF-alpha, IL-1 beta, LPS, or phorbol 12-myristate 13-acetate (PMA). Methodologies for the detection of ICAM-1 included both enzyme-linked immunosorbent assay and immunoprecipitation from biosynthetically labeled cells. The protein kinase inhibitor H-7 blocked induction of ICAM-1 by all of the activators; nonlinear regression analysis revealed 50% inhibitory concentration (IC50) values of 6-10 microM. Another kinase inhibitor, HA1004, did not block expression of the adhesion molecule at concentrations up to 50 microM. In contrast, the kinase inhibitor staurosporine dose dependently inhibited ICAM-1 expression triggered by PMA (IC50 67 +/- 4 nM) but, at similar concentrations, did not inhibit ICAM-1 expression induced by the other inflammatory stimuli. The divalent cation ionophore ionomycin (0.5 microM) interacted synergistically with PMA but not with cytokines or LPS in upregulating ICAM-1. We conclude from these data that although PMA-induced ICAM-1 expression may be triggered through activation of protein kinase C, ICAM-1 induction by IL-1 beta, TNF-alpha, or LPS may involve distinct regulatory pathway(s).

The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.

We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.

High-power Nd:YAG laser end pumped by a cw, 10 mm x 1 microm aperture, 10-W laser-diode bar.

A Nd:YAG laser is end pumped with a 10-W laser-diode bar to produce 1.9 W of TEM(00)-mode power at 1.064 microm. The 10 mm x 1 microm bar is imaged into a 1.8 mm x 390 microm elliptical laser mode. Q-switched operation results in pulse energies of 160 microJ at a 10-kHz repetition rate, which rises to 250 microJ at 5 kHz. Frequency doubling in KTP produces 75-microJ pulses at 10 kHz, corresponding to 750 mW of average power at 532 nm and a nonlinear conversion efficiency of 47%.

Broadly tunable high-power operation of an all-solid-state titanium-doped sapphire laser system.

Broadly tunable and high-power operation of a titanium-doped sapphire laser is obtained with a diode-laser-pumped frequency-doubled Nd:YAG laser as the pump source. A maximum broadband (FWHM = 25 nm) output pulse energy of 720 microJ at 795 nm in a TEM(00) mode is obtained for 1850 microJ of energy of 532-nm pump light. A minimum pulse duration of 7 ns is obtained from a 40-mm-long cavity. With the use of an intracavity prism, the Ti:sapphire laser is tunable continuously over the 696-1000-nm spectral range (with three different mirror sets).

Efficient second-harmonic conversion of cw single-frequency Nd:YAG laser light by frequency locking to a monolithic ring frequency doubler.

Efficient second-harmonic conversion of the 1064-nm output of a diode-pumped cw single-frequency Nd:YAG laser to 532 nm was obtained by frequency locking the laser to a monolithic ring resonator constructed of magnesium-oxide-doped lithium niobate. The conversion efficiency from the fundamental to the second harmonic was 65%. Two hundred milliwatts of cw single-frequency 532-nm light were produced from 310 mW of power of 1064-nm light. This represents a conversion efficiency of 20% from the 1-W diode laser used to pump the Nd:YAG laser to single-frequency 532-nm output. No signs of degradation of the 532-nm power or photo-refractive damage in the crystal were observed for over 500 h of operation of the system at green output powers greater than 100 mW.

Phosphorylation by protein kinase C inactivates an inositol 1,4,5-trisphosphate 3-kinase purified from human platelets.

An inositol 1,4,5-trisphosphate 3-kinase purified from human platelets contains two major components, 53 and 36 kDa polypeptides. Each polypeptide expresses Ca2+/calmodulin-dependent enzymatic activity and is phosphorylated by an unidentified protein kinase in the enzyme preparation. The 36-kDa polypeptide may be further phosphorylated on serine residues by protein kinase C to a stoichiometry of 0.8 mole phosphate per mole of protein. Phosphorylation of the 36-kDa component is correlated with inhibition of the kinase activity; the inhibitory effect is dependent upon Ca2+ and phosphatidylserine/diolein and may be blocked by a selective peptide inhibitor of protein kinase C. Phosphorylation by protein kinase C decreases the Vmax of the enzyme from 160 to 28 nmol/mg/min; the Km (0.76 microM) is not altered. These data suggest that protein kinase C may negatively regulate inositol 1,4,5-trisphosphate 3-kinase activity in the human platelet.

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.