Resistance of Leishmania donovani to sodium stibogluconate is related to the expression of host and parasite gamma-glutamylcysteine synthetase.

Sequencing studies showed that the gamma-glutamylcysteine synthetase (gamma-GCS) heavy chain genes from sodium stibogluconate (SSG)-resistant (SSG-R) and SSG-susceptible (SSG-S) Leishmania donovani strains were identical, indicating that SSG resistance was related to quantitative differences in gamma-GCS expression rather than gene interstrain polymorphisms. In vitro infection of murine macrophages with the SSG-R strain, but not the SSG-S strain, down regulated expression of host gamma-GCS, which would result in a reduction in intramacrophage glutathione (GSH) levels and promote an oxidative intramacrophage environment. This would inhibit, or minimize, the reduction of SSG pentavalent antimony to its more toxic trivalent form. Macrophage studies showed that the SSG-R strain expressed higher levels of gamma-GCS compared to the SSG-S strain, which would result in higher GSH levels, giving increased protection against oxidative stress and facilitating SSG efflux. However a similar differential effect on host and parasite gamma-GCS expression was not obtained when using tissues from infected mice. In this case gamma-GCS expression was organ and strain dependent for both the host and the parasite, indicating that environmental conditions have a profound effect on gamma-GCS expression. Consistent with the proposed mechanism from in vitro studies, increasing tissue GSH levels in the presence of SSG by cotreatment of L. donovani-infected mice with SSG solution and GSH incorporated into nonionic surfactant vesicles was more effective in reducing liver, spleen, and bone marrow parasite burdens than monotherapy with SSG. Together, these results indicate that SSG resistance is associated with manipulation of both host and parasite GSH levels by L. donovani.

Efficacy of Fungicides for Control of Sclerotinia Stem Rot of Canola.

Sclerotinia stem rot (SSR), incited by Sclerotinia sclerotiorum, causes yield reductions to canola (Brassica napus) grown in North Dakota and Minnesota. Field trials were conducted in North Dakota and Minnesota from 2000 to 2004 to evaluate the effect of foliar fungicides on SSR and canola yield. Levels of SSR varied among years and location. In general, fungicides that consistently reduced SSR incidence compared with an untreated control were azoxystrobin, benomyl, boscalid, iprodione, prothioconazole, tebuconazole, thiophanate-methyl, trifloxystrobin, and vinclozolin. Significant reductions in SSR incidence with fungicides did not always translate into differences in canola yield, however. Inconsistent results were observed with different timings of applications based on percent bloom. This indicates that application timing should be based on factors in addition to percent bloom.

Response of Canola Cultivars to Sclerotinia sclerotiorum in Controlled and Field Environments.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, can be a devastating disease of canola (Brassica napus) in the northern United States. No canola cultivars are marketed as having resistance to SSR. Field trials were established in Red Lake Falls, MN (2001, 2003, and 2004) and Carrington, ND (2001, 2002, 2003, and 2004) to evaluate canola cultivars for resistance to SSR. These cultivars also were evaluated for resistance to SSR under controlled conditions using the following methods: petiole inoculation technique (PIT), detached leaf assay (DLA), and oxalic acid assay (OAA). Significant (P ≤ 0.05) differences were detected among cultivars for SSR and yield in the field trials, with SSR levels varying from low to high among years and locations. Cultivars with consistent high levels and low levels of SSR in the field trials were identified. Significant (P ≤ 0.05) differences were detected among cultivars for SSR using the PIT and OAA methods, but not the DLA method. No significant (P ≤ 0.05) correlations between SSR levels in the controlled studies with SSR levels in the field trials were detected; however, significant negative correlations were detected between SSR area under the disease process curve values from the PIT method and yield from Carrington, ND in 2001 and 2002. Although the PIT and OAA methods differentiated cultivars, neither method was able to predict the reaction of cultivars to SSR in the field, indicating that field screening for SSR resistance is still critical for the development of resistant cultivars.

Insulin resistance induced by sucrose feeding in rats is due to an impairment of the hepatic parasympathetic nerves.

AIMS/HYPOTHESIS: A considerable proportion of whole-body insulin-stimulated glucose uptake is dependent upon the hepatic insulin-sensitising substance (HISS) in a pathway mediated by the hepatic parasympathetic nerves (HPNs). We tested the hypothesis that a high-sucrose diet leads to the impairment of the HPN-dependent component of insulin action. METHODS: We quantified insulin sensitivity using the rapid insulin sensitivity test, a modified euglycaemic clamp. Quantification of the HPN-dependent component was achieved by administration of a muscarinic receptor antagonist (atropine, 3 mg/kg). RESULTS: Insulin sensitivity was higher in standard-fed than in sucrose-fed Wistar rats (305.6+/-34.1 vs 193.9+/-13.7 mg glucose/kg body weight; p<0.005) and Sprague-Dawley rats (196.4+/-5.9 vs 95.5+/-16.3 mg glucose/kg body weight; p<0.01). The HPN-independent component was similar in the two diet groups. Insulin resistance was entirely due to an impairment of the HPN-dependent component in both Wistar rats (164.3+/-28.1 [standard-fed] vs 26.5+/-7.5 [sucrose-fed] mg glucose/kg body weight; p<0.0001) and Sprague-Dawley rats (111.7+/-9.5 vs 35.3+/-21.4 mg glucose/kg body weight; p<0.01). Furthermore, HPN-dependent insulin resistance in Sprague-Dawley rats was already evident after 2 weeks of a high-sucrose diet (28.5+/-7.6 [2 weeks], 35.3+/-21.4 [6 weeks], 17.9+/-5.4 [9 weeks] mg glucose/kg body weight) and was independent of the nature of sucrose supplementation (12.3+/-4.7 [solid] and 17.9+/-5.4 [liquid] mg glucose/kg body weight). CONCLUSIONS/INTERPRETATION: Our results support the hypothesis that insulin resistance caused by sucrose feeding is due to an impairment of the HPN-dependent component of insulin action, leading to a dysfunction of the HISS pathway.

Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting.

In response to insulin, a hormone [hepatic insulin sensitizing substance (HISS)] is released from the liver to stimulate glucose uptake in skeletal muscle but not liver or gut. The aim was to characterize dynamic control of HISS action in response to insulin and regulation of release by hepatic parasympathetic nerves. Insulin action was assessed by the rapid insulin sensitivity test, where the index is the glucose required (mg/kg) to maintain euglycemia after a bolus of insulin. Blocking HISS release by interruption of the hepatic parasympathetic nerves by surgical denervation, atropine, or blockade of hepatic nitric oxide synthase produced similar degrees of insulin resistance and revealed a similar dynamic pattern of hormone action that began 3--4 min after, and continued for 9--10 min beyond, insulin action (50 mU/kg). HISS action accounted for 56.5 +/- 3.5% of insulin action at insulin doses from 5 to 100 mU/kg (fed). We also tested the hypothesis that HISS release is controlled by the feed/fast status. Feeding resulted in maximal HISS action, which decreased progressively with the duration of fasting.

Multidrug resistance and ABC transporters in parasitic protozoa.

Drug resistance is an important problem in parasitic protozoa. We review here the role of ABC transporters in drug resistance in parasites. We have concentrated on gene and gene products for which there is a strong evidence for their role in resistance.

The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase.

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein were fused at the COOH terminus of PGPA. Immunofluorescence, confocal, and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PGPAHA transfectants was performed to further characterize this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmol/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transports thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.

ABC proteins of Leishmania.

ABC proteins were first characterized in the protozoan parasite Leishmania while studying mechanisms of drug resistance. PGPA is involved in resistance to arsenite and antimonite and it most likely confers resistance by sequestering metal-thiol conjugates into an intracellular vesicle. PGPA is part of gene family with at least four more members which are in search of a function. Leishmania also contains a P-glycoprotein, homologous to the mammalian MDR1, that is involved in multidrug resistance. The ongoing genome project of Leishmania has pinpointed several novel ABC transporters and experiments are carried out to study the function of the ABC proteins in drug resistance and in host-pathogen interactions.

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

Rapid insulin sensitivity test (RIST).

A rapid insulin sensitivity test (RIST) was recently introduced to assess insulin action in vivo (H. Xie, L. Zhu, Y.L. Zhang, D.J. Legare, and W.W. Lautt. J. Pharmacol. Toxicol. Methods, 35: 77-82. 1996). This technical report describes the current recommended standard operating procedure for the use of the RIST in rats based upon additional experience with approximately 100 tests. We describe the manufacture and use of an arterial-venous shunt that allows rapid multiple arterial samples and intravenous administration of drugs. The RIST procedure involves determination of a stable arterial glucose baseline to define the ideal euglycemic level to be maintained following a 5-min infusion of insulin, with the RIST index being the amount of glucose required to be infused to maintain euglycemia over the test period. Insulin administration by a 5-min infusion is preferable to a 30-s bolus administration. No significant difference was determined between the use of Toronto pork-beef or human insulin. Four consecutive RISTs were carried out in the same animal over 4-5 h with no tendency for change with time. The RIST index is sufficiently sensitive and reproducible to permit establishment of insulin dose-response curves and interference of insulin action by elimination of hepatic parasympathetic nerves, using atropine. This technical report provides the current recommended standard operating procedure for the RIST.

ABC transporters in Leishmania and their role in drug resistance.

ABC transporters have been found in several parasitic protozoa including Leishmania. At least two Leishmania ABC transporters are involved in drug resistance. One is PgpA, which is involved in resistance to arsenic and antimony-containing compounds. Antimonials are the drug of choice against Leishmania infections. Transfection and biochemical studies suggest that PgpA recognizes metals conjugated to thiols. The second ABC transporter is closely related to mammalian P-glycoproteins and confers resistance to anticancer drugs by a mechanism that remains to be elucidated. Additional ABC transporters are likely to be present in Leishmania and these are discussed in relation to the phenomenon of antimony resistance.

Efflux systems and increased trypanothione levels in arsenite-resistant Leishmania.

The mechanism of resistance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mechanism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania.

Insulin sensitivity tested with a modified euglycemic technique in cats and rats.

A new insulin sensitivity test (IST) is described using a modified euglycemic clamp in cats and rats. The IST uses the amount of glucose required to be infused to maintain euglycemia over a 30-min period in rats and 60 min in cats following a bolus administration of insulin as the index of insulin sensitivity. Glucose levels are determined at short time intervals (2-5 min), and variable glucose infusion is used to hold glucose levels within a few percentage points of the basal pre-test glucose level. A new blood sampling procedure is described that allows each IST to be carried out using a total of only 0.5 mL of blood. The IST is sensitive and allows clear insulin dose effects to be demonstrated with 100 mU/kg requiring 355.0 +/- 14.3 mg/kg over 30 min and 50 mU/kg requiring 198.7 +/- 11.1 mg/kg. Five consecutive tests were reproducibly carried out (%CV = 3.0 +/- 0.5) over a 12-hr period in the cat with insulin, glucagon, and glucose levels remaining stable prior to each IST. Glucagon and norepinephrine plasma concentrations do not change significantly during the IST. The IST is sufficiently sensitive to allow demonstration of dose-response relationships for atropine-induced insulin resistance. The IST is thus sensitive, reproducible, and able to demonstrate acute insulin resistance in anesthetized cats and rats. The test is demonstrated in fed (rats) and fasted (cats) state.

Evaluation of vascular tone in portacaval shunts comparing the index of contractility and resistance in cats.

A model of prehepatic chronic portal hypertension in cats was used to determine portacaval shunt responses to infused norepinephrine and to possible transmitter overflow into portal blood from nerves supplying the gut. Responses are compared using a new index of contractility. Four weeks after application of a slowly constricting occluder, the portal vein was completely occluded and acute experiments were carried out under pentobarbital anesthesia. Portal pressure was elevated to 15.0 +/- 0.9 mmHg and all portal flow passed through the shunts. In response to intraportal norepinephrine (0.25, 0.5 and 1.25 micrograms.min-1.kg-1) shunt resistance rose by 6% +/- 3%, 19% +/- 4% and 26% +/- 5%, respectively, whereas the index of contractility rose (by 22% +/- 8%, 46% +/- 10% and 89% +/- 20%, respectively), the distending blood pressure also rose (5% +/- 1%, 7% +/- 1% and 14% +/- 3%, respectively). The difference in percentage increase of resistance and the index of contractility is a result of the passive dilator effect of the elevated distending pressure acting on the distensible shunt vessels. Stimulation of mesenteric nerves caused the mesenteric artery to constrict, but the shunt vessels showed no effect. In conclusion, the shunt vessels respond actively to norepinephrine and passively to altered distending pressure. However, transmitter overflow from nerves supplying the intestines is unlikely to play a role in determining resistance in the shunts. Vascular resistance is affected by both active and passive effects, so that the active contractile responses are best evaluated using the index of contractility, which is not altered passively.

The P-glycoprotein-related gene family in Leishmania.

P-glycoprotein gene amplification has been described in several drug-resistant parasitic protozoa. The first P-glycoprotein related gene described in Leishmania was ltpgpA, a gene frequently amplified in arsenite resistant Leishmania. Hybridization experiments indicated that ltpgpA was part of a gene family. In addition to ltpgpA, four novel genes were cloned that are present in two loci: ltpgpB and ltpgpC tandemly linked to ltpgpA on a 800-kb chromosome; and ltpgpD and ltpgpE closely linked on a chromosome ranging from 950 kb to 1400 kb, depending on the Leishmania species. Another P-glycoprotein gene, homologous to the more recently described ldmdr1, was linked to ltpgpD and ltpgpE. Nucleotide sequencing of ltpgpB and ltpgpE revealed that the Leishmania P-glycoprotein-related genes have diverged considerably from the main branch of P-glycoproteins and are more homologous to the recently described multidrug resistance-associated protein found in multidrug-resistant human lung cancer cell lines. Cross-resistance studies and gene transfection experiments indicated that under the conditions tested only ltpgpA and ldmdr1 are involved in resistance to arsenite and antimonials or hydrophobic drugs such as vinblastine respectively.