Temperature- and sex-related effects of serine protease alleles on larval development in the Glanville fritillary butterfly.

The body reserves of adult Lepidoptera are accumulated during larval development. In the Glanville fritillary butterfly, larger body size increases female fecundity, but in males fast larval development and early eclosion, rather than large body size, increase mating success and hence fitness. Larval growth rate is highly heritable, but genetic variation associated with larval development is largely unknown. By comparing the Glanville fritillary population living in the Åland Islands in northern Europe with a population in Nantaizi in China, within the source of the post-glacial range expansion, we identified candidate genes with reduced variation in Åland, potentially affected by selection under cooler climatic conditions than in Nantaizi. We conducted an association study of larval growth traits by genotyping the extremes of phenotypic trait distributions for 23 SNPs in 10 genes. Three genes in clip-domain serine protease family were associated with larval growth rate, development time and pupal weight. Additive effects of two SNPs in the prophenoloxidase-activating proteinase-3 (ProPO3) gene, related to melanization, showed elevated growth rate in high temperature but reduced growth rate in moderate temperature. The allelic effects of the vitellin-degrading protease precursor gene on development time were opposite in the two sexes, one genotype being associated with long development time and heavy larvae in females but short development time in males. Sexually antagonistic selection is here evident in spite of sexual size dimorphism.

Sterile leukocyturia is associated with interstitial fibrosis and tubular atrophy in kidney allograft protocol biopsies.

Kidney allograft interstitial fibrosis and tubular atrophy (IF/TA) is associated with a poorer renal function and outcome. In the current clinical practice, an early diagnosis can only be provided by invasive tests. We aimed to investigate the association of sterile leukocyturia with Banff criteria histological findings in kidney allograft protocol biopsies. We studied 348 allograft biopsies from two different European countries performed at 8.5 + 3.5 months after transplantation. In these cases, the presence of sterile leukocyturia (Leuc+, n = 70) or no leukocyturia (Leuc-, n = 278) was analyzed and related to Banff elementary lesions. Only IF/TA was significantly different between Leuc+ and Leuc- groups. IF/TA was present in 85.7% of Leuc+ and 27.7% of Leuc- patients (p < 0.001). IF/TA patients had higher serum creatinine and presence of proteinuria (p < 0.05). Independent predictors of IF/TA were donor age, donor male sex, serum creatinine and Leuc+ (hazard ratio 18.2; 95% confidence interval, 8.1-40.7). The positive predictive value of leukocyturia for predicting IF/TA was 85.7% whereas the negative predictive value was 72.3%. These studies suggest that leukocyturia is a noninvasive and low-cost test to identify IF/TA. An early diagnosis may allow timely interventional measures directed to minimize its impact and improve graft outcome.

Current characteristics and outcome of cytomegalovirus infections after kidney transplantation.

INTRODUCTION: The clinical course of cytomegalovirus (CMV) infections in the current era is poorly described. We characterized the symptoms and outcome of all CMV infections in a large cohort of kidney transplant recipients. Among 1129 kidney transplant recipients transplanted between 2004 and 2011 in Charité Universitätsmedizin Berlin and Helsinki University Hospital, 297 patients with CMV infection were characterized. RESULTS: CMV disease occurred in 217/1129 patients (19.2%), and CMV infection in 297/1129 (26.3%). Gastrointestinal symptoms were recorded in 58% and fever in 47% patients with primary CMV disease, compared to 46% and 27% patients with symptomatic CMV reactivation, whereas leukopenia or thrombocytopenia were seen in only 17-28% patients, and malaise in 9-10%. Tissue-invasive CMV gastroenteritis was confirmed in 11% and CMV pneumonia in only 1% of patients with CMV disease. Only 1 patient died because of CMV infection (mortality 0.3%). Virus-related factors or the use of secondary prophylaxis did not predict the risk of recurrence, which occurred in 33% patients. CONCLUSION: In conclusion, CMV disease remains a common problem after kidney transplantation. Gastrointestinal symptoms were common, especially in patients with primary CMV infection, whereas bone marrow suppression, hepatopathy, or malaise were seen less frequently.

Differential effects of pharmacological HIF preconditioning of donors versus recipients in rat cardiac allografts.

Ischemia-reperfusion injury (IRI) induces hypoxia-inducible factor-1 (HIF-1) in the myocardium, but the consequences remain elusive. We investigated HIF-1 activation during cold and warm ischemia and IRI in rat hearts and cardiac syngrafts. We also tested the effect of HIF-α stabilizing prolyl hydroxylase inhibitor (FG-4497) on IRI or allograft survival. Ex vivo ischemia of the heart increased HIF-1α expression in a time- and temperature-dependent fashion. Immunohistochemistry localized HIF-1α to all cardiac cell types. After reperfusion, HIF-1α immunoreactivity persisted in smooth muscle cells and cardiomyocytes in the areas with IRI. This was accompanied with a transient induction of protective HIF-1 downstream genes. Donor FG-4497 pretreatment for 4 h enhanced IRI in cardiac allografts as evidenced by an increase in cardiac troponin T release, cardiomyocyte apoptosis, and activation of innate immunity. Recipient FG-4497 pretreatment for 4 h decreased infiltration of ED1(+) macrophages, and mildly improved the long-term allograft survival. In syngrafts donor FG-4497 pretreatment increased activation of innate immunity, but did not induce myocardial damage. We conclude that the HIF-1 pathway is activated in heart transplants. We suggest that pharmacological HIF-α preconditioning of cardiac allografts donors would not lead to clinical benefit, while in recipients it may result in antiinflammatory effects and prolonged allograft survival.

Metformin vs. insulin in gestational diabetes. A randomized study characterizing metformin patients needing additional insulin.

AIMS: We compared metformin with insulin as treatment of gestational diabetes mellitus (GDM). Furthermore, we aimed to characterize metformin-treated patients needing additional insulin to achieve prespecified glucose targets. METHODS: We conducted a single centre randomized controlled study with non-inferiority design comparing metformin and insulin in the treatment of 217 GDM patients having birth weight as primary outcome variable. RESULTS: There were no significant differences in mean birth weight expressed in grams [+15 (90% confidence interval (CI): -121 to 89)] or SD units [+0.04 (90% CI: -0.27 to 0.18)] between the metformin and insulin groups. There were no significant differences in neonatal or maternal data between the groups. Only 23 (20.9%) of the 110 patients in the metformin group needed additional insulin. Compared with the patients on metformin only, those needing additional insulin were older (p = 0.04), their oral glucose tolerance test had been performed earlier and diabetes therapy started earlier in gestation (p = 0.01 and p = 0.004, respectively). The risk for additional insulin was 4.6-fold in women with baseline serum fructosamine concentration above median compared with those below median. CONCLUSIONS: Metformin is an effective alternative to insulin in the treatment of GDM patients. Serum fructosamine may help in predicting the adequacy of metformin treatment alone.

Diet and blood lipids in 1-4 year-old children.

BACKGROUND AND AIM: Early nutrition may programme blood lipid levels and thereby later cardiovascular health of children. The objective here was to evaluate the effects of maternal dietary counselling during pregnancy and breastfeeding on dietary intakes and blood lipid values in 1-4 year-old children. Further, the nutritional determinants of children's lipid profiles were assessed. METHODS AND RESULTS: Mothers were randomised into dietary counselling or control groups at the first trimester of pregnancy. Their children were followed up clinically at 1, 2 and 4 years of age, by three-day food records and analyses of total cholesterol, HDL cholesterol and apolipoproteins A-I and B as well as lipoprotein (a). In general, the mean intake of saturated fatty acids as a proportion of total energy intake (E%) was higher than the recommended, while the mean intake of polyunsaturated fatty acids was low in children's diet. Over the first years, girls had higher concentration of non-HDL cholesterol than boys; 2.64 mmol/l (95% CI 2.54-2.74) vs. 2.49 (2.38-2.60); p = 0.038. Maternal dietary counselling was not reflected in the children's lipid values. Children's monounsaturated fatty acid intake (E%) correlated with apoA-I (p = 0.048) and, furthermore, there was a negative correlation between polyunsaturated fatty acid intake (E%) and apoB (p = 0.046). CONCLUSION: Children's dietary fatty acid intake, but not maternal dietary counselling was shown to be related to blood apolipoproteins in children.

Lipid profile characterization of wastewaters from different origins.

Lipids in wastewaters are potential raw material for renewable diesel, but may complicate biological treatment of wastewaters. The lipid composition of palm oil mill effluent (POME), chemithermomechanical pulp mill (CTMP) wastewater and municipal wastewater (MWW) was studied with a combination of thin-layer chromatography and nuclear magnetic resonance. Gravimetrically determined content of extracted lipids from the solids of POME and CTMP wastewater were 8.4 ± 1.2 g/L (19.6 ± 0.8% of dry weight) and 0.17-0.23 g/L (12.4-18.5%), respectively, while MWW contained 0.021 ± 0.002 g/L (9.3 ± 1.4%) of lipids. All lipid extracts contained mono-, di- and triacylglycerols (TAGs) and free fatty acids (FFAs). In POME, lipids were mostly TAGs (11.5 ± 0.2 µmol/10 mg of lipid extract). In CTMP and MWW lipid composition was more diverse than in POME containing also sterol derivatives and fatty acid methyl esters and the main lipids were FFAs.

Soluble urokinase-type plasminogen activator receptor in patients with suspected infection in the emergency room: a prospective cohort study.

OBJECTIVES: The soluble form of urokinase-type plasminogen activator (suPAR) was evaluated as an early prognostic marker of sepsis in patients with suspected infection. DESIGN: A single-centre prospective cohort study. METHODS: The cohort comprised 539 patients in the emergency department with suspected infection: 59 without systemic inflammatory response syndrome (SIRS) and without bacterial infection (group 1), 68 with bacterial infection and without SIRS (group 2), 54 with SIRS and without bacterial infection (group 3), 309 with sepsis (SIRS and bacterial infection) and without organ failure (group 4) and 49 with severe sepsis (SIRS, bacterial infection and organ failure) (group 5). suPAR was measured on admission using a commercial solid-phase enzyme-linked immunosorbent assay. RESULTS: The median soluble form of the receptor (suPAR) concentrations in groups 1-5 were 4.7, 5.0, 4.4, 4.8 and 7.9 ng mL(-1) , respectively (P < 0.001). The levels were significantly higher in nonsurvivors compared with survivors (8.3 vs. 4.9 ng mL(-1) , P < 0.001) and in patients with severe sepsis (group 5) compared with those in the other groups (7.9 vs. 4.8 ng mL(-1) , P < 0.001). Area under the receiver operating characteristics curve (AUC(ROC) ) for the prediction of case fatality was 0.79 (95% confidence interval [CI]: 0.72-0.86, P < 0.0001) and 0.75 for severe sepsis (95% CI: 0.68-0.81, P < 0.0001). At a cut-off level of 6.4 ng mL(-1) , suPAR had 76% sensitivity and 69% specificity for fatal disease; at a cut-off level of 6.6 ng mL(-1) , the sensitivity and specificity for severe sepsis were 67% and 72%, respectively. In multivariate models, high suPAR remained an independent predictor of case fatality and severe sepsis after adjusting for potential confounders. CONCLUSIONS: A high suPAR level predicts case fatality and severe sepsis in patients with suspected infection.

Primary CMV infections are common in kidney transplant recipients after 6 months valganciclovir prophylaxis.

Prolonging cytomegalovirus (CMV) prophylaxis in CMV seronegative recipients of a kidney from CMV seropositive donor (D+/R-) may reduce the incidence of late infections. We analyzed late-onset primary CMV infections after 6 months valganciclovir prophylaxis. Data from all CMV D+/R- kidney transplant recipients between January 2004 and December 2008 at our center were analyzed. Patients with a functioning graft at 6 months after transplantation who received 6 months of valganciclovir prophylaxis 900 mg once daily were included (N = 127). CMV was diagnosed with quantitative PCR. Prophylaxis was completed in 119 patients. Prophylaxis was stopped at 3-5 months due to leukopenia or gastrointestinal side effects in eight patients. Late-onset primary CMV infection developed in 47/127 (37%) patients median 244 days after transplantation (range 150-655) and median 67 days after the cessation of prophylaxis (range 1-475). Four infections were asymptomatic. In others, symptoms included fever (N = 28), gastrointestinal symptoms (nausea, vomiting, diarrhea) (N = 24), respiratory tract symptoms (N = 12), and hepatopathy (N = 6). Median peak viral load was 13500 copies/mL (range 400-2,831,000). Recurrent CMV infection developed in 9/47 (19%) patients. No significant risk factors for CMV infection were identified. Symptomatic primary CMV infections were commonly detected also after prolonged valganciclovir prophylaxis.

Association of immune cell function assay with protocol biopsy findings and viral infections in well matched kidney transplant recipients.

BACKGROUND: Cylex Immune Cell Function Assay (ICFA) is in clinical use, but little is known about its association with screening of viral infections or findings in protocol biopsies. PATIENTS AND METHODS: We analyzed ICFA in our well-matched kidney transplant population. Helsinki University Hospital patients who received a kidney transplant after July 2007 were analyzed. Patients with at least 6 months follow-up and ICFA measured together with the screening for cytomegalovirus (CMV), or polyomaviruses from urine or plasma, or patients with a protocol biopsy at 6 or 12 months taken at the time of the ICFA were included (n = 27). Immunosuppression was usually implemented with mycophenolate mofetil (MMF), steroids and cyclosporine A (CyA). Biopsies were analyzed with chronic allograft damage index (CADI). RESULTS: Mean immune response in 61 samples was 368 +/- 179 ATP ng/ml. Immune response was lower during BK virus (BKV) or CMV viremia compared to no viremia (p = 0.009 and p = 0.017), and no viremia was seen if immune response was > 380. BK or JC viruria was not associated with low immune response. Immune responses did not differ between patients with high or low CADI scores or between patients with immune activation or no immune activation in biopsies. Immune response in our population was higher than previously reported without increased risk of rejections. ICFA correlated with viremia but not with findings in protocol biopsies. CONCLUSION: The optimal immune response in our population needs further studies.

Alternative forms of Max as enhancers or suppressors of Myc-ras cotransformation.

Max is a basic-helix-loop-helix-leucine zipper protein capable of forming sequence-specific DNA binding complexes with Myc proteins. An alternatively spliced messenger RNA has been identified that encodes a form of Max truncated at the COOH-terminus. This delta Max protein retained the ability to bind to the CACGTG motif in a complex with c-Myc but lacks the nuclear localization signal and the putative regulatory domain of Max. When tested in a myc-ras cotransformation assay in rat embryo fibroblasts, Max suppressed, whereas delta Max enhanced, transformation. Thus, the max gene may encode both a negative and a positive regulator of c-Myc function.

Is Cystatin C more sensitive than creatinine in detecting early chronic allograft nephropathy?

BACKGROUND: Cystatin C (CyC) has been suggested as a more accurate indicator of renal function than creatinine (Crea). CyC performance against graft histopathology has not been investigated. AIM: To compare CyC and Crea-based methods as predictors of chronic allograft damage index (CADI). MATERIAL AND METHODS: 105 protocol biopsies obtained at 6 months post-transplantation were classified with Banff'97 and CADI. CyC and Crea were measured concomitantly. Histology was correlated to CyC, Crea, their reciprocals, CyC-estimated GFR (Larsson), Cockroft and Gault (C&G) and abbreviated MDRD using Kendall's Tau. The area under ROC curve (ROC-auc),sensitivity/specificity, positive and negative predictive values were calculated at CADI cut-off of 2. RESULTS: Mild histological changes were best revealed by Crea, although with modest sensitivity/ specificity. A Crea threshold of 111 micromol/l distinguished 74% of the patients with CADI > 2 and excluded this condition in 66%. For Crea, ROC-auc was 0.72 (p < 0.001). Crea and 1/Crea correlated best to CADI, chronic allograft nephropathy, chronic inflammation, tubular atrophy, vascular changes and glomerulopathy. Neither C&G nor MDRD improved Crea performance alone. CyC and Larsson formula performed the same (ROC-auc 0.67). A CyC threshold of 1.12 mg/l distinguished 69% of the patients with CADI > 2 and excluded it in 60%. Significant Tau correlation was found between CyC, 1/CyC and Larsson with CADI, chronic inflammation, tubular atrophy and chronic vascular changes. CONCLUSIONS: CyC, 1/CyC and Larsson-estimated GFR did not offer significant advantages over Crea in predicting mild histological allograft changes. Protocol biopsy provides information that cannot be sensitively predicted by biochemical measurements used in clinical practice.

Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat.

The role of nitric oxide in obliterative bronchiolitis development, i.e., chronic rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in the intragraft inducible nitric oxide synthase (iNOS) mRNA and mononuclear inflammatory cell iNOS immunoreactivity was demonstrated during progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared to syngeneic grafts. In nontreated allografts, however, intragraft nitric oxide production was decreased, most likely because of loss of iNOS epithelial expression. Treatment with aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase, was associated with enhanced proliferation of alpha-smooth muscle actin immunoreactive cells and the intensity of obliterative bronchiolitis early after transplantation. Aminoguanidine treatment did not affect iNOS mRNA synthesis or intragraft nitric oxide production, but decreased iNOS immunoreactivity in smooth muscle cells. Treatment with L-arginine, a precursor of nitric oxide, significantly reduced obliterative changes. L-arginine supplementation enhanced intragraft iNOS mRNA synthesis and iNOS immunoreactivity in capillary endothelial and smooth muscle cells as well as intragraft nitric oxide production. Immunohistochemical analysis of allografts showed that neither iNOS inhibition nor supplementation of the nitric oxide pathway affected the number of graft-infiltrating CD4+ and CD8+ T cells, ED1+ and ED3+ macrophages, immune activation with expression of IL-2R or MHC class II, or production of macrophage or Th1 cytokines. In contrast, L-arginine treatment was associated with increased staining for Th2 cytokines IL-4 and IL-10. In conclusion, this study demonstrates that nitric oxide has a protective role in obliterative bronchiolitis development in this model, and suggests that nitric oxide either directly or indirectly inhibits smooth muscle cell proliferation and modulates immune response towards Th2 cytokines.

The effect of platelet-derived growth factor ligands in rat cardiac allograft vasculopathy and fibrosis.

BACKGROUND: In chronic rejection, parenchymal fibrosis and cardiac allograft vasculopathy (CAV) characterized by neointimal growth are the leading causes of graft loss for heart transplant recipients. During alloimmune responses a variety of cytokines, adhesion proteins, and growth factors, such as platelet-derived growth factor (PDGF), are up-regulated. The PDGF family (AA, AB, BB, CC, DD), which acts mainly on connective tissue cells, is considered to be a potent mitogenic and chemotactic factor for fibroblasts and vascular smooth muscle cells. In this study, we evaluated the effects of PDGF ligands in chronic rejection. METHODS: Heterotopic heart transplantations were performed between fully major histocompatability complex-mismatched Dark Agouti to Wistar Furth rats receiving cyclosporine immunosuppression. Allograft coronary arteries were perfused with a recombinant adeno-associated virus (AAV) encoding enhanced green fluorescence protein (EGFP) as a control gene or PDGF-A, -B, -C, -D. Allografts were harvested at 100 days for morphometric analysis of CAV and fibrosis. RESULTS: AAV-mediated transgene expression was detected by EGFP immunoreactivity across the graft section (at 100 days) in AAV EGFP-perfused allografts. AAV PDGF-A, -C, and -D perfusion resulted in accelerated CAV and fibrosis. In contrast, AAV PDGF-B perfusion did not induce arteriosclerotic changes or fibrosis in cardiac allografts. CONCLUSIONS: AAV PDGF-A, -C, and -D overexpression accelerated the development of chronic rejection, whereas PDGF-B did not. Our results suggested that more targeted therapy with monoclonal antibodies blocking the active sites of PDGF-A, -C, and -D may produce beneficial effects on heart transplant survival.

Vascular endothelial growth factor plays a major role in development of experimental obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. The exact molecular and cellular mechanisms contributing to obliterative lesion formation are unknown. Pathological characteristics of OB are epithelial damage, peribronchial inflammation, and increasing obliteration of bronchioli. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that exerts proinflammatory effects by increasing endothelial permeability and inducing expression of endothelial adhesion molecules. We investigated the role of VEGF in the development of OB in rat tracheal allografts and the role of VEGF receptors (VEGFR)-1 and -2 in the development of OB in mouse tracheal allografts. In nontreated allografts, with increasing loss of epithelium and airway occlusion, VEGF messenger RNA (mRNA) and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene led to a decrease in epithelial necrosis but increased luminal occlusion by >50% compared with AdLacZ-treated rat tracheal allografts. When compared with the control immunoglobulin (Ig)G group, simultaneous treatment with antibodies against VEGFR-1 and -2 significantly lowered the degree of luminal occlusion of mouse tracheal allografts.

Effect of graft preservation and acute rejection on hypoxia-inducible factor-1 in rat cardiac allografts.

Hypoxia plays an integral part in cardiac transplantation as prolonged graft preservation is an individual risk factor for the development of cardiac allograft vasculopathy (CAV). In this study we characterized the role of hypoxia-inducible factor-1 (HIF-1) during prolonged graft preservation, ischemia-reperfusion (I/R), acute rejection, and chronic rejection. Heart transplantations were performed from Dark Agouti (DA) to Wister-Furth (allo) or DA to DA (syn) rats, without immunosuppression (acute rejection model, harvested at day 5) or with cyclosporine (chronic rejection model, harvested at day 60). To study the effect of preservation on HIF-1 regulation, normal DA hearts were subjected to different cold ischemia times with or without 45 minutes of additional warm ischemia. The role of I/R was studied by harvesting syngrafts at different time points after reperfusion. Real-time reverse-transcriptase polymerase chain reaction quantified total HIF-1 mRNA, while enzyme-linked immunosorbent assay and immunohistochemistry quantified and localized HIF-1 protein. Our results show that HIF-1 nuclear immunoreactivity is increased during graft preservation and I/R leads to loss of nuclear HIF-1 immunoreactivity. Acute rejection induced HIF-1 in mRNA level. Our findings thus indicated that HIF-1 is activated during transplantation and suggested that manipulation of the HIF-1 pathway might reveal new therapeutic options to manage CAV.

Alternative mRNA forms and open reading frames of the max gene.

The max gene encodes a heterodimeric partner of Myc. We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination. Here we have studied the expression of human max mRNAs by Northern blotting analysis. In addition to the major 2.3-kb mRNA form, four bands were identified. Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames. In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure. Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay. Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium. Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins.

Determination of sequences responsible for the differential regulation of Myc function by delta Max and Max.

The DNA-binding, transcriptional activation and transforming activities of the Myc protein require dimerization with Max. Max can form also homodimers which are able to bind the same DNA sequence as Myc/Max heterodimers and suppress Myc-induced transcription and transformation. We have recently identified a naturally occurring truncated form of Max, delta Max, which in a rat embryo fibroblast enhances transformation by Myc and Ras. Like Max, this delta Max protein contains a b-HLH-Zip domain, except that the end of the leucine zipper is replaced by five delta Max-specific amino acid residues. Delta Max also lacks the C-terminal sequences of Max including a nuclear localisation signal. Here we have dissected the regions responsible for the specific effects of Max and delta Max in Ras-Myc cotransformation of rat embryo fibroblasts. Our results indicate that the suppressive activity of Max requires C-terminal acidic and basic regions and an intact leucine zipper. Replacement of the end of the leucine zipper with the delta Max-specific sequence is responsible for the enhancement of transformation by delta Max. Surprisingly, delta Max does not require the DNA-binding basic region for enhancement of transformation and has no effect on Myc-induced transcription activation from Myc/Max-binding site-containing promoter construct.

Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.

We have cloned a novel quail cDNA with strong homology to the pim family of proto-oncogenes. The deduced amino acid (aa) sequence of the cDNA, named qpim, is more closely related to Xenopus Pim and to the recently identified rat Pim-3 than to human or rodent Pim-1 or Pim-2. The protein encoded by the qpim cDNA can autophosphorylate itself and share substrates with murine Pim-1, suggesting functional redundancy to other Pim family serine/threonine kinases. We have compared the expression of qpim in avian embryos to mouse pim-1, -2 and -3 by in situ hybridization. qpim shows a highly dynamic expression pattern, particularly at early developmental stages. Surprisingly, its expression pattern is not identical to any of the murine pim genes, which show complementary and/or partially overlapping expression sites both in- and outside of the hematopoietic system. Altogether, our results suggest novel functions for Pim family kinases during embryonic development, in particular in epithelia and in the central nervous system.

The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway.

We have examined potential mechanisms by which the Pim-1 kinase acts as a hematopoietic cell survival factor. Enforced expression of the wild type 33 kd (FD/hpim33) and 44 kd (FD/mpim44) Pim-1 proteins in murine factor-dependent FDCP1 cells prolonged survival after withdrawal of IL-3, while expression of a dominant negative Pim-1 protein (FD/pimNT81) shortened survival. Following removal of IL-3 FDCP1 cells exhibited loss of mitochondrial transmembrane potential and production of reactive oxygen species, as determined by flow cytometry analysis. The wild type Pim-1 proteins decreased these changes while the dominant negative protein enhanced mitochondrial dysfunction. The antiapoptotic activity of the kinases could not be attributed to modulation of glutathione, catalase, or superoxide dismutase activities. Both the FD/hpim33 and FD/mpim44 cells maintained expression of bcl-2 mRNA following cytokine removal, while a substantial decrease was seen in FD/neo cells. To modulate Bcl-2 protein levels, a bcl-2 antisense RNA construct was coexpressed with the wild type pim-1 cDNAs. FD/hpim33 cells with low cellular Bcl-2 protein levels had shortened cytokine-independent survival compared with FD/hpim33 clones with high Bcl-2 expression. However survival of FD/mpim44 cells after IL-3 withdrawal was substantially independent of cellular Bcl-2 protein levels. The 33 kd protein delayed, and the 44 kd protein completely prevented enhanced cell death associated with enforced expression of human Bax protein however. Our results suggest that the 33 kd Pim-1 kinase may enhance cell survival through cooperation with and regulation of bcl-2. In addition the 44 kd kinase may regulate the expression or activity of other pro- and anti-apoptotic members of the bcl-2 family.