Pathways to paediatric urology subspecialisation: a study of casemix, incumbent attitudes and opinions.

OBJECTIVE: To identify any self-reported differences or attitudes towards certification, publication, or practice patterns between adult urology and paediatric general surgery-trained paediatric urology providers. There are no known published differences in clinical/operative/research outcomes in either group. METHODS: An 18-item cross-sectional survey was compiled through the EAU Young Academic Urologists (YAU) office and disseminated to a trans-Atlantic convenience sample of current practising paediatric urologists. This was created using a mini-Delphi method to provide current semi-quantitative data relating to current opinions and attitudes of this cohort. RESULTS: A total of 228 respondents completed the survey, with female respondents representing 37% and 34% for urology and paediatric general surgery, respectively. Nearly 90% overall respondents felt that a full 2-year paediatric fellowship program was very important and 94% endorsed a collaborative dedicated paediatric urology on call service, with 92% supporting the joint development of transitional care. Urology managed higher numbers of bedwetting (p = 0.04), bladder bowel dysfunction (p = 0.02), endourological procedures (p = 0.04), and robotics (p = 0.04). Paediatric general surgery managed higher numbers of laparoscopic reconstruction (p = 0.03), and posterior urethral valve ablation (p = 0.002). CONCLUSION: This study represents the first time that a cross-sectional cohort of paediatric urologists from different training backgrounds were compared to assess their productivity, practice patterns and attitudes. Paediatric urology is in a unique position to have two contributing specialities, with the ability to provide optimal transitional and lifelong care. We believe that there should be a strong emphasis on collaboration and to remove any historically-created barriers under policies of equity, diversity and inclusivity.

Neuropsychiatric Developmental Disorders in Children Are Associated With an Impaired Response to Treatment in Bladder Bowel Dysfunction: A Prospective Multi-Institutional European Observational Study.

PURPOSE: Bladder and bowel dysfunction is a common but underdiagnosed pediatric entity which may represent up to 47% of pediatric urology consults. The objectives of this observational study were to determine functional 1-year outcomes following standard treatment of bladder and bowel dysfunction in both control and neuropsychiatric developmental disorder groups using validated questionnaires, and to perform an initial cost analysis. MATERIALS AND METHODS: This was a prospective observational study conducted across a number of academic European centers (July 2020-November 2022) for new bladder and bowel dysfunction patients. Parents completed a sociodemographic survey, information pertaining to prior neuropsychiatric developmental disorder diagnoses, as well as a number of validated functional scores. RESULTS: A total of 240 patients were recruited. In the control bladder and bowel dysfunction group, the baseline Dysfunctional Voiding Scoring System and Childhood Bladder and Bowel Dysfunction Questionnaire scores were 20% and 17.% lower, respectively, after 1 year compared to the neuropsychiatric developmental disorder group. The change in improvement was diminished for the neuropsychiatric developmental disorder cohort in both Dysfunctional Voiding Scoring System and Childhood Bladder and Bowel Dysfunction Questionnaire scores. The odds ratio of full symptom resolution was 5.7 in the control cohort compared to the neuropsychiatric developmental disorder cohort. A cost analysis on prescribed medications at referral led to a total cost of €32,603.76 (US $35,381.00) in the control group and €37,625.36 (US $40,830.00) in the neuropsychiatric developmental disorder group. CONCLUSIONS: This study demonstrates that pediatric patients with a neuropsychiatric developmental disorder exhibit more severe bladder and bowel dysfunction at baseline and throughout treatment with a lower overall quality of life, as well as 15.4% higher medication costs at referral. It is also important that parents' and caregivers' expectations are managed regarding higher levels of treatment resistance for functional bladder and bowel issues.

A cross-sectional analysis of paediatric urologists' current practices, opinions and areas of perceived importance in the delivery of adolescent & transitional care.

INTRODUCTION: Complex urological anomalies often require continued care as patients reach adulthood. Adequate transition for adolescents with ongoing urological care needs is critical to allow for seamless care in adult hospitals. Studies have shown that this can lead to improved patient and parental satisfaction, and lower utilisation of unplanned inpatient beds and emergency department visits. There is currently no ESPU-EAU consensus on the adequate mechanism and very few individual papers examining the role of urological transition for these patients in a European setting. This study aimed to identify current practice patterns in paediatric urologists providing adolescent/transitional care, to assess their opinions towards formal transition and to look for variations in care. This has implications for long-term patient health and specialist care. METHODS: An 18-item cross-sectional survey was compiled and pre-approved through the EAU-EWPU and ESPU board offices prior to dissemination to all registered ordinary members affiliated with the ESPU. This was created using a mini-Delphi method through the EWPU research meetings to provide current semi-quantitative data relating to current opinions and attitudes of this cohort. RESULTS: A total of 172 respondents (55% paediatric general surgery; 45% urology) across 28 countries completed the survey. The majority of respondents were in practice >10 years and spent >80% time in paediatric urology. There was no formal transition process according to 50% respondents and over half of those that did have less than 1/month, with <10% using validated questionnaires. More than two-thirds respondents continued to provide care after transition, as >70% units had no designated corresponding adult service. Furthermore, 93% paediatric believe a formal transition service to be very important, using a multidisciplinary framework. A pareto chart demonstrated 10 specific conditions to be of most interest in transition to adulthood. CONCLUSION: This is the first study to assess the requirements of paediatric urologists for adequate transitional care, however due to the nature of the survey's distribution, this was a non-scientific poll based on a convenience sample of respondents. It is critical that dual-trained or adult-trained urologists with a specific interest in paediatric urology work with current paediatric urologists in a multidisciplinary fashion to facilitate early transition based on the adolescent's developmental and biopsychosocial requirements. National urological and paediatric surgical societies need to make transitional urology a priority. The ESPU and EAU should collaboratively consider developing transitional urology guidelines to allow a framework by which this can occur.

Genetic variability of procolipase associates with altered insulin secretion in non-diabetic Caucasians.

AIMS: Procolipase (CLPS) is secreted from the exocrine pancreas into the gastrointestinal tract and becomes proteolytically cleaved into colipase and the pentapeptide enterostatin. While colipase is an indispensable cofactor for pancreatic lipase, enterostatin acts as a hormone that inhibits insulin secretion and confers satiety signals to the brain, thereby restricting further food intake in animal models. As both high fat diet and obesity contribute to insulin resistance, we investigated whether genetic variability of CLPS associates with metabolic traits in non-diabetic humans at diabetes risk. METHODS: Tagging single nucleotide polymorphisms (SNPs) in the human CLPS locus on chr6p21.1 were selected using HapMap data. 498 humans, phenotyped for different glucose and lipid metabolic traits, were genotyped by bidirectional sequencing and multivariate linear regression analyses were undertaken. RESULTS: 2 tagging SNPs (rs3748050 in the Kozak sequence: A/G and rs3748051 in intron 1: A/G), covering 100% of CLPS variability including 8 kb of its promoter, were genotyped for association analyses. The minor alleles of both tagging SNPs associated significantly with a reduced insulin secretion (-20.2%, both SNPs) in various estimation models derived from the oral glucose tolerance test (OGTT; rs3748050/51: 30 min C-peptide levels: p=0.001/0.01, insulinogenic index: p=0.02/0.02, AUC C-peptide/AUC glucose: p=0.01/0.01) after adjustment for relevant covariates. No significant associations with fasting total cholesterol (c), HDL-c, LDL-c, triglycerides and free fatty acids were found (all p > 0.11). CONCLUSIONS: CLPS genetic variability associates with insulin secretory function in non-diabetic humans and may represent a novel candidate gene for development of type 2 diabetes.

The Par6alpha/aPKC complex regulates Akt1 activity by phosphorylating Thr34 in the PH-domain.

A single nucleotide polymorphism in the partitioning defective protein-6alpha (Par6alpha) promoter is coupled with lower Par6alpha expression and better insulin sensitivity, whereas overexpression of Par6alpha in C2C12 myoblasts inhibits insulin-induced protein kinase B/Akt1 activation and glycogen synthesis. Here we show that a direct interaction of Par6alpha with atypical protein kinase C (aPKC) is crucial for this inhibition. A DeltaPB1-Par6alpha deletion mutant that does not interact with aPKC neither increased aPKC activity nor interfered with insulin-induced Akt1 activation in C2C12 cells. Further, T34 phosphorylation of Akt1 through aPKC is important for inhibition of Akt1. When Par6alpha was overexpressed, activation of wild-type Akt1 (-59.3%; p=0.049), but not T34A-Akt1 (+2.9%, p=0.41) was reduced after insulin stimulation. The resistance of T34A-Akt1 to Par6alpha/aPKC-mediated inhibition was also reflected by reconstitution of insulin-induced glycogen synthesis. In summary, Par6alpha-mediated inhibition of insulin-dependent glycogen synthesis in C2C12 cells depends on the direct interaction of Par6alpha with aPKC and on aPKC-mediated T34 phosphorylation of Akt1.

A novel functional polymorphism (-336A/G) in the promoter of the partitioning-defective protein-6alpha gene is associated with increased glucose tolerance and lower concentrations of serum non-esterified fatty acids.

AIMS/HYPOTHESIS: Partitioning-defective protein-6alpha (Par6alpha) has recently been demonstrated to negatively regulate insulin signalling in murine myoblasts. To address whether Par6alpha plays a role in human physiology, the present study investigated whether mutations exist in the Par6alpha gene and whether these mutations, if present, are associated with pre-diabetic phenotypes in non-diabetic subjects. METHODS: The complete gene (part of the promoter [2.1 kb], all exons/introns and the 3' untranslated region) encoding Par6alpha was analysed in 664 non-diabetic subjects. We investigated possible associations between single nucleotide polymorphisms and percentage of body fat, glucose tolerance (as determined by OGTT), serum NEFA concentrations and whole-body insulin sensitivity (estimated during the OGTT, and for a subgroup of 242 subjects determined by the euglycaemic-hyperinsulinaemic clamp). RESULTS: A rare A/G polymorphism was found 336-bp upstream of the translational start codon (allele frequency 0.03). The data for subjects homozygous and heterozygous for -336G (R/G, n=43) were combined and compared with those for subjects homozygous for -336A (A/A, n=621). Subjects with the R/G genotype had lower fasting (4.84+/-0.09 mmol/l, means+/-SEM, p=0.049) and 2-h (5.50+/-0.02 mmol/l, p=0.050) plasma glucose concentrations than subjects with the A/A genotype (5.02+/-0.02 and 5.94+/-0.06 mmol/l, respectively). Subjects with the R/G genotype also had lower fasting (448+/-31 micromol/l, p=0.018) and 2-h serum NEFA concentrations (61+/-7 micromol/l, p=0.015) than subjects with the A/A genotype (529+/-9 and 75+/-2 micromol/l, respectively), adjusted for age, sex and percentage of body fat. There were no differences in adiposity or whole-body insulin sensitivity between the two genotype groups (all p>0.36). A luciferase reporter gene assay revealed that the -336G promoter variant had a significantly lower (-22.8%, p=0.006) transcriptional activity in transfected C2C12 murine myoblasts than the -336A promoter variant. CONCLUSIONS/INTERPRETATION: A novel functional variant in the promoter of the Par6alpha gene is associated with reduced fasting glycaemia, increased glucose tolerance and reduced serum NEFA concentrations.

Induction of adiponectin gene expression in human myotubes by an adiponectin-containing HEK293 cell culture supernatant.

AIMS/HYPOTHESIS: Adiponectin, an adipocytokine known to be down-regulated in obesity-linked disorders, is considered to be a potential key mediator of insulin sensitivity. In this study, we asked whether adiponectin is able to regulate ten selected genes possibly associated with insulin sensitivity in human skeletal muscle cells. METHODS: To this end, we treated in vitro differentiated human myotubes with the culture supernatant of HEK293 cells stably transfected with human recombinant adiponectin and assessed gene expression by RT-PCR. Intracellular adiponectin protein was quantified by radioimmunoassay and visualized by Western blotting. RESULTS: In contrast to the control supernatant, the adiponectin-containing supernatant consistently induced expression of adiponectin mRNA in human myotubes from eight different donors (mean increase: 90-fold over control; n=8, p<0.001). This increase in mRNA was paralleled by a rise in intracellular adiponectin protein (mean increase: 8.3-fold over control; n=4, p<0.05). Expression of the other nine candidate genes was not altered. In human skin fibroblasts and HepG2 cells, the adiponectin-enriched supernatant did not induce relevant amounts of adiponectin mRNA. CONCLUSIONS/INTERPRETATION: In conclusion, we show here that adiponectin gene expression is specifically inducible in skeletal muscle cells.

Polymorphism of the CNS active drug Org 13011: the application of high temperature analysis to detect new polymorphs.

The polymorphism of the CNS active compound Org 13011 was studied using different crystallisation methods (i.e. different solvents and cooling rates). The samples were analysed by Raman, solid state NMR, X-ray powder diffraction (XRPD) and thermal analysis. This led to the characterisation of two crystalline forms A and B. Further high temperature analysis using Raman, XRPD, solid state NMR and DSC revealed another two (high temperature) crystalline forms C and D. The transitions to the high temperature crystalline forms occur at temperatures of about 60 degrees C. This study shows that the application of high temperature experiments is useful and can lead to the discovery of new crystalline forms.

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance.

AIMS/HYPOTHESIS: Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain. METHODS: This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046-38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain. RESULTS: The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. CONCLUSION/INTERPRETATION: In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance.

Comparison of test characteristics of urine dipstick and urinalysis at various test cutoff points.

STUDY OBJECTIVE: We compare the test characteristics of urine dipstick and urinalysis at various test cutoff points in women presenting to emergency departments and an intermediate care center with symptoms of urinary tract infection. METHODS: This was a prospective, observational study of adult women presenting to 1 of 2 community hospital EDs or an intermediate care center with dysuria, urgency, or urinary frequency on history, or suprapubic or costovertebral angle tenderness on examination. Patients who had taken antibiotics in the past 72 hours, had indwelling Foley catheters, symptomatic vaginal discharge, diabetes mellitus, immunodeficiency disorders, or were unable to provide a reliable history were excluded. The patient's clean-catch or catheterized urine specimen was tested immediately by a nurse using a Multistix 9 SG reagent strip. A second aliquot was sent within 1 hour of collection to the hospital laboratory, where a semiautomated microscopic urinalysis and a urine culture were performed. A positive urine culture was defined as more than 100,000 colonies of 1 or 2 uropathogenic bacteria per mL of urine at 48 hours. Dipstick and urinalysis data were compared with urine culture results. Sensitivity, specificity, and predictive values were calculated at various definitions of a positive test, or "test cutoff points," for combinations of leukocyte esterase, nitrite, and blood on dipstick and for RBCs and WBCs on urinalyses. The probability of an erroneous decision to withhold treatment on the basis of a negative test result was defined as "undertreatment," or 1 minus the negative predictive value. "Overtreatment" was defined as 1 minus the positive predictive value. RESULTS: Three hundred forty-three patients were enrolled in this study. Twelve patients were withdrawn because of missing laboratory results. Forty-six percent (152/331) of patients had positive urine cultures. If urine dipstick results are defined as positive when leukocyte esterase or nitrite is positive or blood is more than trace, the overtreatment rate is 47% (156/331) and the undertreatment rate is 13% (43/331). If urinalysis results are defined as positive when WBCs are more than 3 per high-power field or RBCs are more than 5 per high-power field, the overtreatment rate is 44% (146/331) and the undertreatment rate is 11% (36/331). Matched pairs of test characteristics were identified when the analysis was repeated using more than 10,000 colonies per mL as a positive culture. CONCLUSION: In this patient population, similar overtreatment and undertreatment rates were identified for various test cutoff points for urine dipstick tests and urinalysis. Although a urine dipstick may be equivalent to a urinalysis for the diagnosis of urinary tract infection, the limitations in the diagnostic accuracy of both tests should be incorporated into medical decisionmaking.

Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1.

Germline mutations in the Ret protooncogene give rise to the inherited endocrine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid carcinoma. Although it is well accepted that the constitutive active tyrosine kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potential modulators of the tyrosine kinase activity of Ret could be tyrosine phosphatases that are expressed in human thyroid tissue. Therefore, we investigated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplication in the cysteine-rich domain (codons 634-636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret-9bp reduced the autophosphorylation of Ret-9bp by 19 +/- 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coimmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of the Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (protein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphorylation led to reduced phosphorylation of the transcription factor jun-D. Finally, the inhibitory effect on Ret-9bp signaling resulted in a 40-60% reduction of [(3)H]thymidine incorporation and in reduced ability of NIH3T3 cells to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a strong suppression of the Ret oncogene activity.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Bacterial counts in experimental, contaminated crush wounds irrigated with various concentrations of cefazolin and penicillin.

The objective of this study was to determine if three different concentrations of cefazolin and penicillin irrigation solutions reduce quantitative bacterial counts in experimental crush wounds contaminated with multiple species of bacteria. The design used was a randomized, blinded, experimental animal study. An animal bite wound model was created by innoculating crushed incisions with three species of bacteria. Four paravertebral incisions extending to deep fascia were created in each of twelve anesthetized albino guinea pigs. Wound edges were clamped with a hemostat for five seconds to create crushed, devitalized tissue within each wound. Wounds were inoculated with 0.4 mL of a standard solution of Staphylococcus aureus, Bacterioides fragilis, and Pasturella multocida and covered. Four hours after inoculation, each wound was scrubbed for 30 seconds with 20% poloxamer 188 and then irrigated with 100 mL of one of four solutions: normal saline solution (control); cefazolin (CZ) 2 mg/mL, plus penicillin G (PCN) 200 units/mL (low dose); CZ 10 mg/mL, plus PCN 2,000 units/mL (intermediate dose); and CZ 50 mg/mL, plus PCN 20, 000 units/mL (high dose). Investigators were blinded to the solutions used. Wounds were covered with a vapor-permeable dressing. Six days after treatment, each wound was examined for signs of infection and then excised for quantitative bacteriologic analysis. Colony counts were reported as counts per gram of tissue. Wounds in the four irrigation solution groups were compared using ANOVA. A log difference of 3 was considered significant. The average log total bacteria/gram tissue for the four groups were: control, 4.35 (95% CI; 1.01); low dose, 4.09 (95% CI; 1.42); intermediate dose, 4.47 (95% CI; 1.27); and high dose, 3.45 (95% CI; 1.33). No wounds in the high-dose group had any clinical signs of infection, whereas 50% of wounds in the intermediate dose group, 42% in the low dose group, and 33% in the control group had either erythema, induration, or purulence. There were no statistically significant differences in the bacterial counts/gram tissue or clinical infection rates in any of the groups. A formal trend analysis failed to find a significant linear trend for decreasing bacterial counts for either antibiotic. In this experimental bite wound model containing contaminated, crushed tissue, irrigation with various solutions of cefazolin plus penicillin G did not reduce quantitative bacterial counts more than 3.1 log total bacteria/gram tissue.

Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells.

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited disease caused by mutations of the Ret proto-oncogene. Although many different Ret mutations have been described, little is known about the signaling pathways triggered by the Ret oncogene. In this study, we have determined the signaling properties of a Ret-9bp duplication encoding amino acids 634-636, which was recently identified in a patient with all clinical features of the MEN 2A syndrome. The Ret-9bp duplication leads to constitutive activation of the Ret tyrosine kinase. Furthermore, Ret-9bp increased mitogenic and transforming activity demonstrated by thymidine incorporation as well as colony formation in soft agar. Studying intracellular signaling pathways, which may be involved in malignant transformation of Ret-9bp expressing NIH3T3 cells, we could demonstrate Ret-9bp dependent phosphorylation of insulin receptor substrate-2 (IRS-2) with consecutive activation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT). Moreover, Ret-9bp induces phosphorylation of SHC resulting in growth factor receptor binding protein-2 (Grb-2) binding and activation of the mitogen activating protein (MAP) kinase pathway. In addition to these postreceptor cytoplasmic signaling events, we have studied nuclear signal by Ret-9bp and found activation of c-jun and jun-D, two members of the jun/AP-1 family of transcription factors. In summary, an oncogenic 9bp duplication of Ret causes Ret dimer formation and ligand independent activation of the tyrosine kinase. Besides the signaling steps leading to MAPK activation, we could demonstrate that Ret-9bp induced constitutive activation of a signaling pathway involving IRS-2, PI 3-kinase and PKB/AKT which could transduce the oncogenic Ret signal to increased gene transcription via activation of the jun/AP-1 transcription factor family.

Global water resources: vulnerability from climate change and population growth.

The future adequacy of freshwater resources is difficult to assess, owing to a complex and rapidly changing geography of water supply and use. Numerical experiments combining climate model outputs, water budgets, and socioeconomic information along digitized river networks demonstrate that (i) a large proportion of the world's population is currently experiencing water stress and (ii) rising water demands greatly outweigh greenhouse warming in defining the state of global water systems to 2025. Consideration of direct human impacts on global water supply remains a poorly articulated but potentially important facet of the larger global change question.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta.

AIMS/HYPOTHESIS: Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor beta-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity. METHODS: Tyrosine kinase assay and transfection studies. RESULTS: In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms beta2 and theta. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C. CONCLUSION/INTERPRETATION: The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms beta2 and theta on insulin receptor autophosphorylation.

The carboxyl-terminal tyrosine residue of protein-tyrosine phosphatase alpha mediates association with focal adhesion plaques.

The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.

The kinesin-like motor protein KIF1C occurs in intact cells as a dimer and associates with proteins of the 14-3-3 family.

Proteins of the kinesin superfamily are regulated in their motor activity as well as in their ability to bind to their cargo by carboxyl-terminal associating proteins and phosphorylation. KIF1C, a recently identified member of the KIF1/Unc104 family, was shown to be involved in the retrograde vesicle transport from the Golgi-apparatus to the endoplasmic reticulum. In a yeast two-hybrid screen using the carboxyl-terminal 350 amino acids of KIF1C as a bait, we identified as binding proteins 14-3-3 beta, gamma, epsilon, and zeta. In addition, a clone encoding the carboxyl-terminal 290 amino acids of KIF1C was found, indicating a potential for KIF1C to dimerize. Subsequent transient overexpression experiments showed that KIF1C can dimerize efficiently. However, in untransfected cells, only a small portion of KIF1C was detected as a dimer. The association of 14-3-3 proteins with KIF1C could be confirmed in transient expression systems and in untransfected cells and was dependent on the phosphorylation of serine 1092 located in a consensus binding sequence for 14-3-3 ligands. Serine 1092 was a substrate for the protein kinase casein kinase II in vitro, and inhibition of casein kinase II in cells diminished the association of KIF1C with 14-3-3gamma. Our data thus suggest that KIF1C can form dimers and is associated with proteins of the 14-3-3 family.