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Abstract Background and Aims In the United States, the opioid epidemic has led many young people who use opioids to initiate injection drug use, putting them at risk for hepatitis C virus (HCV) infection. However, community surveys to monitor HCV prevalence among young people who inject drugs (YPWID) are rare. Methods As part of Staying Safe (Ssafe), a trial to evaluate an HCV‐prevention intervention, a community‐recruited sample of 439 young people who use opioids (ages 18−30) in New York City (NYC) were screened from 2018 to 2021. Screening procedures included a brief verbal questionnaire, a visual check for injection marks, onsite urine drug testing, rapid HCV antibody (Ab) testing, and dried blood spot (DBS) collection. DBS specimens were sent to a laboratory for HCV RNA testing and phylogenetic analysis to identify genetic linkages among HCV RNA‐positive specimens. Multivariable logistic regression was used to assess associations between HCV status (Ab and RNA) and demographics and drug use patterns. Results Among the 330 participants who reported injecting drugs (past 6 months), 33% (n = 110) tested HCV Ab‐positive, 58% of whom (n = 64) had HCV RNA‐positive DBS specimens, indicating active infection. In multivariable analysis, visible injection marks (AOR = 3.02; p

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2. The track diffusivity distributions of these two populations of single-particle tracks differ significantly, demonstrating that labeling method can be an important determinant of diffusive behavior. We also applied perturbation expectation maximization (pEMv2) (Koo and Mochrie in Phys Rev E 94(5):052412, 2016), which sorts trajectories into the statistically optimum number of diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the tracks into two diffusive states: an essentially immobile state and a more mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much smaller ([Formula: see text]) than the mobile fraction of TRAP-labeled Pma1 tracks ([Formula: see text]). In addition, the diffusivity of Pma1-mEos3.2's mobile state is several times smaller than the diffusivity of TRAP-labeled Pma1's mobile state. Thus, the two different labeling methods give rise to very different overall diffusive behaviors. To critically assess pEMv2's performance, we compare the diffusivity and covariance distributions of the experimental pEMv2-sorted populations to corresponding theoretical distributions, assuming that Pma1 displacements realize a Gaussian random process. The experiment-theory comparisons for both the TRAP-labeled Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach., (© 2023. The Author(s), under exclusive licence to EDP Sciences, SIF and Springer-Verlag GmbH Germany, part of Springer Nature.)

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)(70) = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

Purpose: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity., Experimental Design: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts., Results: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P ≤ 0.001)., Conclusions: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression., (©2012 AACR.)

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

The time course of insulin sensitivity, skeletal muscle glycogen and GLUT4 content, and glycogen synthase (GS) activity after a single bout of intense exercise was examined in eight horses. On separate days, a euglycemic-hyperinsulinemic clamp (EHC) was undertaken at 0.5, 4, or 24 h after exercise or after 48 h of rest [control (Con)]. There was no increase in mean glucose infusion rate (GIR) with exercise (0.5-, 4-, and 24-h trials), and GIR was significantly decreased at 0.5 h postexercise (GIR: 8.6 +/- 2.7, 6.7 +/- 2.0, 9.0 +/- 2.0, and 10.6 +/- 2.2 mg.kg(-1).min(-1) for Con and at 0.5, 4, and 24 h, respectively). Before each EHC, muscle glycogen content (mmol glucosyl units/kg dry muscle) was higher (P < 0.05) for Con (565 +/- 102) than for other treatments (317 +/- 84, 362 +/- 79, and 382 +/- 74 for 0.5, 4, and 24 h, respectively) and muscle GLUT4 content was unchanged. Pre-EHC active-to-total GS activity ratio was higher (P < 0.05) at 0.5, 4, and 24 h after exercise than in Con. Post-EHC active GS and GS activity ratio were higher (P < 0.05) in Con and at 24 h. There was a significant inverse correlation (r = -0.43, P = 0.02) between glycogen content and GS activity ratio but no relationship between GS activity and GIR. The lack of increase in insulin sensitivity, determined by EHC, after exercise that resulted in a significant reduction in muscle glycogen content is consistent with the slow rate of muscle glycogen resynthesis observed in equine studies.

Reasons for Performing Study: Exercise is an important stimulus for the regulation of numerous metabolic functions in muscle, but there has been little investigation of the mechanism of exercise-enhanced glucose utilisation., Objective: To examine the effects of exercise on the signalling components Akt and glycogen synthase kinase-3 (GSK3) involved in insulin regulation of glycogen synthase (GS) activity. It was hypothesised that hyperinsulinaemia and prior exercise would alter the extent of Akt phosphorylation and GSK3beta deactivation and increase insulin stimulation of GS activity in skeletal muscle., Methods: Eight Standardbred horses completed a euglycaemic-hyperinsulinaemic clamp (EHC) either without prior exercise (NonEX) or at 0.5, 4 or 24 h post exercise (0.5 PostEX, 4 PostEX, and 24 PostEX, respectively). Muscle samples for determination of Akt and GSK3 serine phosphorylation, and GS fractional velocity (GS(FV)) were collected 5 min before (Pre-INS) and at the end (Post-INS) of each EHC (pre- and post insulin stimulation). Protein was separated by SDS-PAGE on polyacrylamide gels, transferred to nitrocellulose membranes and incubated with antibodies against phospho-Akt Ser473 and GSK3alpha/beta Ser21/9., Results: GS(FV) was increased (P<0.05) 2-fold at 0.5 PostEX, 4 PostEX and 24 PostEX when compared to NonEX in Pre-INS and in Post-INS was increased 33 and 28% in NonEX and at 24PostEX (P<0.05), respectively, when compared to Pre-INS in these trials. Phospho-Akt Ser473 was increased at 0.5 PostEX only (P<0.05) in Pre-INS but increased in all trials in Post-INS. Phospho-GSK3alpha (Ser21) was also greater (P<0.05) at 0.5 PostEX in Pre-INS than in NonEX and 24 PostEX and in Post-INS increased (P<0.05) in NonEX and at 24 PostEX but not at 0.5 PostEX or 4 PostEX post exercise. There was no effect of time or treatment on GSK3, (Ser9) phosphorylation or on total GSK3., Conclusions: Elevated plasma insulin and prior exercise were associated with Akt activation and GSK3alpha deactivation (0.5 PostEX) indicating that an effect of exercise is evident in the early post exercise period and supporting a physiological role for GSK3alpha in the regulation of GS activity. The changes are consistent with insulin signalling downstream from these molecules.

Reasons for Performing Study: There is evidence that adaptation to diets rich in nonstructural carbohydrates (NSC) contributes to the development of insulin resistance in horses. To date, however, no study in horses has examined the effects of physical conditioning on diet-associated alterations in insulin sensitivity and glucose tolerance., Objectives: To examine the effects of adaptation to concentrate feeds rich in NSC or fat on insulin sensitivity and glucose tolerance in horses, both in the sedentary state and after a subsequent period of physical conditioning., Methods: Fourteen mature Standardbred horses underwent both a euglycaemic-hyperinsulinaemic clamp (EHC) and oral glucose tolerance test (OGTT) after each of the following phases: Baseline - fed only forage cubes for 3 weeks; Diet horses were randomly assigned to receive either a high NSC (S) concentrate or a high fat concentrate (F) with forage cubes for 6 weeks; and Diet x Exercise - horses remained on the assigned ration and underwent a 7 week period of physical conditioning. An incremental exercise test was performed before, and after, the Diet x Exercise phase for measurement of the peak rate of oxygen consumption (VO2peak)., Results: In both diet groups, there was an approximately 10% increase in mean VO2peak after physical conditioning. The mean rate of glucose disposal (M) per unit of serum insulin (I) during the EHC [M/I ratio] in S horses was 30% lower (P<0.05) in the Diet phase when compared to Baseline, but not different from Baseline after physical conditioning. The S diet also resulted in a greater (P<0.05) OGTT insulin response (area under the insulin vs. time curve, AUC(INS)) in both Diet and Diet x Exercise phases when compared to Baseline. In F, insulin sensitivity (mean M/I ratio) and glucose tolerance were unchanged during the study., Conclusions and Potential Relevance: Feeding a diet rich in NSC for 6 weeks resulted in decreased insulin sensitivity and impaired glucose tolerance. Physical conditioning lessened the effects of the high NSC diet on insulin sensitivity, as evidenced by the return to baseline M/I, but did not mitigate the impaired glucose tolerance. Decreased insulin sensitivity has been implicated in the development of obesity and laminitis in horses and the present findings provide support for avoidance of concentrates with high NSC in the dietary management of horses at risk for the development of these conditions.

Both the euglycemic-hyperinsulinemic clamp (EHC) and minimal model analysis of the frequently sampled intravenous glucose tolerance test (FSIGT) have been applied for measurement of insulin sensitivity in horses. However, no published data are available on the reproducibility of these methods. Therefore, the objective of this study was to evaluate the variation and repeatability of measures of glucose dynamics and insulin sensitivity in horses derived from minimal model analysis of the FSIGT and from the EHC method. Six healthy horses underwent both the FSIGT and EHC on 2 occasions over a 4-week period, with a minimum of 5 days between tests. Coefficient of variation (CV) and intraclass correlation coefficient (ICC) were calculated for measures of glucose metabolism and insulin sensitivity derived from each test. In the EHC, insulin sensitivity, expressed as the amount of metabolized glucose (M) per unit of serum insulin (I) (M/I ratio), averaged 0.19 +/- 0.06 x 10(-4) mmol/kg/min x (pmol/L)(-1) with an average interday CV of 14.1 +/- 5.7% (range, 7-20%) and ICC of 0.74. Minimal model analysis of the FSIGT demonstrated mean insulin sensitivity (Si) of 0.49 +/- 0.17 x 10(-4)/min x (pmol/L)(-1) with an average interday CV of 23.7 +/- 11.2% (range, 9-35%) and ICC of 0.33. Mean CV and ICC for minimal model glucose effectiveness (Sg) and acute insulin response (AIRg) were, respectively, 26.4 +/- 11.2% (range 13-40%) and 0.10 and 11.7 +/- 6.5% (range 7-21%) and 0.98. Insulin sensitivity measured by the EHC has lower interday variation when compared with the minimal model estimate derived from the FSIGT.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.

Aims: Using tissue microarrays, this study analysed the expression of the multidrug resistance protein, MRP2, by immunohistochemistry with two different MRP2 antibodies. This is the first study to address the expression of MRP2 in various common human neoplasms., Methods and Results: Immunohistochemistry was performed on zinc formalin-fixed tissue to evaluate normal tissues and carcinomas using two antibodies against MRP2 (EAG5, a polyclonal antibody, and M2-lll-6, a monoclonal antibody). Immunostaining was localized in neoplastic cells mainly on the cell membrane with M2-lll-6 and cell membrane and cytoplasm with EAG5. In normal tissues MRP2 was expressed in liver, gastrointestinal tract, and kidney tubular epithelial cells with both antibodies. MRP2 was seen in nine of 22 renal cell carcinomas, eight of 13 gastric carcinomas, 25 of 49 breast carcinomas, 14 of 32 lung carcinomas, 39 of 50 colon carcinomas, and 16 of 17 ovarian carcinomas. There was < 10% variability between the two antibodies. MRP2 expression was highest in moderate to poorly differentiated tumours from colon, lung, gastric, and ovarian carcinomas and in grade 2 and 3 breast and renal carcinomas., Conclusion: The expression of MRP2 in many solid human tumours indicates that inherent drug resistance may play an important role as a biomarker for predictive chemotherapy treatment.

The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.

In serum starved V79 Chinese hamster lung fibroblast cells, replacement of D-glucose with D-allose resulted in a significant 38 +/- 18% (P < 0.05) reduction of 2-deoxy-D-glucose (2-DG) transport. Similarly, in a respiration-deficient mutant cell line (V79-G14), which has elevated 2-DG transport activity, D-allose reduced 2-DG transport by 59 +/- 18% (P < 0.05). [3H]D-allose uptake by V79 cells occurred slowly and was not inhibited by cytochalasin B, suggesting diffusion as the mode of D-allose entry. Western blot analysis using a rabbit polyclonal antibody to the human erythrocyte glucose transporter (GT) demonstrated that, in both cell lines, GT content and GT subcellular distribution were not significantly different in D-glucose vs. D-allose-treated cells. delta-Antibody, which has been shown to bind to exofacial epitopes of the GT (Harrison et al., 1990, J. Biol. Chem., 265:5793-5801), did not demonstrate any differences in surface binding to D-glucose vs. D-allose-treated intact V79 cells. D-allose treatment of 3T3 fibroblasts resulted in a similar decrease (72%) of 2-DG transport, however D-allose had no apparent effect on basal sugar transport in 3T3 adipocytes. These results suggest that D-allose reduces sugar transport through a modulation of the intrinsic activity of the GT, and that D-allose may act in a tissue-specific manner.

Sugar transport regulation was characterized in terms of the expression and subcellular distribution of the glucose transporter (GT) in the V79 hamster fibroblast cell line and in a respiration deficient mutant (G14) of the V79 cell line. Comparison of GT content in V79 and G14 cells and cell fractions revealed that the 3-fold elevation in basal sugar transport observed in the G14 cell line did not coincide with any significant difference in either the whole cell or plasma membrane GT content when compared to the V79 parental cell line. Determination of delta-antibody binding to intact cell monolayers supported the finding that the two cell lines demonstrate equivalent plasma membrane GT content. Further, D-glucose inhibitable cytochalasin B binding to total cell membranes indicates that additional, unrecognized GT isoforms do not occur in either cell line. A higher average molecular weight GT was detected in the G14 cell line, and treatment of GT enriched preparations with endoglycosidase F established that the G14 cell line exhibits a hyperglycosylated form of the same core GT protein expressed in V79. These results suggest that an enhancement of the intrinsic activity of the GT expressed in G14 is responsible for its increased sugar transport capabilities and this may be related to differences in GT post-translational processing.

We have recently demonstrated that exposure of MCF7 cells to antiestrogens in vitro results in both accumulation of IGF-BP3 in media and reduced mitogenic responsivity to IGFs (Cancer Res. 53:5193-5198). We show here that MCF7 cell proliferation in steroid-stripped, serum containing media is significantly attenuated by rhIGF-BP3 (p < 0.05), with a maximal 40% inhibition of serum stimulated growth achieved by 6.25nM IGF-BP3. 10(-10) M estradiol (E2) significantly stimulated MCF7 proliferation, and co-incubation of estrogen containing cultures with 50nM IGF-BP3 resulted in significant attenuation of the estrogen-stimulated proliferation ([3H]thymidine incorporation: E2, 147 +/- 18% of control; E2 +/- IGF-BP3, 111 +/- 18% of control, p < 0.05). These results demonstrate antagonism of steroid stimulated proliferation by an IGF binding protein and are compatible with the hypothesis that antiestrogen-induced accumulation of IGF-BP3 in the conditioned media of MCF7 cells contributes to the cytostatic action of these drugs.

Many neoplastic cell lines secrete insulin-like growth factor binding proteins (IGFBPs). The physiological role of these proteins is incompletely characterized; under various conditions IGFBPs have been observed to either enhance or inhibit the biological activity of insulin-like growth factors. MCF7 human breast cancer cells are known to be mitogenically responsive to insulin-like growth factors and estrogens, to secrete several IGFBPs, including BP-2, BP-4 and BP-5, and to be growth inhibited by antiestrogens. We report here that the pure antiestrogen ICI 182,780 and, to a lesser extent, the commonly used drug tamoxifen significantly increase levels of a M(r) 43,000-46,000 IGFBP (BP-3) and significantly reduce levels of a M(r) 24,000 IGFBP (BP-4) in the conditioned medium of MCF7 cells. Effects of estradiol and antiestrogens on M(r) 30,000 and M(r) 36,000 IGFBPs are also described. The effects of ICI 182,780 on IGFBPs in the conditioned medium of MCF7 cells may contribute to the remarkable ability of this compound to attenuate insulin-like growth factor I stimulated MCF7 cell proliferation.

The prevalence of Dirofilaria immitis and Dipetalonema reconditum infections in Missouri dogs was investigated by means of a statewide veterinary mail survey and a study of shelter dogs from central Missouri and Mississippi River communities. The veterinary survey revealed microfilariae of D immitis in 447 of 11,823 (3.8%) dogs, microfilariae of D reconditum in 14 (0.1%), and microfilariae of both species in 8 (less than 0.1%). Of 493 shelter dogs examined, using the modified Knott test, 23 (4.7%) were positive for D immitis, 23 (4.7%) for D reconditum, and 2 (0.4%) for both helminths. The prevalence of infection with both D immitis (9.4%) and D reconditum (6.3%) was higher in shelter dogs from the Mississippi River communities than in those from central Missouri (1.7% for D immitis, 3.7% for D reconditum). The microfilariae of D immitis in shelter dogs ranged in length from 255 to 343 micrometer (mean, 314 micrometer) and in width from 5.0 to 7.5 micrometer (mean, 6.5 micrometer); those of D reconditum ranged in length from 238 to 295 micrometer (mean, 257 micrometer) and in width from 3.2 to 6.5 micrometer (mean, 4.9 micrometer). Buttonhook tails were seen in 41% of D reconditum. In the final analysis, the shape of the anterior end was considered the most reliable criterion for differentiation.

The relative efficacies of pyrantel tartrate and of pyrantel citrate against Oesophagostomum sp in swine were evaluated in a controlled-critical study and the efficacy of pyrantel citrate in a field trial. In the controlled-critical study, pigs naturally infected with Oesophagostomum dentatum were either not treated or were treated with pyrantel citrate or pyrantel tartrate at a dosage of 510 mg of free pyrantel base/kg of feed. Six days later, the pigs were necropsied, adult O dentatum was recovered and counted, and fecal samples were examined for helminth eggs. The efficacies of pyrantel citrate and pyrantel tartrate were each 100% based on fecal egg counts and numbers of adults at necropsy. The field trial was conducted in a similar manner except that pyrantel citrate only was tested against a control group on the basis of fecal egg counts made both at the beginning and at the termination of the trial. In this study pyrantel citrate was found to reduce Oesophagostomum sp egg counts by 89.4%.

A paste formulation of 3.4% febantel and 0.34% praziquantel was given in soft food at a rate of 0.29 g/kg of body weight, once daily for 3 days to dogs and cats. Eleven of 12 dogs were naturally infected with Ancylostoma caninum, 10 with Trichuris vulpis, 2 with Toxocara canis, 5 with Taenia sp, and 3 with Dirofilaria immitis. Ten of 11 cats were naturally infected with Toxocara cati, 2 with Ancylostoma tubaeforme, 3 with Taenia sp, 5 with Capillaria aerophila, and 1 with Paragonimus kellicotti. Efficacy was 100% for the removal of A caninum, T canis, and Taenia sp and 99.9% in clearance of T vulpis in dogs, and 100% in removal of A tubaeforme, T cati, and Taenia sp in cats. In dogs, Dirofilaria appeared unaffected, and in cats, there was no apparent effect on Capillaria or on Paragonimus. The paste formulation given in soft food was well tolerated and without observable adverse effects.

Fenbendazole was given in the feed to swine at a cumulative dosage of 9 mg/kg of body weight over a period of 3, 6, and 12 days to compare efficacy. Four treatment groups of ten 2- to 3-month-old pigs each, with a mean of 15 kg of body weight per group, received 3 mg of fenbendazole/kg/day for 3 days, 1.5 mg/kg/day for 6 days, 0.75 mg/kg/day for 12 days, and no medication. Medicated feed was scheduled so that all treated pigs reached the last day of treatment on the same day, thus making the time between the last treatment and necropsy equal for all groups. Ascaris suum and Trichuris suis were the target species, their presence before treatment being determined by fecal egg counts and at necropsy by worm counts. At necropsy, 9 control pigs were infected with A suum (mean of 18.0 worms/pig), and all control pigs had T suis infection (mean of 36.5 worms/pig). All 3 treatment schedules were 100% effective in removal of A suum; and for T suis, the 3-day regimen was 100% effective, the 6-day regimen, 99.2%, and the 12-day regimen, 91.0%.

Oxfendazole, a benzimidazole carbamate, was administered as a 0.5% feed additive to 88 pigs naturally infected with two to four nematode species. Dose rates of 1.5 mg/kg of body weight, 3.0 mg/kg, 3.75 mg/kg, 4.5 mg/kg, 6.75 mg/kg, or 9.0 mg/kg were 100% efficacious against Oesophagostomum dentatum and 99.2% to 100% effective against Ascaris suum. Dose rates of 4.5 mg/kg, 6.75 mg/kg, and 9.0 mg/kg were 92.7%, 98.9%, and 99.5% effective, respectively, against mixed populations of Metastrongylus apri and M pudendotectus. Results were variable with Trichuris suis infections. Efficacy was based on the number of nematodes recovered at necropsy. Palatability and acceptability of the feed additive were good, and adverse reactions following administration were not observed.

Febantel was administered as a 3.4% paste to 45 dogs infected with 1 or more nematode species, which included Ancylostoma caninum and Trichuris vulpis. Dosage levels of 5, 10, or 15 mg/kg of body weight daily for 3 consecutive days for a total of 15, 30, or 45 mg/kg were administered. At 15 mg/kg, all dogs were completely cleared of all nematode and cestode infections, including A caninum, T vulpis, Uncinaria stenocephala, Toxocara canis, Toxascaris leonina, Taenia sp, and Dipylidium caninum, except for 1 Physaloptera sp adult. The 2 other dosage levels produced somewhat lower efficacy, with the 5 mg/kg being the least effective. The paste appeared palatable to the dogs and there were no apparent adverse effects.

The translocation mnT12(IV;X) is a fusion of holocentric chromosomes IV and X, the breakpoints occurring near the left end of IV and the right end of X. Animals homozygous for mnT12 are viable and fertile; they contain five pairs of chromosomes rather than the normal set of six pairs. The mnT12 chromosome is larger than all wild-type chromosomes and thus identifies linkage groups IV and X cytologically. Hermaphrodites heterozygous for mnT12 show high frequency meiotic nondisjunction both between mnT12 and the X chromosome, which results in a high incidence of male self progeny (27% compared to the wild-type incidence of 0.2%), and between mnT12 and chromosome IV, which results in a high incidence of self progeny essentially trisomic for chromosome IV (karyotype IV/mnT12/mnT12). The viability of chromosome IV trisomics has been confirmed by constructing animals trisomic for only normal copies of chromosome IV; these animals are morphologically wild type. Meiotic chromosome disjunction in mnT12 homozygotes appears to be normal, although the frequency of recombination between markers that are normally X-linked is significantly reduced. Males of genotype IV/mnT12/0 are fertile. They can be thought of as having a neo-X(mnT12) neo-Y(normal IV) karyotype since it is possible to maintain a male-hermaphrodite stock of C. elegans consisting of such males and hermaphrodites carrying two neo-X chromosomes and no neo-Y; the organism is thus converted from an XO:XX type of sex determination to an XY:XX system.

The physiology of the superior colliculus (SC) implicates it in the visual control of eye movements. In the primate, acute inactivation of the superior colliculus delays the onset of a visually guided saccade, slows its velocity, and shortens its amplitude. Previous research leaves uncertain whether other oculomotor disorders which sometimes follow ablation of this structure are due to tectal pathology, to neural damage surrounding the tectum, or to both causes. In this study, 7 cynomolgus monkeys received SC ablations. In 3 others, control lesions were placed in the pretectal/posterior thalamic region. Both procedures produced a qualitatively similar syndrome of 4 oculomotor changes. Reaction time to initiate saccades to visual targets was slowed. Secondly, the surgery constricted the normal ocular range. At the worst, movement was confined to a radius of 10-12 degrees of primary gaze. The monkeys displayed two kinds of inaccuracies when attempting to foveate stationary visual targets within their surviving ocular range. Saccadic amplitude was reduced, causing the monkeys' initial attempt to fall short of foveating the target. If the target remained lighted there then ensued a series of stepwise corrective saccades toward it. The corrective saccades ceased with the eyes still at a position short of the target. Eye position remained in error for the duration of the trial. The final position was independent of the target's retinal position or the vector of the motor command needed to acquire the target. Rather, the error was related to the angular position of the target about the head ( = desired eye position). The syndrome appeared qualitatively similar whether resulting from tectal or the more rostral pretectal/diencephalic ablation. When occurring along the horizontal axis, the deficits appeared to require damage to the superior colliculus, perhaps combined with pathology of some other structure. The same syndrome along the vertical axis was better correlated with pretectal/diencephalic pathology. Invasion of these areas together with invasion of the transthalamic axons from the frontal eye fields is interpreted as the critical pathology responsible for the syndrome. A similar oculomotor trajectory can be modelled by supposing a loss in the gain of the signal which conveys the target's retinal position, combined with one other fault in the circuit: either a loss in gain of the eye position signal, or the signal representing the target's position in craniocentric coordinates.

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.