In vitro and in vivo assessment of renal drug transporters in the disposition of mesna and dimesna.

Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide- and cisplatin-induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P-glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady-state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug-drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide- and cisplatin-induced toxicities.

Carotid baroreflex function during and following voluntary apnea in humans.

Muscle sympathetic nerve activity (MSNA) and arterial pressure increase concomitantly during apnea, suggesting a possible overriding of arterial baroreflex inhibitory input to sympathoregulatory centers by apnea-induced excitatory mechanisms. Apnea termination is accompanied by strong sympathoinhibition while arterial pressure remains elevated. Therefore, we hypothesized that the sensitivity of carotid baroreflex control of MSNA would decrease during apnea and return upon apnea termination. MSNA and heart rate responses to -60-Torr neck suction (NS) were evaluated during baseline and throughout apnea. Responses to +30-Torr neck pressure (NP) were evaluated during baseline and throughout 1 min postapnea. Apnea did not affect the sympathoinhibitory or bradycardic response to NS (P > 0.05); however, whereas the cardiac response to NP was maintained postapnea, the sympathoexcitatory response was reduced for 50 s (P < 0.05). These data demonstrate that the sensitivity of carotid baroreflex control of MSNA is not attenuated during apnea. We propose a transient rightward and upward resetting of the carotid baroreflex-MSNA function curve during apnea and that return of the function curve to, or more likely beyond, baseline (i.e., a downward and leftward shift) upon apnea termination may importantly contribute to the reduced sympathoexcitatory response to NP.

Ectopic expression of Rsu-1 results in elevation of p21CIP and inhibits anchorage-independent growth of MCF7 breast cancer cells.

Signal transduction from tyrosine kinase receptors mediates growth regulation of breast cancer cells in part through the GTPase Ras and downstream kinases. Rsu-1 is a cDNA previously identified as an inhibitor of Ras-induced transformation. An HA-epitope tagged Rsu-1 cDNA was introduced into the MCF7 breast carcinoma cell line. Stable transfectants were selected and used for analysis of Rsu-1 expression on growth control and Ras-dependent kinase pathways. Assessment of biological activity of HA-Rsu-1 transfectants revealed that HA-Rsu-1 clones showed slower anchorage dependent growth rates than control MCF7 cell lines and a significant reduction in anchorage independent growth. Analysis of cell cycle regulatory proteins required for transit through G1 revealed that HA-Rsu-1 transfectant cell lines expressed elevated levels of p21CIP CDK inhibitor. Perturbations in signal transduction pathways which can be activated by Ras were detected in the Ha-Rsu-1 transfectants. Exposure of serum-starved cells to EGF revealed that expression of HA-Rsu-1 increased ERK-2 kinase activation, decreased activation of Jun kinase and inhibited Rho-dependent Rho-alpha kinase (ROK) activity compared to control cells. While serum starvation reduced AKT activity to undetectable levels in HA-Rsu-1 transfectants but not in control MCF7 cells, activation of AKT kinase by serum was unaffected by HA-Rsu-1 expression. Finally, the level of c-myc transcription in HA-Rsu-1 transfectants reached only 60% of the MCF7 control cell line following serum stimulation of starved cells while Fos RNA levels were similar to control cells. These results demonstrate that increased Rsu-1 expression critically altered cell cycle regulation and growth of MCF7 cells as well as signaling pathways in MCF7 cells required for malignant growth.

The ras suppressor, RSU-1, enhances nerve growth factor-induced differentiation of PC12 cells and induces p21CIP expression.

The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21(WAF/CIP) expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells.

Low-level transforming activity of an activated Ras gene under the control of a vaccinia virus p40 promoter is abrogated by truncation of the Ras cDNA.

Many human cancers have been shown to contain activated forms of the Ras proto-oncogene. Mutations comprising amino acid changes at codons 12, 13 and 61 therefore represent unique targets for cancer immunotherapy. Recombinant Vaccinia viruses encoding point mutated Ras oncogenes have raised issues concerning the safety and transforming ability of these recombinant vaccines. Vaccinia virus, a representative of the orthopox virus genus, is a large DNA virus that is cytopathogenic and that replicates in the cytoplasm of the infected cell. However, it remains unclear whether orthopox viruses are capable of genetic interactions with infected cells. Our studies show that DNA isolated from cells infected with a recombinant Vaccinia virus expressing mutated Ras constituted a poor reagent for transfection into NIH3T3 cells for transformation analysis. Stable integration of a recombinant Vaccinia virus expressing mutant Ras DNA was not detected in recipient cells. This study also demonstrates that the crossover plasmids used to generate the recombinant virus where the activated Ras gene is under the control of a Vaccinia virus early promoter had low but detectable transforming efficiency in the NIH3T3 transformation assay. Analysis of the transfected cells indicated that Ras transcription was initiated upstream of the Vaccinia virus promoter. The introduction of wobble mutations as well as the truncation of the Ras protein removed the transforming capabilities of the crossover vector. This study demonstrates the potential problems and solutions in the use of point mutated oncogenes in live vectors for cancer vaccine development.

Retention in substance abuse treatment. Role of psychiatric symptom severity.

The authors investigated the association between psychiatric symptom severity and subsequent treatment retention among substance abusers. The Symptom Check List-90-R was administered, after admission to an addiction treatment facility, to 308 male and 106 female clients with moderate-to-severe substance abuse problems. Mean scores on nine symptom and three summary scales were computed. Controlling for other sociodemographic and treatment variables, somatization was significantly associated with dropout from specialized outpatient and inpatient treatment programs. This study, however, suggests that psychopathologic symptom severity at admission has only limited utility in predicting subsequent treatment retention among substance abusers with overall moderate levels of psychological distress.

Psychosocial outcome of children evaluated for short stature.

OBJECTIVE: To assess the psychosocial functioning of adults who were evaluated as children for short stature and were not treated with human growth hormone. DESIGN: Inception cohort study. SETTING: Hospital-based pediatric endocrinology clinic. PARTICIPANTS: From 1975 to 1980, medical record review indicated that 181 of the children referred to our clinic for concerns about short stature were non-growth hormone deficient. In 1992 and 1993, we were able to recruit 35 of these patients for a follow-up study. Eligible subjects were at least 18 years of age at the time of follow-up. MAIN OUTCOME MEASURES: Standardized self-report questionnaires assessed various domains of psychosocial adjustment. Also, a brief test of intellectual functioning was administered and subjects underwent a semistructured in-person interview to evaluate pragmatic functioning and experiences associated with short stature. RESULTS: Few significant differences between the study sample and standardization samples were found on measures of psychosocial and intellectual functioning. Within-group childhood height during the first evaluation appointment was not significantly associated with most adult measures of psychosocial adjustment. Shorter adult stature was significantly associated with lower educational achievement, lower self-esteem, and greater emotional distress. CONCLUSIONS: The absence of significant psychosocial distress or impairment in these subjects brings into question one basis for hormonal treatment for non-growth hormone deficient short stature; that short stature in childhood is likely to lead to psychological dysfunction in adulthood. The results, however, also suggest that shorter stature in adulthood may constitute a psychosocial stressor, increasing vulnerability across several domains.

Administration of Epoetin alfa. Case study of the anemic patient.

Improving patient outcomes by achieving a stable Hct in the higher end of the target Hct range of 30% to 36% is the primary goal of anemia management and Epoetin alfa therapy. Recently, attention has been focused on the potential differences between subcutaneous (s.c.) and intravenous (i.v.) administration. Although some patients may require less Epoetin alfa when it is administered by the s.c. route, many patients require the same dose or more due to the significant heterogeneity in response. To ensure that therapeutic outcomes are maintained or improved, clinicians should evaluate both staff considerations and individual patient tolerance and response when determining the optimal route of administration.

Behavioural effects in mice of subchronic chlordiazepoxide, maprotiline, and fluvoxamine. I. Social interactions.

The present study compares the effects of subchronic administration (daily. 21 days) of chlordiazepoxide (CD), maprotiline and fluvoxamine on the behavior of male mice during dyadic social interactions. Maprotiline like chlordiazepoxide, stimulated aggression at 4 mg/kg and 2 mg/kg respectively (intermediate dose levels), whereas effects of fluvoxamine (3-8 mg/kg) were mainly sedative. Non-social activity was reduced by CD at 4 and 8 mg/kg and by maprotiline at 0.5 mg/kg. At the highest dose tested (10 mg/kg), maprotiline increased immobility, resembling the effects of fluvoxamine, while at 2 mg/kg, it reduced social investigation. Thus, despite some commonalities, there were several differences in behavioral profile of the compounds tested. Data are discussed in relation to the efficacy of each of these compounds in treating anxiety and depressive disorders.

Behavioural effects in mice of subchronic chlordiazepoxide, maprotiline and fluvoxamine. II. The elevated plus-maze.

In view of apparent commonalities in the aetiology, symptomatology, and pharmacotherapy of anxiety and depressive disorders, the present study compares the effects of the benzodiazepine, chlordiazepoxide (1.0-8.0 mg/kg), the selective noradrenaline (NA) reuptake inhibitor, maprotiline (0.5-10.0 mg/kg), and the serotonin (5-HT)-selective reuptake inhibitor, fluvoxamine (2.0-8.0 mg/kg), on the behaviour of mice in the elevated plus-maze test of anxiety. To more accurately reflect the clinical situation, subjects were treated daily for 21 days prior to testing, and comprehensive behavioural profiles were obtained through the application of an ethological scoring technique. Results show that subchronic treatment with chlordiazepoxide produced clear anxiolytic-like effects at the highest dose tested, coupled with an inhibition of risk assessment over the entire dose range. With the exception of risk assessment measures, anxiolytic-like effects were also seen with a low dose (0.5 mg/kg) of maprotiline: these effects were lost at higher doses. In contrast to these data, fluvoxamine produced minimal behavioural change under present test conditions. Findings are discussed in relation to the relative efficacy of selective monoamine. reuptake inhibitors in the treatment of anxiety disorders, and the nature of anxiety evoked in mice by exposure to the elevated plus-maze.

Behavioural effects in mice of subchronic buspirone, ondansetron and tianeptine. I. Social interactions.

In a continuation of recent work on effects of a benzodiazepine (chlordiazepoxide) and selective monoamine reuptake inhibitors (maprotiline and fluvoxamine), the current study compares effects of the 5-HT1A receptor partial agonist, buspirone (0.75-3.0 mg/kg), the 5-HT3 receptor antagonist, ondansetron (0.1-100 micrograms/kg) and the novel antidepressant, tianeptine (2.5-10.0 mg/kg). Compounds were given daily to mice for 21 days prior to testing and the subsequent behaviour of the animals during social interactions was assessed by ethopharmacological procedures. Buspirone, at 0.75 mg/kg, increased immobility and reduced occurrence of the aggressive act, "attack." At 1.5 and 3.0 mg/kg, it enhanced olfactory exploration of the sawdust substrate, but had no effect on social investigation. Ondansetron increased the duration of environmental exploration at 0.1 microgram/kg, while at 100 micrograms/kg it increased the duration of digging in the substrate. Ondansetron had no effect on the categories of behaviour and failed to induce an anxiolytic-like enhancement of social investigation. Tianeptine produced an anxiogenic-like effect at 10 mg/kg, while at 5 mg/kg it enhanced flight and immobility. The relevance of these findings is discussed in relation of the reported behavioural actions of these compounds and to current pharmacotherapy of anxiety and depression. The apparent anxiogenic effect of tianeptine is a novel finding which requires further study.

Behavioural effects in mice of subchronic buspirone, ondansetron and tianeptine. II. The elevated plus-maze.

In follow-up to recent work on benzodiazepines (chlordiazepoxide) and selective monoamine reuptake inhibitors (maprotiline and fluvoxamine), the present study compares the effects of the 5-HT1A receptor partial agonist, buspirone (0.75-3.0 mg/kg), the 5-HT3 receptor antagonist, ondansetron (0.1-100 micrograms/kg), and the novel antidepressant, tianeptine (2.5-10.0 mg/kg), on the behaviour of mice in the elevated plus-maze test of anxiety. Compounds were administered daily for 21 days prior to testing, and an ethological scoring technique was used to generate comprehensive behavioural profiles. Results show that subchronic treatment with ondansetron failed to influence the behaviour of mice in the plus-maze, while the limited changes induced by buspirone could not be attributed to anxiety-related processes. In contrast, tianeptine produced unambiguous anxiogenic-like effects at the top dose tested (10.0 mg/kg), a profile that was not secondary to changes in general levels of locomotor activity or exploration. Data are discussed in relation to current pharmacotherapy of anxiety and depressive disorders, and the nature of anxiety induced by animal models.

Induction of ductal morphogenesis and lobular hyperplasia by amphiregulin in the mouse mammary gland.

As the juvenile mouse mammary gland matures, it undergoes extensive epithelial proliferation, leading to a network of ductal branching that transverses the organ. Recent evidence suggests that the epidermal growth factor-related peptide amphiregulin (AR) may play multiple roles in the proliferation, differentiation, and neoplastic conversion of the mouse mammary gland. Using a dual approach of recombinant AR in slow-release pellets and retroviral expression of AR, we explored the roles of this growth factor in the mouse mammary gland in vivo. We first noted that recombinant AR can reestablish longitudinal ductal proliferation in growth quiescent mammary glands of ovariectomized mice. Furthermore, retrovirally transduced mammary transplants overexpressing AR developed into hyperplastic tertiary ducts and hyperplastic lobules with increased lateral branching, apparent 9 weeks after transplantation into cleared mammary fat pads. This is the first study to demonstrate that AR can reestablish the early developmental activity of ductal mammary epithelium and induce hyperplasia in vivo. These data, coupled with previous findings that demonstrated nearly universal overexpression of AR in human breast cancer and rodent mammary tumorigenesis, suggest that AR may be an important intermediary in glandular maturation and early malignant progression.

O-linked glycosylation modifies CD44 adhesion to hyaluronate in colon carcinoma cells.

CD44 alternative splicing patterns differ between normal and malignant tissue, and accordingly, modulation of CD44 splicing has received the most attention in studies that have examined the role of CD44 in tumor progression. Many investigators have examined functional differences between individual CD44 alternative splice variants. However, specific CD44 isoforms function uniquely depending on the type of cell on which they are expressed, thereby suggesting that additional tissue-specific mechanisms regulate CD44 function. In the present study we have demonstrated that colon carcinoma cells modify CD44 with O-linked glycosyl groups, and blockade of this glycosylation enhances their CD44-mediated adhesion to hyaluronate. This enhancement is attributable principally to CD44H (CD44s) rather than high molecular weight CD44 variants. Use of site-directed mutant CD44H cDNA transfectants demonstrated that CD44 O-linked glycosylation modulates interaction between hyaluronate and the B loop domain of CD44. The influence of glycosylation on CD44 function in colon carcinoma cells is specific to the presence of O-linked sugars; inhibition of N-linked glycosylation had minimal influence on CD44 function. These findings indicate that O-linked glycosylation may be as important as alternative splicing in the regulation of CD44 function and the broad spectrum of biological processes attributed to it, including normal development, tumor metastases, and lymphocyte function.

Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation.

Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.

Keratan sulfate modification of CD44 modulates adhesion to hyaluronate.

CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function.

CD44 hyaluronate binding influences growth kinetics and tumorigenicity of human colon carcinomas.

CD44 is a cell surface receptor of hyaluronate that is implicated in the regulation of tumor growth and metastatic potential. Transformation of colon mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The functional roles of CD44 isoforms in colon carcinoma tumor progression remain unclear. CD44H expression is downregulated in colon carcinomas compared to paired normal mucosa. In the present study we demonstrate that reintroduction of CD44H back into colon carcinoma cells by stable transfection reduces their in vitro growth rate and tumorigenicity. Examination of several colon carcinoma cell lines and use of mutant CD44H reveal that this in vitro and in vivo growth reduction requires the ability of CD44H to bind hyaluronate. These observations indicate that the CD44H downregulation associated with transformation of mucosa to colon carcinoma may provide the carcinoma cells with a growth advantage. Furthermore, the reduction in tumor growth rate mediated by reintroduction of CD44H into colon carcinoma cells is dependent on its ability to bind hyaluronate.

Restoration of CD44H expression in colon carcinomas reduces tumorigenicity.

OBJECTIVE: The functional consequences of reintroduction of the CD44H cell adhesion molecule into colon carcinomas were investigated. BACKGROUND: CD44 is a cell surface adhesion molecule that is normally present in numerous isoforms as a result of messenger RNA alternative splicing. Individual CD44 isoforms differ in their ability to enhance tumorigenic or metastatic potential when overexpressed on tumor cells. Reverse transcriptase-polymerase chain reaction analysis demonstrates that CD44H is down-regulated during transformation of normal colon mucosa to carcinoma. The functional consequences of CD44H down-regulation in colon carcinomas has not been clarified. METHODS: Tumor cell lines and fresh tissue specimens were examined for CD44 expression by Western blot analysis. CD44H cDNA and site-directed mutants of CD44H cDNA were transfected into colon carcinoma cells. Stable transfectants were examined for adhesion to hyaluronate, in vitro growth, and in vivo growth. RESULTS: CD44H expression was nearly undetectable in primary colon carcinomas and colon carcinoma cell lines. In contrast, normal mucosa expressed high levels of CD44H. When CD44H was reintroduced into colon carcinoma cells, their in vitro and in vivo growth was significantly reduced. This CD44H-mediated growth rate reduction required an intact cytoplasmic domain. CONCLUSIONS: Transformation of normal mucosa to colon carcinoma is associated with a down-regulation of CD44H, which consequently may enhance the growth rate and tumorigenicity.

Effects of oral creatine loading on single and repeated maximal short sprints.

This investigation determined whether oral creatine (Cr) loading could enhance single and repeated short sprint performance. A 1 x 10 s cycle sprint (Study One) and 6 x 6 s (departing every 30 s) repeated cycle sprints (Study two) were the performance tests used. Separate groups of subjects, randomly assigned to either a Cr or placebo (P) group, were used in each study in a double blind design. After performing a familiarization and two baseline tests subjects loaded with 5g of Cr or P four times per day for five days before repeating the tests one and three days post-loading. In Study One (n = 9 in each group), work completed (kJ) at 2, 4, 6, 8 and 10 s and peak power (W) were greater than baseline values in each of the post-loading trials in both the Cr and P groups. There were no between group performance differences. In Study Two (n = 11 in each group) after loading, the Cr group recorded significantly greater scores than the P group in total work (kJ) completed over the 6 sprints, work completed in sprint 1, and peak power (W). Post-exercise blood lactate and pH responses were not different between the Cr and P groups after loading in either study. Although Study One results are equivocal, Study Two results suggest that Cr supplementation can enhance both single (if sprint 1 results are considered in isolation) and repeated short sprint performance.