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S Ritchie

Publications and source records attributed to S Ritchie.

At least 19 recordsLinked to original sources
PubMed2001

A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca(2+)-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

Amino Acid Sequence↗
PubMed2001

Ross River virus transmission, infection, and disease: a cross-disciplinary review.

Ross River virus (RRV) is a fascinating, important arbovirus that is endemic and enzootic in Australia and Papua New Guinea and was epidemic in the South Pacific in 1979 and 1980. Infection with RRV may cause disease in humans, typically presenting as peripheral polyarthralgia or arthritis, sometimes with fever and rash. RRV disease notifications in Australia average 5,000 per year. The first well-described outbreak occurred in 1928. During World War II there were more outbreaks, and the name epidemic polyarthritis was applied. During a 1956 outbreak, epidemic polyarthritis was linked serologically to a group A arbovirus (Alphavirus). The virus was subsequently isolated from Aedes vigilax mosquitoes in 1963 and then from epidemic polyarthritis patients. We review the literature on the evolutionary biology of RRV, immune response to infection, pathogenesis, serologic diagnosis, disease manifestations, the extraordinary variety of vertebrate hosts, mosquito vectors, and transmission cycles, antibody prevalence, epidemiology of asymptomatic and symptomatic human infection, infection risks, and public health impact. RRV arthritis is due to joint infection, and treatment is currently based on empirical anti-inflammatory regimens. Further research on pathogenesis may improve understanding of the natural history of this disease and lead to new treatment strategies. The burden of morbidity is considerable, and the virus could spread to other countries. To justify and design preventive programs, we need accurate data on economic costs and better understanding of transmission and behavioral and environmental risks.

Alphavirus Infections↗
PubMed2000

Transcription of the human c-Src promoter is dependent on Sp1, a novel pyrimidine binding factor SPy, and can be inhibited by triplex-forming oligonucleotides.

The tyrosine kinase pp60(c-src) has been implicated in the regulation of numerous normal physiological processes as well the development of several human cancers. However, the mechanisms regulating its expression have not been addressed. In the present study, we report the presence of two Sp1/Sp3 binding sites and three polypurine:polypyrimidine (Pu:Py) tracts in the c-Src promoter that are essential for controlling expression. We demonstrate that Sp1, but not Sp3, is capable of activating the c-Src promoter and that Sp3 is also capable of inhibiting Sp1-mediated transactivation. The presence of multiple Pu:Py tracts conferred S1 sensitivity on plasmids in vitro, suggesting they are capable of adopting non B-DNA conformations. These tracts specifically bind a nuclear factor we named SPy (Src pyrimidine binding factor), which demonstrates both novel double- and single-stranded binding specificities. Mutations eliminating SPy binding compromised Src transcriptional activity, especially in concert with additional mutations affecting Sp1 binding, suggesting the two factors may cooperate in regulating c-Src expression. Finally, we demonstrate that triplex-forming oligonucleotides designed to target both Sp1 and SPy binding sites can down-regulate c-Src expression in vitro, suggesting a potential therapeutic approach to controlling c-Src expression in diseases where aberrant expression or activity has been documented.

Adenocarcinoma↗
PubMed2000

Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane.

We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5- to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTPgammaS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDPbetaS or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated alpha-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.

Abscisic Acid↗
PubMed2000

Mosquito isolates of Ross River virus from Cairns, Queensland, Australia.

During 1996-1998 60,619 mosquitoes were collected around Cairns, Australia and processed for Alphavirus isolation. Thirty-three isolates of Ross River (RR) virus were made from 9 species, Aedes imprimens, Aedes kochi, Aedes notoscriptus, Aedes vigilax, Culex annulirostris, Culex gelidus, Mansonia septempunctata, Verrallina (formerly Aedes) carmenti, and Verrallina lineatus. Attempts to isolate RR virus from 121 Aedes aegypti were unsuccessful. Twenty-six (79%) of the isolates came from within 1 km of a colony of spectacled flying-foxes, Pteropus conspicillatus. The minimum infection rate for these mosquitoes was 1.0 compared with 0.2 per 1,000 for mosquitoes trapped at all other sites. Ross River virus has not previously been isolated from Ae. imprimens, Cx. gelidus, Ma. septempunctata, Ve. carmenti, or Ve. lineatus. This is also the first isolation of an arbovirus from Cx. gelidus in Australia. In conclusion, the vector status of Ve. carmenti, Ae. aegypti and Ma. septempunctata warrants further study. This study also provides evidence that P. conspicillatus may be a reservoir host.

Animals↗
PubMed2000

Serial renal biopsy in systemic lupus erythematosus.

OBJECTIVE: To examine the role of sequential renal biopsies in patients with systemic lupus erythematosus (SLE), with regard to indications, morphologic change over time, and the clinical utility of repeat biopsies. METHODS: Patients with repeat renal biopsies were identified from the University of Toronto Lupus Clinic Database and their biopsies were reviewed blindly by a committee, using the WHO classification as well as activity and chronicity indices. Reasons for obtaining biopsy were documented, and therapeutic decisions following repeat biopsy were tabulated. RESULTS: Fifty-seven patients (49 F/9 M) had at least 2 renal biopsies between 1970 and 1994. The mean interval between biopsies was 4.2 years. The major reason for obtaining the first biopsy was disease diagnosis (32/57), while the majority of repeat biopsies were performed to discern the cause of increasing proteinuria (45/57). A comparison of the WHO classification of initial and repeat biopsies showed evolution to another class in 23 instances, but more commonly a change within a class was seen. A decrease in proliferative lesions (classes III and IV and subsets of V) was noted on repeat biopsies. The chronicity index increased significantly (p = 0.0001) and the activity index decreased (p = 0.064) between biopsies. Seventy-seven percent of patients had a change in treatment based on biopsy results. CONCLUSION: The major reason for repeat renal biopsy in patients with SLE was proteinuria. Renal morphology in patients with SLE can change with time, particularly in terms of chronicity and activity features. Repeat biopsies in patients with SLE appear to have clinical utility.

Adult↗
PubMed1999

Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity.

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K+ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

Journal Article↗
PubMed1999

The sensitivity of barley aleurone tissue to gibberellin is heterogeneous and may Be spatially determined

In cereals, gibberellin (GA) enhances the synthesis and secretion of hydrolytic enzymes from aleurone cells. These enzymes then mobilize the endosperm storage reserves that fuel germination. The dose-response curve of aleurone protoplasts to GA extends over a range of concentrations from 10(-11) to more than 10(-6) M. One hypothesis is that subpopulations of cells have different sensitivities to GA, with each cell having a threshold concentration of GA above which it is switched on. The dose-response curve therefore reflects a gradual recruitment of cells to the pool exhibiting a full GA response. Alternatively, all cells may gradually increase their responses as the GA level is increased. In the present study we found that at increasing GA concentrations, increasing numbers of barley (Hordeum vulgare) cells showed the enhanced amylase secretion and vacuolation characteristic of the GA response. We also observed that the region of aleurone tissue closest to the embryo contains the highest proportion of cells activated at the GA concentrations thought to occur naturally in germinating grain. These data indicate that an aleurone layer contains cells of varying sensitivities to GA and that recruitment of these differentially responding pools of cells may explain the broad dose response to GA.

Journal Article↗
PubMed1999

Neonatal suck reflex pattern does not predict apnea.

Respiration and suck are gestational age-dependent reflexes modulated in the brain stem. To determine if the suck reflex pattern could be used to predict apnea, the relationship between the two was examined in 28 neonates. The suck reflex was quantified with respect to burst-pause rhythm, amplitude of negative suck pressure, and synchrony of the negative-positive pressure. Apneas were counted 5 days prior to and following measurement of the suck reflex pattern. Increasing gestational age correlated with a lower frequency of apnea (P < .01) and higher suck scores (P < .01). A mature suck reflex pattern, however, failed to predict the occurrence of apnea.

Apnea↗
PubMed1998

Thermodynamic and kinetic studies of the formation of triple helices between purine-rich deoxyribo-oligonucleotides and the promoter region of the human c-src proto-oncogene.

The thermodynamic and kinetic parameters of triplex formation between four purine-rich oligonucleotides and a 22 bp pyrimidine. purine tract in the promoter region of the c-src gene were determined by fluorescence polarization studies. Three of these four oligonucleotides were 11 nt in length, corresponding to the left, central or right portion of the tract, while the fourth was a 22mer covering the whole tract. Binding constants ( Ka) were measured as a function of Mg2+ concentration (0-10 mM) and temperature (0-41 degrees C). In 10 mM Mg2+, K a for the left, central and right 11mers were 0.26, 0.75 and 1.4 x 10(8)/M, respectively, while for the 22mer the value was 1.8 x 10(8)/M at 22 degrees C. Under the same conditions, Ka was estimated by an electrophoretic band shift technique. The agreement between the two methods was acceptable for the 22mer but not for the 11mers. Kinetic measurements demonstrated that the rate of dissociation of the 22mer from the triplex was significantly slower than that of the 11mers, providing an explanation for the observed discrepancy. The entropy and enthalpy of triplex formation were calculated from van't Hoff plots. In all cases the entropy was favourable, especially for the 22mer and for the 11mer with the lowest guanine content. The enthalpy was unfavourable for the 22mer and most favourable for the 11mer with the highest guanine content. These results provide a thermodynamic explanation for length and sequence effects on the formation of purine.pyrimidine.purine triplexes.

Base Sequence↗
PubMed1998

Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity.

The plant hormones abscisic acid (ABA) and gibberellic acid (GA) are important regulators of the dormancy and germination of seeds. In cereals, GA enhances the synthesis and secretion of enzymes (principally alpha-amylases) in the aleurone cells of the endosperm, which then mobilize the storage reserves that fuel germination. ABA inhibits this enhanced secretory activity and delays germination. Despite the central role of ABA in regulating germination, the signal transduction events leading to altered gene expression and cellular activity are essentially unknown. We report that the application of ABA to aleurone protoplasts increased the activity of the enzyme phospholipase D (PLD) 10 min after treatment. The product of PLD activity, phosphatidic acid (PPA), also increased transiently at this time. The application of PPA to aleurone protoplasts led to an ABA-like inhibition of alpha-amylase production, and induction of the ABA up-regulated proteins ASI (amylase subtilisin inhibitor) and RAB (responsive to ABA). Inhibition of PLD activity by 0.1% 1-butanol during the initial 20 min of ABA treatment resulted in inhibition of ABA-regulated processes. This inhibition coincided with the timing of PLD activation by ABA and was overcome by simultaneous addition of PPA. These results suggest that ABA activates the enzyme PLD to produce PPA that is involved in triggering the subsequent ABA responses of the aleurone cell.

1-Butanol↗
PubMed1998

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 mum), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells.

Journal Article↗
PubMed1998

The human c-Src proto-oncogene promoter contains multiple targets for triplex-forming oligonucleotides.

The overexpression and activation of the human c-Src proto-oncogene is closely associated with cancer of the colon and breast. Characterization of the 5' region of the c-Src gene revealed that the promoter is very GC rich, regulated by the Sp family of transcription factors, and contains four perfect homopolypurine/homopolypyrimidine tracts (Pu:Py tracts). These Pu:Py tracts (TC1, TC1.1, TC2, and TC3) are located near or overlap critical Spl binding sites required for full activation of the gene. Triplex-forming oligonucleotides (TFOs) can be targeted to such sequences with high affinity to form intermolecular triple-helical DNA and modulate transcriptional activity. We therefore designed a series of antiparallel purine-based TFOs and measured their ability to form triplexes with the c-Src promoter Pu:Py tracts using comigration, bandshift, and chemical footprint techniques. With one interesting exception, all of the TFOs were found to bind with specificity and high affinity (67 nM-28 nM) to their target sequences at physiologic pH. These results indicate that the c-Src gene can successfully form stable triplexes under physiologic conditions and is, therefore, an excellent candidate for triplex-mediated transcriptional downregulation.

Base Composition↗
PubMed1998

Domestic Aedes aegypti breeding site surveillance: limitations of remote sensing as a predictive surveillance tool.

This project tested aerial photography as a surveillance tool in identifying residential premises at high risk of Aedes aegypti breeding by extending the use of a recently developed, ground-based, rapid assessment technique, the modified Premise Condition Index (PCI2). During 1995, we inspected 360 premises in Townsville, Australia for Ae. aegypti breeding, and PCI2 scores were recorded. The PCI2 values were also estimated from 1:3,000 color and infrared aerial photograph interpretation for the same premises. We found that shade levels can be accurately identified from both color and infrared images, and the PCI2 can be accurately identified from infrared photographs. Yard conditions, however, cannot be accurately identified from either aerial photograph type. The airborne PCI2 did not significantly correlate with breeding measures, and logistic regression further demonstrated that neither aerial photograph type allows the accurate prediction of Ae. aegypti breeding risk. Therefore, the ability of low-level aerial photography to enhance Ae. aegypti breeding site surveillance is at present limited, with ground surveillance remaining our most reliable tool for identifying the probability of Ae. aegypti breeding in the residential environment.

Aedes↗