Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children.

AIMS/HYPOTHESIS: Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes. METHODS: The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence. RESULTS: Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1ß, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls. CONCLUSIONS/INTERPRETATION: These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

Drf1, a novel regulatory subunit for human Cdc7 kinase.

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.

Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors.

TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.

[Ogilvie syndrome after Cesarean section]. / Ogilvies syndrom efter sectio.

INTRODUCTION: Ogilvie's syndrome, acute pseudo-obstruction of the colon, can lead to perforation of the caecum and death. The syndrome is not well known and diagnosis can be difficult to make in time. METHODS: We analysed seven cases of Ogilvie's syndrome with the aim of improving diagnostics. RESULTS: All had prolonged labour before cesarean section, which was complicated by bleeding. All were treated with syntocinon, a hormone that may influence gastrointestinal motility. All patients developed abdominal meteorism within a few days of operation, which increased despite the passing of flatus and stool. Five cases resulted in caecum perforation before the correct diagnosis and treatment were made. Perforation occurred on days 3-4, day 5, or probably days 8-10 after the operation. One of these patients, who suffered from severe adipositas, died. CONCLUSION: It is very important to make an early diagnosis, as the condition can progress quickly. Diagnosis should be made on the history, clinical assessment, and abdominal X-ray. Intermittent flatus and stool are characteristic of this truly non-obstructive condition and should not therefore delay a diagnostic X-ray.

Oct-1 specifically binds the UEF4 site of the human AP1-regulated urokinase enhancer.

The inducible urokinase enhancer contains three essential elements: a combined PEA3/AP1 and a downstream AP1 site, separated by a 74-bp DNA region called COM (cooperation mediator), that is required for the synergism between the three sites. The 5' half of COM (uCOM) forms four retarded complexes with HeLa or HepG2 nuclear proteins (UEF1-4). We now demonstrate that the UEF4 complex is the transcription factor Oct-1. Because of functional redundancy of the UEF sites, single mutations in UEF4 have no phenotype; we have changed UEF4 from a low to a high affinity binding site for Oct-1. In vitro, this mutation increases the DNA binding of Oct-1 and disturbs the binding of the Prep-Pbx complexes to the nearby UEF3 site. In vivo, this mutation reduces the basal transcriptional activity of the urokinase enhancer, while not affecting its phorbol ester inducibility. This is in keeping with the effect of the deletions of the COM region, which result in an increase in the basal level and, as a consequence, in the loss of 4beta-phorbol 12-myristate 13-acetate inducibility. Oct-1 therefore is not involved in the inducibility of the urokinase enhancer but only in determining its basal activity level.

Cooperative interactions between PBX, PREP, and HOX proteins modulate the activity of the alpha 2(V) collagen (COL5A2) promoter.

Cell type-specific expression of the human alpha2(V) collagen (COL5A2) gene depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relative to the transcription start site. The present study focused on the characterization of the FPB-bound complex. DNA binding assays and cell transfection experiments revealed that the bipartite core sequence of FPB (5'-ATCAATCA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOXB1 result in binding of the heterodimers to FPB in vitro. Similarly, overexpression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB-driven transcription. In contrast to the composition of the FPB complex purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously introduced into COL5A2-negative cells cannot stimulate FPB-driven transcription unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions among PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V.

Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function.

The Drosophila Homothorax (HTH) and Extradenticle (EXD) are two homeoproteins required in a number of developmental processes. EXD can function as a cofactor to Hox proteins. Its nuclear localization is dependent on HTH. In this study we present evidence of in vivo physical interaction between HTH and EXD, mediated primarily through an evolutionarily conserved MH domain in HTH. This interaction is essential for the mutual stabilization of both proteins, for EXD nuclear localization, and for the cooperative DNA binding of the EXD-HTH heterodimer. Some in vivo functions require both EXD and HTH in the nucleus, suggesting that the EXD-HTH complex may function as a transcriptional regulator.

Impaired testicular function in patients with carcinoma-in-situ of the testis.

PURPOSE: To elucidate the biologic association between germ cell neoplasia and testicular dysfunction, through investigation of Leydig cell function and semen quality in men with carcinoma-in-situ (CIS) of the testis. PATIENTS AND METHODS: We examined two groups of men, unilaterally orchidectomized for testicular cancer. Biopsy of the contralateral testis had showed CIS in a group of 24 patients and no evidence of CIS in the other group of 30 patients. Semen quality and serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared in these two groups of men after orchidectomy but before further treatment for testicular cancer. RESULTS: Significantly higher LH levels (median, 8.1 IU/L v 4.8 IU/L; P < .001) and generally lower testosterone levels (median, 12.5 nmol/L v 15.5 nmol/L; P = .13) were found in the CIS group. The proportion of patients with Leydig cell dysfunction was higher in the group of patients with CIS (11 of 24) than in the group of patients without (two of 30) (P = .01). Sperm concentration and total sperm count were significantly lower (P < .001) in patients with CIS (median, 0.03 x 10(6)/mL and 0.10 x 10(6), respectively) than in patients without (median, 9.1 x 10(6)/mL and 32 x 10(6), respectively), whereas the levels of FSH were significantly higher (P < .001) in the former group of men (median, 19.6 IU/L v 9.0 IU/L). CONCLUSION: Not only spermatogenesis but also Leydig cell function is impaired in testes with CIS. This impairment could be due to common factors in the pathogenesis of germ cell neoplasm and testicular dysfunction. Alternatively, CIS cells may have a negative impact on Leydig cell function.

The PBX-regulating protein PREP1 is present in different PBX-complexed forms in mouse.

Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.

Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export.

Prep1, a novel functional partner of Pbx proteins.

The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.

The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Prevalence of carcinoma in situ of the testis in 207 oligozoospermic men from infertile couples: prospective study of testicular biopsies.

OBJECTIVE: To investigate the prevalence of carcinoma in situ of the testis in a group of oligozoospermic men from infertile couples. DESIGN: A consecutive group of oligozoospermic men from infertile couples were offered bilateral testicular biopsy. The observed prevalence of carcinoma in situ was compared with the expected prevalence of testicular cancer in a corresponding age matched population of Danish men, assuming all untreated cases of carcinoma in situ progress to tumour stage. This calculation was based on data from the Danish Cancer Registry. SUBJECTS: 207 men aged 18-50 years who had sperm density below 10 million/ml in two samples within the previous 2 years or sperm density below 20 million/ml in two samples within the previous 2 years and a history of cryptorchidism or one or two atrophic testicles (orchidometer volume less than 15 cm3), or both. INTERVENTIONS: Bilateral testicular biopsies. MAIN OUTCOME MEASURES: Carcinoma in situ in the biopsy specimen. RESULTS: No case of carcinoma in situ was found among the 207 men. The expected number in a normal age matched population of corresponding size was 0.8. CONCLUSIONS: There is no increase in risk of carcinoma in situ of the testis in moderately oligozoospermic men of couples referred because of infertility.

Characterization of UEF-4, a DNA-binding protein required for transcriptional synergism between two AP-1 sites in the human urokinase enhancer.

The enhancer of the inducible urokinase gene depends on three essential but not sufficient transactivating elements, an upstream PEA3/AP-1A and a downstream AP-1B site. Enhancer activity also requires the interposed 74-base pair-long cooperation mediator (COM) region that allows transcriptional synergism between the transactivating sites. The 5'-half of COM (uCOM) forms four retarded complexes with HeLa or Hep-G2 nuclear proteins (UEF-1-4). We have identified the binding sequence for UEF-4 and generated uCOM elements uniquely mutated in the UEF-4-binding site or uniquely binding UEF-4. Introduction of these and other mutations in the context of the urokinase enhancer showed that all uCOM sites are important for enhancer activity but that UEF-4 and UEF-1 plus UEF-2/3 can substitute for each other, suggesting functional redundancy of urokinase enhancer factors. UEF-4 was purified from HeLa nuclear extract by affinity chromatography and shown to contain two polypeptides of 105 and 65 kDa, respectively, of which at least the former was endowed with DNA binding activity.

Cloning and expression of human NF-YC.

The CCAAT box is an important element in eukaryotic promoters and NF-Y (CBF) is a conserved heterotrimeric protein binding to it. Two subunits, NF-YB and NF-YC, contain a histone-like motif. We cloned the complete cDNA coding for the human NF-YC gene. The ORF codes for a 335 aa protein that shows virtual identity to the rat sequence, confirming the stunning invariance of NF-Y genes across species. We expressed and purified the yeast homology domain of NF-YC in bacteria and performed EMSA together with the corresponding conserved domains of NF-YA and NF-YB, obtaining a CCAAT-binding mini-NF-Y. We evaluated the expression of NF-YC and found that mRNA levels are similar in different human tissues except in testis.

Purification and characterization of UEF3, a novel factor involved in the regulation of the urokinase and other AP-1 controlled promoters.

Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.

Acute effects of graded alcohol intake on glucose, insulin and free fatty acid levels in non-insulin-dependent diabetic subjects.

Alcohol-induced hypoglycaemia is a well-known phenomenon in insulin-treated diabetic subjects. Less attention has been paid to the impact of alcohol on blood glucose and insulin responses in non-insulin dependent diabetic subjects. The aim of this study was to investigate the acute metabolic effects of different alcohol contents added to a non-alcohol beer in 10 non-insulin-dependent diabetes mellitus (NIDDM) subjects. The patients received 500 ml non-alcohol beer with an alcohol percentage (v/v) of 0 (A), 2.7 (B), and 5.4 (C), implying identical contents of ingredients except for alcohol. Blood glucose (mean +/- SE) responses were similar in the three situations (395 +/- 59, 365 +/- 86 and 261 +/- 26 mmol/l x 240 min). In contrast, the incremental insulin response areas increased dose dependently to alcohol (5430 +/- 1158, 9336 +/- 2172 and 12336 +/- 2922 pmol/l x 240 min) and showed a linear correlation (r = 0.39; P < 0.03). The average suppression of serum free fatty acid was similar in the three situations (72.4 +/- 4.4%, 76.3 +/- 6.0% and 68.2 +/- 6.3%). In conclusion, intake of small amounts of alcohol does not acutely deteriorate the glycaemic control in NIDDM. The fact that alcohol results in a dose-related elevation in insulin levels with unaltered blood glucose and free fatty acid responses in NIDDM points to an aggravation of insulin resistance.

Risk of bilateral testicular germ cell cancer in Denmark: 1960-1984.

The incidence of a second primary testicular germ cell cancer among 2850 (96.6% of eligible) men with a histologically verified first primary germ cell cancer diagnosed in the period 1960-1979 in Denmark was established. Of these 2850 men, 73 (2.6%) developed a contralateral testicular cancer. In five of these patients (0.18%), the tumors were synchronous. The cumulative risk of developing a contralateral cancer 25 years after diagnosis of the first testicular germ cell cancer was 5.2% according to a Kaplan-Meier estimate. It was higher among men with a nonseminoma as the first tumor (8.4%) than among men with a seminoma as the first tumor (3.6%). Of the second tumors, 12% were stage II and 17% were stage III at the time of diagnosis. Based on 24,588 person-years at risk and 68 nonsimultaneously occurring bilateral testicular germ cell cancers, the overall relative risk (RR) of developing a second primary cancer in the contralateral testicle following a first germ cell cancer was found to be 24.8 (95% confidence interval = 19-38). Among men with a nonseminoma, the risk was higher (RR = 27.1) than among men with a seminoma (RR = 22.5). The excess risk was not affected by age at diagnosis, calendar period, or time since diagnosis. Close surveillance by screening for and treatment of carcinoma in situ of the remaining testicle in testicular cancer patients are advised.