Reversal of chloroquine resistance in Plasmodium falciparum by verapamil.

The parasite Plasmodium falciparum, like neoplastic cells, develops resistance to multiple structurally unrelated drugs. If the mechanisms by which P. falciparum and neoplastic cells become resistant are similar, then it may be possible to reverse the resistance in the two types of cells by the same pharmacological agents. Verapamil, a calcium channel blocker, completely reversed chloroquine resistance in two chloroquine-resistant P. falciparum clones from Southeast Asia and Brazil. Verapamil reversed chloroquine resistance at the same concentration (1 X 10(-6)M) as that at which it reversed resistance in multidrug-resistant cultured neoplastic cells. This same concentration of verapamil had no effect on chloroquine-sensitive parasites. Hence, chloroquine resistance in P. falciparum may fit the criteria for the multidrug-resistant phenotype.

Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance.

Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptible parasites, and this is thought to be the basis of their resistance. However, the reason for the lower accumulation of chloroquine was unknown. The resistant parasite has now been found to release chloroquine 40 to 50 times more rapidly than the susceptible parasite, although their initial rates of chloroquine accumulation are the same. Verapamil and two other calcium channel blockers, as well as vinblastine and daunomycin, each slowed the release and increased the accumulation of chloroquine by resistant (but not susceptible) Plasmodium falciparum. These results suggest that a higher rate of chloroquine release explains the lower chloroquine accumulation, and thus the resistance observed in resistant Plasmodium falciparum.

Genetic Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae.

ABSTRACT Phytophthora sojae, which causes Phytophthora root and stem rot of soybean, is a serious disease worldwide and is managed primarily by deploying cultivars with resistance. Thirty-two soybean plant introductions (PIs), all but three of which were from South Korea, were proposed as new sources of single-gene resistance to P. sojae. The objective of this study was to characterize the inheritance of resistance to P. sojae in these PIs. Twenty-two soybean populations from crosses of these PIs and the susceptible cv. Williams were inoculated with P. sojae OH17 (vir 1b, 1d, 2, 3a, 3b, 3c, 4, 5, 6, 7), and OH25 (vir 1a, 1b, 1c, 1k, 7). These isolates were selected because they are virulent on soybeans with all known Rps genes and many Rps gene combinations. Thirteen of the twenty-two populations had consistent segregation responses following inoculations between the two generations. In two PIs, resistance was conferred by two genes to OH17 and three genes to OH25. Resistance to both isolates was conferred by a single gene in PI 398440 although the individual families were not resistant to the same isolates. The data suggest that six of the populations have three-Rps gene combinations as previously proposed, while another four may have either a novel Rps gene or a four-Rps gene combination. Based on this phenotypic analysis, novel and uncharacterized Rps genes may be present in this material. More importantly, these PIs may serve as sources of novel Rps genes that can be used to more effectively manage Phytophthora root and stem rot.

Molecular Marker Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae.

ABSTRACT Molecular analysis of sources of resistance to plant pathogens should expedite and confirm novel gene discovery and consequently the development of disease resistant cultivars. Recently, soybean plant introductions (PIs) were identified that contain putative novel Rps genes for resistance to Phytophthora sojae. The number of resistance genes that confer resistance to P. sojae isolates OH17 (1b,1d,2,3a,3b,3c,4,5,6,7) and OH25 (1a,1b,1c,1k,7) was then determined in several of the PIs. The objective of this study was to determine if the Rps genes present in these PIs were associated with eight described Rps loci that have been mapped on soybean molecular linkage groups F, G, J, and N. Nine F(2:3) soybean populations were genotyped with simple sequence repeat (SSR) markers linked to previously mapped Rps loci. The nine PI populations all had SSR markers associated (P < 0.01) with resistance to P. sojae isolate OH17 in the Rps1 region. Rps1c is a likely candidate in eight PIs but novel genes may also be possible, while novel genes may confer resistance in one PI to P. sojae isolate OHI7. Two or more Rps genes, including some that are potentially novel, confer resistance to P. sojae isolate OH25 in eight of the populations. However, based on the response to these two isolates, virulence already exists for at least some of the novel genes identified in this study.

Assessing Heterodera glycines-Resistant and Susceptible Cultivar Yield Response.

The soybean cyst nematode Heterodera glycines (SCN) is of major economic importance and widely distributed throughout soybean production regions of the United States where different maturity groups with the same sources of SCN resistance are grown. The objective of this study was to assess SCN-resistant and -susceptible soybean yield responses in infested soils across the north-central region. In 1994 and 1995, eight SCN-resistant and eight SCN-susceptible public soybean cultivars representing maturity groups (MG) I to IV were planted in 63 fields, either infested or noninfested, in 10 states in the north-central United States. Soil samples were taken to determine initial SCN population density and race, and soil classification. Data were grouped for analysis by adaptation based on MG zones. Soybean yields were 658 to 3,840 kg/ha across the sites. Soybean cyst nematode-resistant cultivars yielded better at SCN-infested sites but lost this superiority to susceptible soybean cultivars at noninfested sites. Interactions were observed among initial SCN population density, cultivar, and location. This study showed that no region-wide predictive equations could be developed for yield loss based on initial nematode populations in the soil and that yield loss due to SCN in our region was greatly confounded by other stress factors, which included temperature and moisture extremes.

Radical prostatectomy for clinically localized prostate cancer: long-term results of 1,143 patients from a single institution.

PURPOSE: To determine the efficacy and complication rate of radical prostatectomy (RP) as a treatment option for clinically localized prostate cancer (clinical stage < or = T2c). METHODS: The study was a retrospective analysis of 1,143 consecutive patients (median age, 64 years; range, 38 to 79 y) who underwent RP at one institution (mean follow-up time, 9.7 years). Complications for this study population were compared with those of a contemporary group of 1,000 consecutive patients. RESULTS: Of 1,143 patients, 83 (7%) had a low clinical stage (T1) and 160 (14%) had a low histologic grade (Gleason score < or = 3); 648 (57%) had a high clinical stage (T2b or T2c) and 204 (18%) had a high histologic grade (Gleason score > or = 7). Only 113 (10%) died of prostate cancer, and 177 (15%) developed metastasis. Adjuvant treatment (androgen deprivation or radiation therapy) was given in 197 (17%) patients (> or = pT3) and provided virtually identical results as without adjuvant treatment. The 10- and 15-year crude survival rates for 1,143 patients were 75% +/- 1.5% (SE) and 60% +/- 2.2%, respectively; the cause-specific survival rates were 90% +/- 1.1% and 83% +/- 1.9%, respectively; and the metastasis-free survival rates were 83% +/- 1.3% and 77% +/- 1.9%, respectively (398 men at risk at 10 years and 138 men at risk at 15 years). The 10-year survival rate for patients with Gleason score > or = 7 was 74% +/- 3.9%. Only tumor grade was a significant predictor for disease outcome. The hospital mortality rate decreased from 0.7% for the 1,143 study patients to 0% for the more recent 1,000 patients. Severe incontinence declined to 1.4% for the more recent 1,000 patients. Most patients who underwent RP were healthy (Charlson comorbidity index). CONCLUSION: Survival at 15 years was similar to the expected survival rate. Current morbidity and mortality rates associated with RP were extremely low. Thus, RP has been a viable management option for men with clinically localized prostate cancer who have a life expectancy of more than 10 years.

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine-inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca(2+)-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-beta-D-ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.

Expression of PC3, carboxypeptidase E and enkephalin in human monocyte-derived macrophages as a tool for genetic studies.

Circulating monocytes in human peripheral blood are readily available, easily obtained, and can be cultured in vitro. Once plated, the monocytes spontaneously differentiate into macrophages. Undifferentiated human monocytes do not express carboxypeptidase E (CPE), prohormone convertase 3 (PC3/PC1) or proenkephalin (ENK), suggesting that gene induction during differentiation results in the expression of these genes. RT-PCR of human monocyte-derived macrophage (HMDM) mRNA showed detectable levels of ENK mRNA at 48 h after plating, followed by PC3 and CPE mRNAs at 72 h. PC3 expression was confirmed by Western blotting in THP-1 cells. Similarities in expression of enzymes involved in the conversion of neuroendocrine precursors, such as proenkephalin, into functionally mature peptides underscores the close association HMDMs may share with the neuroendocrine system. Because of this, HMDMs may prove to be a valuable, non-surgical source of human tissue for clinical genetic diagnosis of some convertase disorders.

Regulation by glucose of the biosynthesis of PC2, PC3 and proinsulin in (ob/ob) mouse islets of Langerhans.

The prohormone convertases PC2 and PC3 have been shown to catalyze the processing of proinsulin to insulin in pancreatic beta-cells. In these studies we have compared the effects of glucose on PC2 and PC3 biosynthesis in freshly isolated islets from normal and hyperglycemic (ob/ob) mice. In contrast to normal islets [Alarcón, et al. (1993) J. Biol. Chem. 268, 4276] the biosynthesis of both PC2 and PC3 is stimulated by glucose, parallel to the stimulation of proinsulin in the (ob/ob) islets. Inhibition of PC2 biosynthesis by glucose in normal islet non beta-cells may obscure stimulation of PC2 biosynthesis in normal islet beta-cells.

Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens.

An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 microg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref-ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 microg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n=22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

Medium-intensity light produces circadian rhythm adaptation to simulated night-shift work.

STUDY OBJECTIVES: To assess the effect of nocturnal light intensity on circadian adaptation to simulated night work. SETTING AND PARTICIPANTS: Normal young men and women, simulated night work, home sleep. DESIGN AND MEASUREMENTS: We compared temperature rhythm phase shifts following timed exposure to high (approximately 5700 lux 3 hours/day), medium (approximately 1230 lux 3 hours/day) or constant low-intensity (< 250 lux) light during consecutive night shifts. Subjects (n = 35) followed a schedule of 7 days baseline, 6 days of 8-hour night shifts (with day sleep delayed 10 hours from baseline sleep), and 4 days of recovery. Subjects wore dark sunglasses while outdoors during daylight. Sleep logs were completed after each 8-hour sleep/dark period. Night work fatigue was rated by questionnaire. RESULTS: During the 3rd through 5th days of night work, most subjects in the high and medium groups (100% and 85%) exhibited phase delays large enough that their body temperature minima occurred within the daytime sleep/dark period. Only 42% of subjects in the low group exhibited phase delays large enough to meet this criterion of circadian adaptation. The phase shifts of the high and medium groups were not significantly different, and were significantly different from the low group. Larger phase shifts were correlated with more sleep and less fatigue. CONCLUSIONS: Extremely "bright" light may not be necessary for circadian adaptation in shift work situations similar to our study protocol (e.g., regular daytime sleep/dark periods, sunglasses).

Use of pharmacological agents to implicate a role for phosphoinositide hydrolysis products in malaria gamete formation.

The kinetics of phosphoinositol 4,5 bisphosphate hydrolysis products in activated Plasmodium falciparum gametocytes suggests a role for inositol trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) in the signal transduction pathway of malaria gametocytes. To investigate further this role, compounds that have an effect on the metabolism and biologic functions of these second messengers were tested in an in vitro system. Gentamycin, 2,3 diphosphoglycerate (2,3 DPG) and magnesium ion (Mg2+), inhibitors of Ins(1,4,5)P3 5' phosphatase, all stimulated gametocytes to exflagellate in suspended animation buffer, pH 7.4, at room temperature. In addition, methylxanthines, caffeine and theobromine, calcium ionophore (A-23187), and external calcium also stimulated exflagellation. In contrast, neomycin, an aminoglycoside that inhibits phospholipase C activity, and heparin, an antagonist of Ins(1,4,5)P3 binding to its receptor, inhibited microgamete formation. Quinine and chloroquine which can inhibit both phospholipase A and C activity also inhibited gametocyte exflagellation. The consistent manner in which these various compounds affect gametocyte activation further implicates phosphoinositol turnover in the signal transduction pathway of falciparum gametocytes.

Biochemical diagnosis of fatty acid oxidation disorders by metabolite analysis of postmortem liver.

At least 12 fatty acid oxidation disorders are known to be responsible for cases of sudden and unexpected death in early childhood. A specific diagnosis of these disorders is essential for genetic counseling and for the screening of siblings potentially at risk for life-threatening episodes of fasting intolerance. Postmortem blood and urine samples often are not available for further biochemical studies, and currently only medium-chain acyl-CoA dehydrogenase (MCAD) deficiency can be diagnosed by the molecular analysis of tissues. We developed a postmortem screening method for fatty acid oxidation disorders by the simultaneous measurement of C8-C20 fatty acids, glucose, lactate, and other metabolites from the methanol wash of a pellet obtained by ultracentrifugation of liver homogenate. Cis-4-decenoic acid was present in five confirmed cases with MCAD deficiency and in one case with glutaric aciduria type II and was absent in 97 of 100 randomly chosen sudden death cases, at least 81 of which were diagnosed as sudden infant death syndrome (SIDS). C14-C18 monounsaturated fatty acids were significantly elevated in the one examined case affected with long-chain acyl-CoA dehydrogenase (LCAD) deficiency. The metabolite profiles in two cases with carnitine uptake deficiency were less informative, but they shared with all the other disease controls a very low glucose concentration, a finding compatible with premortem hypoglycemia. This method is proposed as a simple and practical means of biochemical screening to follow up the postmortem finding of liver fat infiltration.

Cloning, expression, and chromosomal mapping of a novel human CC-chemokine receptor (CCR10) that displays high-affinity binding for MCP-1 and MCP-3.

Chemokines mediate their chemotactic, proinflammatory effects by binding to and activating a variety of specific receptors belonging to the G protein-coupled superfamily of seven-transmembrane serpentine receptors. We report the cloning, chromosomal localization, expression, and ligand binding of a novel CC chemokine receptor, CCR10. CCR10 is expressed primarily in placenta and fetal liver, and binds two of the CC chemokines, monocyte chemoattractant protein (MCP)-1 and MCP-3, with highest affinity. The KD for MCP-3 binding was 1 nM, and MCP-1 competed for MCP-3 binding with an IC50 of 1.2 nM. The CC chemokines MCP-4 and RANTES competed for MCP-3 binding with IC50 values of 7.5 and 5.4 nM, respectively. The chromosomal location of CCR10 was determined to coincide with the CC chemokine receptor cluster on chromosome 3 (3p21.31-3p21.32). These results indicate that CCR10 is a novel CC chemokine receptor with a unique expression pattern that would be consistent with a role in placental immunity or hematopoiesis.

Cultivation of Plasmodium falciparum parasites in a serum-free medium.

The elimination of serum from Plasmodium falciparum culture media could decrease costs, enhance procurement, and improve the feasibility of large-scale production of parasite material. We provide a semi-defined, serum-free formulation, of commercially available constituents that supports P. falciparum parasite growth at rates comparable with those obtained with serum-supplemented media. The medium is composed of RPMI 1640 to which HEPES, extra glucose, bicarbonate, and hypoxanthine have been added. Bovine albumin and serum-derived, lipids-cholesterol-rich mixture are then used in place of serum.

Factors affecting exflagellation of in vitro-cultivated Plasmodium falciparum gametocytes.

The environment of Plasmodium falciparum gametocytes changes when they make the transition from the vertebrate to the invertebrate host. Gametocytes of this species cultivated in vitro were used to evaluate the effect of serum, pH, pCO2 tension, bicarbonate ion, and temperature on gamete formation. Temperature was the only factor responsible for keeping P. falciparum gametocytes in the inactivated state. Mature gametocytes held at temperatures above 30 degrees C remained quiescent in 10% serum, even at low ambient pCO2 tension, alkaline pH, and in the presence of 25 mM bicarbonate ion. When the temperature of the medium was allowed to drop below 30 degrees C, gametocytes emerged from the red blood cells and microgametocytes consistently exflagellated at pH 7.4, even in the absence of bicarbonate ion. With regard to bicarbonate ion, exflagellation in P. falciparum is similar to P. berghei and different from P. gallinaceum gametocytes, which have an obligate requirement for bicarbonate ion.

Determination of fifty percent inhibitory concentrations (IC50) of antimalarial drugs against Plasmodium falciparum parasites in a serum-free medium.

Plasmodium falciparum chemosensitivity to the various antimalarial drugs is presently determined in the laboratory by setting up multiple microcultures of the parasite and estimating the amount of growth inhibition caused by known concentrations of drug. Parasite growth inhibition is assessed either by microscopy, radiolabeled substrate uptake, or calorimetrically. The obligate requirement for serum in this assay presents difficulties in the direct comparison of results among laboratories. We now have evidence that antimalarial drug sensitivity assays can be reliably performed in a serum-free medium. The overall comparison of 50% inhibitory concentration (IC50) values obtained with serum-free media (bovine albumin, Cohn fraction V [BAM] and BAM combined with glucose and lipids-cholesterol-rich mixture) and those obtained in serum-supplemented medium was r = 0.56; n = 60; P < 0.01.

Ultrastructural study of the effects of chloroquine and verapamil on Plasmodium falciparum.

Verapamil, a calcium antagonist, has recently been shown to reverse chloroquine resistance in malarial parasites in vitro. We report the first ultrastructural morphological changes associated with this phenomenon using chloroquine-sensitive and -resistant clones of Plasmodium falciparum. While the administration of 6.3 x 10(-8) M chloroquine had little morphological effect on the chloroquine-resistant strain, the combination of chloroquine and verapamil resulted in typical chloroquine-related food vacuolar swelling with increased amounts of granular matrix. Secondary morphological changes included degeneration of nuclei, mitochondria, and other organelles. These effects appeared similar to those in the chloroquine-sensitive strain of P. falciparum treated with chloroquine alone or with the chloroquine/verapamil combination. Furthermore mild food vacuolar changes were seen in a small number of parasites (from both chloroquine-sensitive and -resistant groups) exposed to high concentrations (1 x 10(-4) M) of verapamil alone.

Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites.

Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.

Anatomic site-specific positive margins in organ-confined prostate cancer and its impact on outcome after radical prostatectomy.

OBJECTIVES: The impact of a positive surgical margin in otherwise confined prostate cancer after radical prostatectomy remains unclear. We analyzed the outcome of a large number of patients with organ-confined prostate cancer according to the presence and anatomic site of margin positivity. METHODS: We evaluated 2712 prostatectomy patients with Stage pT2N0 cancer (ie, no evidence of extra-prostatic disease, seminal vesicle or regional node involvement) and no prior therapy who were treated by radical prostatectomy between 1987 and 1995 at Mayo Clinic. A total of 697 patients (26%) had positive margins. To assess the effect of margin status in the absence of treatment, 378 patients with postoperative adjuvant therapy were not considered for the study group: the final group consisted of 2334 patients. RESULTS: Overall, 253 (58%) tumors were positive at the apex and/or urethra, 85 (19%) at the prostate base, 11 (2.5%) at the anterior prostate, and 174 (40%) at the posterior prostate; 89 (20%) had at least two margins involved and 21 (8.3%) had more than two involved. The apex/urethra was the only positive anatomic site in 183 (42%). Five-year survival free of clinical recurrence or prostate-specific antigen (PSA) biochemical failure (postoperative serum PSA of 0.2 ng/mL or more) for patients with a single positive margin was 79% for apex or urethra, 78% for anterior/posterior, and 56% for prostate base. Five-year survival free of clinical recurrence or PSA (biochemical) failure was slightly higher for those with one versus two margin-positive regions (77% versus 68%, respectively). Multivariate analysis revealed that positive surgical margins were a significant predictor of clinical recurrence and PSA (biochemical) failure (relative risk [95% confidence interval]: 1.65 [1.24, 2.18]) after controlling for Gleason grade, preoperative PSA, and deoxyribonucleic acid (DNA) ploidy. The effect of margin positivity on recurrence at a specific anatomic site (versus negative margins or positive at a different anatomic site) revealed the prostate base to be the only significant anatomic site when adjusted for grade, PSA, and ploidy. Five-year survival free of the combined clinical or PSA failure end point for those with versus those without positive margins at the prostate base was 56% versus 85%, respectively (P < 0.0001). CONCLUSIONS: Positive surgical margins are a significant predictor of recurrence in Stage pT2N0 cancer, which is independent of grade, PSA, and DNA ploidy. The impact of positive margin status on recurrence-free survival appears to be anatomic and site-specific, with prostate base positivity significantly associated with poor outcome. The benefit of adjuvant therapy based on anatomic site-specific margin positivity remains to be tested in order to optimize recurrence-free survival.