Who cares? The impact on caregivers of suspected mining-related lung cancer.

Background: In the present study, we investigated the emotional, physical, financial, occupational, practical, and quality-of-life impacts on caregivers of patients with mining-related lung cancer. Methods: This concurrent, embedded, mixed-methods study used individual in-depth qualitative interviews and the 36-item Short Form Health Survey (version 2: RAND Corporation, Santa Monica, CA, U.S.A.) quality-of-life measure with 8 caregivers of patients with suspected mining-related lung cancer who had worked in Sudbury or Elliot Lake (or both), and sometimes elsewhere. Individuals who assist workers in filing compensation claims were also interviewed in Sudbury and Elliot Lake. Interviews (n = 11) were transcribed and analyzed thematically. Results: Caregiver themes focused on the long time to, and the shock of, diagnosis and dealing with lung cancer; not much of a life for caregivers; strong views about potential cancer causes; concerns about financial impacts; compensation experiences and long time to compensation; and suggestions for additional support. Quality-of-life scores were below the norm for most measures. Individuals who assist workers in preparing claims were passionate about challenges in the compensation journey; the requirement for more and better family support; the need to focus on compensation compared with cost control; the need for better exposure monitoring, controls, resources, and research; and job challenges, barriers, and satisfaction. Conclusions: Caregivers expressed a need for more education about the compensation process and for greater support. Worker representatives required persistence, additional workplace monitoring and controls, additional research, and a focus on compensation compared with cost control. They also emphasized the need for more family support.

DNMT3A mutant transcript levels persist in remission and do not predict outcome in patients with acute myeloid leukemia.

We investigated the prognostic impact of minimal residual disease (MRD) monitoring in acute myeloid leukemia patients harboring DNA methyltransferase 3A-R882H/-R882C mutations (DNMT3Amut). MRD was determined by real-time quantitative PCR (RQ-PCR) in 1494 samples of 181 DNMT3Amut patients. At the time of diagnosis, DNMT3Amut transcript levels did not correlate with presenting clinical characteristics and concurrent gene mutations as well as the survival end points. In Cox regression analyses, bone marrow (BM) DNMT3Amut transcript levels (log10-transformed continuous variable) were not associated with the rate of relapse or death. DNMT3Amut transcript levels were significantly higher in BM than in blood after induction I (P=0.01), induction II (P=0.05), consolidation I (P=0.004) and consolidation II (P=0.008). With regard to the clinically relevant MRD time points, after two cycles of induction and at the end of therapy, DNMT3Amut transcript levels had no impact on the end point remission duration and overall survival. Of note, only a minority of the patients achieved RQ-PCR negativity, whereas most had constantly high DNMT3Amut transcript levels, a finding which is consistent with the persistence of clonal hematopoiesis in hematological remission.

Syndesmosis injuries in the pediatric and adolescent athlete: an analysis of risk factors related to operative intervention.

PURPOSE: To review all paediatric ankle syndesmotic injuries occurring at our institution and identify risk factors associated with operative intervention. METHODS: Among 22 873 evaluations for ankle trauma, we found 220 children suffering from syndesmotic injuries (incidence: 0.96%). We recorded demographic data, details of the injury, features on examination and treatment variables. Univariable and multivariable logistic regression modelling was performed to identify risk factors associated with operative intervention. RESULTS: The mean age at injury was 15.8 years (8.9 to 19.0) with a median follow-up of 13 weeks (IQR 5 to 30 weeks). A sports-related injury was most common (168/220, 76%). A total of 82 of 220 (37%) patients underwent operative fixation, of which 76 (93%) had an associated fibular fracture. Patients undergoing surgery had a higher incidence of swelling and inability to weight bear (p < 0.001). Statistically significant differences were recorded in tibiofibular (TF) clear space, TF overlap and medial clear space (MCS) between the operative and non-operative cohorts (6.0 vs 4.6 mm (p = 0.002), 5.4 vs 6.9 mm (p = 0.004) and 6.4 vs 3.5 mm (p < 0.001)). Multivariable analysis revealed patients with a fracture of the ankle had 44 times the odds of surgical intervention, patients with a closed physis had over five times the odds of surgical intervention and patients with a medial clear space greater than 5 mm had nearly eight times the odds of requiring surgical intervention. CONCLUSIONS: Operative ankle syndesmotic injuries in the paediatric population are often associated with a closed distal tibial physis and concomitant fibular fracture.

En Bloc Liver Kidney Transplantation Using Donor Splenic Artery as Inflow to the Kidney: Report of Two Cases.

The number of simultaneous liver-kidney transplants has been increasing. This surgery is associated with an increased risk of complications, longer duration of surgery and longer ischemia time for the renal allograft. Two patients listed for liver-kidney transplant at our center underwent en bloc combined liver-kidney transplantation using donor splenic artery as inflow. Patient 1 previously underwent cardiac catheterization that was complicated by a bleeding pseudoaneurysm of the right external iliac artery that required endovascular stenting of the external iliac artery and embolization of the inferior epigastric artery. Patient 2 was on vasopressor support and continuous renal replacement therapy at the time of transplant. In this paper, we described a novel technique of en bloc liver-kidney transplant with simultaneous reperfusion of both allografts using the donor splenic artery for renal inflow. This technique is useful for decreasing cold ischemia time and total operative time by simultaneous reperfusion of both allografts. It is a useful technical variant that can be used in patients with severe disease of the iliac arteries.

Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium.

Biopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

A pro-apoptotic function of iASPP by stabilizing p300 and CBP through inhibition of BRMS1 E3 ubiquitin ligase activity.

The p53 family and its cofactors are potent inducers of apoptosis and form a barrier to cancer. Here, we investigated the impact of the supposedly inhibitory member of the apoptosis-stimulating protein of p53, iASPP, on the activity of the p53 homolog TAp73, and its cofactors p300 and CBP. We found that iASPP interacted with and stabilized the histone acetyltransferase p300 and its homolog CBP upon cisplatin treatment. Vice versa, iASPP depletion by shRNA resulted in decreased amounts of p300 and CBP, impaired binding of p300 and TAp73 to target site promoters, reduced induction of pro-apoptotic TAp73 target genes, and impaired apoptosis. Mechanistically, we observed that the p300-regulatory E3 ubiquitin ligase BRMS1 could rescue the degradation of p300 and CBP in cisplatin-treated, iASPP-depleted cells. This argues that iASPP stabilizes p300 and CBP by interfering with their BRMS1-mediated ubiquitination, thereby contributing to apoptotic susceptibility. In line, iASPP overexpression partially abolished the interaction of BRMS1 and CBP upon DNA damage. Reduced levels of iASPP mRNA and protein as well as CBP protein were observed in human melanoma compared with normal skin tissue and benign melanocytic nevi. In line with our findings, iASPP overexpression or knockdown of BRMS1 each augmented p300/CBP levels in melanoma cell lines, thereby enhancing apoptosis upon DNA damage. Taken together, destabilization of p300/CBP by downregulation of iASPP expression levels appears to represent a molecular mechanism that contributes to chemoresistance in melanoma cells.

Laboratory diagnosis of paraneoplastic pemphigus.

BACKGROUND: Paraneoplastic pemphigus (PNP) is a multiorgan disease characterized by antibodies against plakins, desmogleins and the α2-macroglobulin-like-1 (A2ML1) protein, in association with an underlying neoplasm. Accurate diagnosis relies on the demonstration of these autoantibodies in serum. OBJECTIVES: To evaluate the value of different laboratory techniques in the serological diagnosis of PNP. METHODS: We performed immunoblotting, envoplakin (EP) enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IIF) on rat bladder, radioactive immunoprecipitation and a nonradioactive combined immunoprecipitation-immunoblot assay. Additional assays included BP180 ELISA and BP230 ELISA. We included the sera of 19 patients with PNP and 40 control subjects. RESULTS: The sensitivities were 63% for anti-EP ELISA, 74% for rat bladder IIF, 89% for immunoblotting, 95% for radioactive immunoprecipitation and 100% for nonradioactive immunoprecipitation. Specificities ranged from 86% to 100%. The BP180 and BP230 ELISAs had low sensitivity and specificity for PNP. The combination of rat bladder IIF and immunoblot showed 100% sensitivity and specificity. The analysis of sequential PNP sera showed that antibody titres may decrease over time, possibly resulting in negative outcomes for EP ELISA and rat bladder IIF studies. CONCLUSIONS: The detection of autoantibodies against EP and periplakin, or A2ML1 by immunoprecipitation is most sensitive for PNP. The combination of rat bladder IIF and immunoblotting is equally sensitive and highly specific, and represents an alternative valuable and relatively easy approach for the serological diagnosis of PNP.

[Normalisation of screening HASE for 4- to 4½-year-old children]. / Normierung des Screening HASE für 4- bis 4 ½-jährige Kinder.

For reasons of prevention medical examinations of school beginners are brought forward up to 2 years before school enrollment. In Baden-Württemberg HASE is used as screening for risks in the acquisition of language and in learning to read and write. Up to now norms were insufficient for the age-group of 4;0-4;5 years. Based on the results of 3 354 children the norms for this age-group could be recalculated and are now available as percentile ranks, T-scores, and C-scores.

Influence of anthropometric features on graft diameter in ACL reconstruction.

BACKGROUND: There is a paucity of literature regarding the influence of anthropometric features on the hamstring graft obtained in ACL reconstruction. This study was undertaken to assess the influence of anthropometric measurements on the graft diameter obtained at ACL reconstruction surgery within the European population. We hypothesise that anthropometric features do influence graft thickness in ACL reconstruction. MATERIALS AND METHODS: Data from 121 consecutive patients who had undergone ACL reconstruction by the same surgeon using quadruple hamstring grafts were analysed. The body mass index (BMI), height and weight of these patients were correlated with the graft diameter obtained during surgery. Regression analysis was undertaken to assess the influence of individual anthropometric variables on the graft diameter. RESULTS: There were 121 patients with mean age of 32 years (14-55). There was a statistically significant positive correlation individually between the height and graft diameter (r = 0.38, p < 0.01) as well as between the body weight and graft diameter (r = 0.29, p < 0.01). However, when the body mass index was calculated, the correlation was not statistically significant (r = 0.08, p > 0.1). Regression analysis confirmed that BMI was not statistically significant as a predictor of hamstring graft diameter whereas height was statistically the most important predictor (F = 20.1; p < 0.01).This yielded the predictive equation, graft diameter = 4.5 + 0.02 x Ht (in cm). CONCLUSION: Although body mass index did not significantly correlate, body height may be a predictive variable in predicting the graft diameter in ACL reconstruction and provide useful pre operative information.

A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes.

BACKGROUND: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies. METHODS: In this study, we describe the development of high resolution melting (HRM) technology-based assays with direct sequencing confirmation possibility for mutation screening of the EGFR gene (exons 19, 20 and 21) in routine diagnostic specimens, and compared assay findings to those of conventional nested-PCR following cycle-sequencing. RESULTS: In reconstruction experiments, each HRM assay following sequencing demonstrated a sensitivity of < or =5% of mutated DNA in a background of wild-type DNA. The panel of EGFR HRM assays following sequencing applied to a series of genomic DNA samples isolated from 68 FFPE NSCLC specimens correctly identified all EGFR mutations that were previously found by nested-PCR following cycle-sequencing. The HRM approach additionally scored two mutations not detected by the conventional assay. Complementary HRM following sequencing for K-ras revealed three mutations. EGFR and K-ras mutations were mutually exclusive. CONCLUSIONS: The panel of designed HRM assays with direct reflex sequencing possibility provides an effective method for mutation screening of EGFR and K-ras genes in routine diagnostic specimens, thereby allowing the selection of the treatment of choice in clinical practice.

Polyamine biosynthesis impacts cellular folate requirements necessary to maintain S-adenosylmethionine and nucleotide pools.

Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.

A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.

BACKGROUND: Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed. METHODS: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype. RESULTS: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5-2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing. CONCLUSIONS: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.

Kidney allocation to liver transplant candidates with renal failure of undetermined etiology: role of percutaneous renal biopsy.

The feasibility, value and risk of percutaneous renal biopsy (PRB) in liver transplant candidates with renal failure are unknown. PRB was performed on 44 liver transplant candidates with renal failure of undetermined etiology and glomerular filtration rate (GFR) <40 mL/min/1.73 m(2) (n = 37) or on renal replacement therapy (RRT) (n = 7). Patients with >or=30% interstitial fibrosis (IF), >or=40% global glomerulosclerosis (gGS) and/or diffuse glomerulonephritis were approved for simultaneous-liver-kidney (SLK) transplantation. Prebiopsy GFR, urinary sodium indices, dependency on RRT and kidney size were comparable between 27 liver-transplant-alone (LTA) and 17 SLK candidates and did not relate to the biopsy diagnosis. The interobserver agreement for the degree of IF or gGS was moderate-to-excellent. After a mean of 78 +/- 67 days, 16 and 8 patients received LTA and SLK transplants. All five LTA recipients on RRT recovered kidney function after transplantation and serum creatinine was comparable between LTA and SLK recipients at last follow-up. Biopsy complications developed in 13, of these, five required intervention. PRB is feasible in liver transplant candidates with renal failure and provides reproducible histological information that does not relate to the pretransplant clinical data. Randomized studies are needed to determine if PRB can direct kidney allocation in this challenging group of liver transplant candidates.

Association between dense CADM1 promoter methylation and reduced protein expression in high-grade CIN and cervical SCC.

We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the CADM1 promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. CADM1 protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and CADM1 gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as >or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased CADM1 protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in >or= CIN3 compared with or= CIN3.

Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation.

AIMS: Inhibition of apoptosis is important in the pathogenesis of lymphomas. c-FLIP, a regulator of caspase 8-mediated apoptosis, plays an important role in protecting normal B and T cells from apoptosis and possibly also in lymphomas. Because of contradictory reports about immunohistochemical detection of c-FLIP expression, the aim was to test the specificity of four antibodies in c-FLIP-transfected cells and subsequently to investigate expression of c-FLIP in different types of lymphoma. METHODS AND RESULTS: Two of four antibodies were specific. In primary lymphomas c-FLIP expression was restricted to Hodgkin's lymphomas (> 90%) and diffuse large B-cell lymphomas (44%). Burkitt lymphomas and indolent B-cell lymphomas were negative in all cases. No expression was detected in primary T-cell lymphomas, although expression was observed in one relapsed ALK+ anaplastic large cell lymphoma. Expression of c-FLIP was inversely correlated with caspase 8 activation. CONCLUSIONS: c-FLIP is important in escape of B cells from apoptosis during normal follicle centre cell reaction and may thus be an important early event in the development of B-cell-derived lymphomas. Moreover, non-specific staining of frequently used antibodies might explain discrepancies in different reports of c-FLIP expression.

Polyamine metabolism and tumorigenesis in the Apc(Min/+) mouse.

While polyamine homoeostasis is clearly important in maintenance of normal cell function, the roles of these cations, as well as the enzymes that regulate their metabolism, in the neoplastic process are not clear. In particular, the polyamine catabolic enzyme SSAT (spermidine/spermine N(1)-acetyltransferase) seems to have different roles in tumorigenesis, depending upon the particular system being analysed. In attempts to clarify the function of SSAT in tumour development, we have utilized the Apc(Min/+) mouse, which carries a mutant allele of the Apc (adenomatous polyposis coli) gene, rendering it susceptible to the formation of multiple adenomas in the small intestine and colon. Using genetically engineered animals (i.e. transgenic and knockout mice), we have shown that SSAT acts as a tumour promoter in the Apc(Min/+) model. Modulation of tumorigenesis is not associated with changes in tissue levels of either spermidine or spermine. These findings, along with those made in other animal models of cancer, have prompted us to propose that metabolic flux through the polyamine biosynthetic and catabolic pathways, and the consequent changes in levels of various metabolites within the cell (i.e. the metabolome), is critical to tumour development. The metabolic flux model represents a novel way of thinking about the role of polyamines in cell physiology and the neoplastic process.

Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells.

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

[Heat shock protein 90 alpha und beta are overexpressed in multiple myeloma cells and critically contribute to survival]. / Die Hitzeschockproteine 90 alpha und beta sind im mutiplen Myelom überexprimiert und tragen zum Tumorzellüberleben bei.

HSP90's are overexpressed in different cancer types and they probably are required to sustain aberrant signalling in malignant cells. Recently, pharmacological inhibition of HSP90 was found to suppress growth of myeloma cell lines and in primary myeloma cells. Therefore, we wanted to investigate the role of HSP90alpha and HSP90beta in the pathogenesis of malignant myeloma (MM) in more detail. Immunohistochemistry was employed to examine the expression of HSP90alpha and HSP90beta in MM. The importance of HSP90 for survival of MM -cells was investigated by SiRNA-mediated knockdown of HSP90 and blockade of the IL-6R/STAT3 and the MAPK signaling pathways in vitro. HSP90alpha and HSP90beta were overexpressed in majority of investigated MM cases, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of HSP90 or treatment with the novel HSP90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. The knockdown of HSP90alpha was sufficient to induce apoptosis. This effect was strongly increased when both HSP90s were targeted, indicating a cooperation of both. HSP90 critically contributes to myeloma survival in the context of its microenvironment and therefore strengthen the potential value of HSP90 as a therapeutic target.

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells.

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation.

AIM: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. METHODS: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). RESULTS: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. CONCLUSION: Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.