The KASH-containing isoform of Nesprin1 giant associates with ciliary rootlets of ependymal cells.

Biallelic nonsense mutations of SYNE1 underlie a variable array of cerebellar and non-cerebellar pathologies of unknown molecular etiology. SYNE1 encodes multiple isoforms of Nesprin1 that associate with the nuclear envelope, with large cerebellar synapses and with ciliary rootlets of photoreceptors. Using two novel mouse models, we determined the expression pattern of Nesprin1 isoforms in the cerebellum whose integrity and functions are invariably affected by SYNE1 mutations. We further show that a giant isoform of Nesprin1 associates with the ciliary rootlets of ependymal cells that line brain ventricles and establish that this giant ciliary isoform of Nesprin1 harbors a KASH domain. Whereas cerebellar phenotypes are not recapitulated in Nes1gSTOP/STOP mice, these mice display a significant increase of ventricular volume. Together, these data fuel novel hypotheses about the molecular pathogenesis of SYNE1 mutations and support that KASH proteins may localize beyond the nuclear envelope in vivo.

Early onset of inflammation during ontogeny of bipolar disorder: the NLRP2 inflammasome gene distinctly differentiates between patients and healthy controls in the transition between iPS cell and neural stem cell stages.

Neuro-inflammation and neuronal communication are considered as mis-regulated processes in the aetiology and pathology of bipolar disorder (BD). Which and when specific signal pathways become abnormal during the ontogeny of bipolar disorder patients is unknown. To address this question, we applied induced pluripotent stem cell (iPSC) technology followed by cortical neural differentiation on adipocyte-derived cells from BD type I patients (with psychotic episodes in psychiatric history) and healthy volunteers (controls). RNA sequencing in iPSC and cortical neural stem cell (NSC) lines were used to examine alterations between the transcriptomes from BD I and control samples during transition from the pluripotent stage towards the neural developmental stage. At the iPSC stage, the most highly significant differentially expressed gene (DEG) was the NLRP2 inflammasome (P=2.66 × 10-10). Also among 42 DEGs at the NSC stage, NLRP2 showed the strongest statistical significance (P=3.07 × 10-19). In addition, we have also identified several cytoskeleton-associated genes as DEGs from the NSC stage, such as TMP2, TAGLN and ACTA2; the former two genes are recognised for the first time to be associated with BD. Our results also suggest that iPSC-derived BD-cortical NSCs carry several abnormalities in dopamine and GABA receptor canonical pathways, underlining that our in vitro BD model reflects pathology in the central nervous system. This would indicate that mis-regulated gene expression of inflammatory, neurotransmitter and cytoskeletal signalling occurs during early fetal brain development of BD I patients.

Calcium-dependent intracellular signal pathways in primary cultured adipocytes and ANK3 gene variation in patients with bipolar disorder and healthy controls.

Bipolar disorder (BD) is a chronic psychiatric disorder of public health importance affecting >1% of the Swedish population. Despite progress, patients still suffer from chronic mood switches with potential severe consequences. Thus, early detection, diagnosis and initiation of correct treatment are critical. Cultured adipocytes from 35 patients with BD and 38 healthy controls were analysed using signal pathway reporter assays, that is, protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), Myc, Wnt and p53. The levels of activated target transcriptional factors were measured in adipocytes before and after stimulation with lithium and escitalopram. Variations were analysed in the loci of 25 different single-nucleotide polymorphisms (SNPs). Activation of intracellular signals in several pathways analysed were significantly higher in patients than in healthy controls upon drug stimulation, especially with escitalopram stimulation of PKC, JNK and Myc, as well as lithium-stimulated PKC, whereas no meaningful difference was observed before stimulation. Univariate analyses of contingency tables for 80 categorical SNP results versus diagnoses showed a significant link with the ANK3 gene (rs10761482; likelihood ratio χ(2)=4.63; P=0.031). In a multivariate ordinal logistic fit for diagnosis, a backward stepwise procedure selected ANK3 as the remaining significant predictor. Comparison of the escitalopram-stimulated PKC activity and the ANK3 genotype showed them to add their share of the diagnostic variance, with no interaction (15% of variance explained, P<0.002). The study is cross-sectional with no longitudinal follow-up. Cohorts are relatively small with no medication-free patients, and there are no 'ill patient' controls. It takes 3 to 4 weeks of culture to expand adipocytes that may change epigenetic profiles but remove the possibility of medication effects. Abnormalities in the reactivity of intracellular signal pathways to stimulation and the ANK3 genotype may be associated with pathogenesis of BD. Algorithms using biological patterns such as pathway reactivity together with structural genetic SNP data may provide opportunities for earlier detection and effective treatment of BD.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan-Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

[The IGF system: summary and recent data]. / Le système IGF: synthèse et données récentes.

We are entitled to state that our knowledge about the IGF system has literally exploded in the last years. Having been considered for some time merely as trophic and mitogenic factors, the IGFs now appear as molecules essential for the differentiation of many cell types, and even more so, as powerful protective agents for the nervous and the cardiovascular systems. However, these properties so beneficial in normal physiological conditions, are subverted by the cancerous cells who use them to extend their life span and resist therapy. The IGFs did not live up to expectations in the treatment of diabetes; however, today their capacity to improve the condition of patients suffering from severe neurological, renal or muscle diseases is tested. The IGF system might also be targetted by the anticancer treatments. In the following paper we have briefly summarized our knowledge on the IGF system, and presented in more detail the recent data.

Cloning of breakpoints in and downstream the IGF2 gene that are associated with overexpression of IGF2 transcripts in colorectal tumours.

The human IGF2 gene belongs to a group of imprinted genes clustered on the short arm of chromosome 11, band p15.5. It contains 9 exons and spans over 30 kb. IGF2 mRNA overexpression has been reported in human tumours and in some inherited growth disorders. It was recently demonstrated that IGF2 mRNA overexpression contributes to tumour progression and that loss of parental imprinting as well as altered transcription factors are contributing to this overexpression. We have reported structural alterations in the 3' region of the IGF2 gene in two colorectal tumours that overexpressed the IGF2 transcript by 200- and 800-fold. We cloned by the vectorette-PCR strategy, genomic DNA fragments containing the breakpoints from these tumours. The sequencing of these fragments positioned the breakpoint 2 kb downstream the IGF2 gene in one tumour, and in exon 9 in the second. Both breakpoints occurred in regions containing repetitive elements: a TGGA repeat we have identified downstream the gene, and the (CA)n repetition in exon 9. We hypothesize that a negative regulatory element, located downstream the IGF2 gene, has been deleted following these structural alterations and leads to IGF2 gene overexpression.

A new cis element is involved in the HER2 gene overexpression in human breast cancer cells.

The HER2 proto-oncogene product is overexpressed in 30% of breast cancers, and this correlates with poor prognosis. Increased levels of HER2 mRNA in breast cancer cell lines result from increased gene transcription. We report the identification of a new 17-bp-long cis sequence located between positions -506 and -489 from the transcription start site. This sequence is recognized by a trans-activating factor that we tentatively named HER2 transcription factor (HTF). This factor, involved in the increased transcription of the HER2 gene in the BT-474 mammary tumor cells, has a molecular weight of about Mr 50,000. HTF can also bind, but with a lower affinity, to a related cis sequence present in the epidermal growth factor receptor promoter. Interestingly, the HTF binding activity is high in nuclear extracts from several mammary tumor cells overexpressing the HER2 gene.

IGF-II in primary human colorectal tumors: peptide level, activated promoters, parental imprinting and gene rearrangement.

IGF-II is a polypeptide growth factor with growth and differentiation promoting activities, involved in human development. We have reported previously IGF-II mRNA and peptide overexpression in primary human colon cancers. Here we show that the IGF-II peptide content is increased in six primary colon cancers compared to the corresponding healthy tissues. The IGF-II transcripts in healthy and cancerous colon tissues were identified by Northern blotting and RT-PCR. Promoters P3 and P4 were active in most tissues. Relaxation of parental imprinting was observed in two tumors and one healthy tissue, without any correlation with the IGF-II transcript levels. Rearrangements of the IGF-II gene in two tumors containing very high amounts of IGF-II mRNA are described. Fragments containing the breakpoints were cloned by the vectorette-PCR strategy. In both tumors, the breakpoints occurred in repetitive sequences. In one tumor (T11), the breakpoint was localized 2 kb downstream the end of exon 9. The second tumor (T18) contains two modified alleles. In one rearranged allele the breakpoint is located in exon 9. The exact position of the breakpoint in the second rearranged allele has not been identified. In future experiments, the correlation between the gene rearrangements and IGF-II mRNA overexpression will be studied.

Histopathologic findings of placenta and arcuate artery Doppler velociometry in preeclampsia and intrauterine growth retardation.

The aim was to analyze the histopathologic changes of placentas and to compare them to the results of arcuate artery color Doppler velocimetry. Fifty four placentas were from pregnancies complicated with pre-eclampsia that ranged from mild forms to convulsions (group 1), 42 from pregnancies complicated with intrauterine growth retardation (IUGR) (group 2) and 40 from uncomplicated pregnancies (group 3). The arcuate artery resistance index (AARI) was increased in 66.66% in group 1 and 59.52% in group 2 (NS). In all uncomplicated pregnancies (group 3) AARI was normal. In group 2, increased AARI was significantly more frequently associated with minimal hypoxic damage (MHD) of placental tissue than in group 1 (p < 0.005), whereas multiple infarcts were more common in group 1 than in group 2 (p < 0.005). At normal AARI multiple infarcts were significantly more frequently found in group 1 than in groups 2 and 3 (p < 0.005), whereas normal placental findings were significantly more common in group 3 than in groups 1 and 2 (p < 0.001). Hypoxic lesions were significantly more often associated with increased AARI (p < 0.01). The positive predictive value of arcuate artery color Doppler velocimetry for hypoxic placental lesions was 93%, and negative predictive value was 10%. Sensitivity and specificity of the method in the prediction of hypoxic placental lesions was 62% and 91%, respectively.

Characterization of the IGF system and analysis of the possible molecular mechanisms leading to IGF-II overexpression in a mesothelioma.

The expression of members of the IGF system in a mesothelioma from a patient suffering from hypoglycemia, in term placenta and HT29 colon adenocarcinoma cells were compared. Very high levels of IGF-II mRNA and protein were detected in the mesothelioma. Moreover, half of the IGF-II protein took the high-molecular-weight form. We also analyzed the parental imprinting status and the promoter usage of the IGF-II gene. Our results showed loss of imprinting (LOI) in the mesothelioma while the imprinting was maintained in HT29 cells, expressing moderate levels of the transcript. Promoter P4 was active in the three tissues we analyzed, whereas IGF-II mRNA transcription from promoter P3 was only detected in the mesothelioma and the placenta, expressing comparably high levels of the transcript. IGF-II gene structure was identical in the analyzed tissues and cells. The type-I receptor mRNA expression was very low in the tumor. IGFBP-2, -4 and -5 mRNAs were detected in the mesothelioma, while IGFBP-2, -3 and -5 transcripts were detected in the placenta. IGFBP-1 and -6 transcripts were not detected.

Possible influence of expatriation on perinatal outcome.

BACKGROUND: In order to test the hypothesis of possible influence of environmental stress on the length of gestation the data on deliveries in the Maternity Unit, Zagreb University School of Medicine in three six months periods; May-October 1991 (active war in Croatia), May-October 1990 (pre-war period), and the same period in 1992 are analysed. METHODS: Deliveries of 7845 women from free areas of Croatia (non-displaced population) and deliveries from 712 women from occupied areas of Croatia, as well of 593 Croatian refugees from Bosnia and Herzegovina and Serbia (expatriated population) were compared. The duration of pregnancy, fetal weight, immediate neonatal condition, mode of delivery and perinatal outcome in non-displaced and expatriated population were compared using chi-square test in statistical analysis. RESULTS: During 1992 and 1991, there was a slight increase in total number of deliveries in comparison to 1990. The number of deliveries by displaced women more than doubled. The incidence of major pregnancy complications was almost the same for both groups in all three time periods. The two populations were comparable regarding their age, parity and previous obstetric history. Slight increase in preterm delivery rate (7.7% in l990, 8.7% in 1991 and 9.4% in 1992), and a subsequent slight decrease in birth weight was found in all women. There was no significant change in the proportion of growth-retarded newborns. Expatriated women both in 1990 and in 1991 delivered twice as often prematurely in comparison to non-displaced women (17.5% and l4.3% deliveries), respectively. Birth weight of their infants was significantly more often under 2500 grams. Slight increase in overall perinatal mortality was observed. Perinatal mortality in the experiated population was significantly higher than in the non-displaced population. Increase in perinatal mortality could be attributed exclusively to increase in prematurity rate. CONCLUSION: Our results support the concept of possible influence of stress, fear, exile and inadequate antenatal surveillance on the length of gestation.

Antenatal fetal demise in multiple gestation--the outcome of surviving fetus at one to four years of age.

The outcome of 26 multifetal pregnancies with single fetal demise after 12 weeks of pregnancy cared for at the University Medical School of Zagreb from 1986 to 1991 is presented. Growth retardation of dead and surviving infant and pathohistological findings consistent with reduced uterine blood flow to cotyledonary space was the most frequent complication, irrespective of the type of placentation. Perinatal infection was the most frequent neonatal complication. Neonatal asphyxia was observed in only one infant. The outcome at 1-4 years of age was perfect in 14 out of 15 surviving infants for whom data are available. An early diagnosis of multifetal pregnancy, based essentially on ultrasonography and appropriate monitoring of the mother and the survivor, with cesarean delivery carried out at first signs of imminent asphyxia, is the best guarantee for a favorable outcome.

[The effect of chronic kidney disease on the course and outcome of pregnancy]. / Utjecaj kronicnih bubreznih bolesti na tijek i ishod trudnoce.

In order to estimate the effect of renal disease on the pregnancy, and the effect of pregnancy on the natural course of renal disease, the course and outcome of the 37 pregnancies was analyzed in gravidas with chronic renal disease. The women were cared for between 1978 and 1990 at the Department of Obstetrics, School of Medicine University of Zagreb. Analysis of the results in this article is retrospective. Thirty-six pregnancies finished by vaginal or cesarean delivery or by abortion, while one pregnancy was ectopic and ended by laparotomy during the 20th week. There were 31 live births from 36 fetuses (86.1%), 5 stillbirths (13.19%), of which 2 pregnancies ended in fetal death (abortion). In addition, there were 6 instances of neonatal death (8.3%). Twenty-five percent of pregnancies finished before 37 weeks of gestation. In 6 of 34 (17.6%) deliveries amniotic fluid was meconium stained, and 5 of 31 (16.1%) infants were born hypoxic. There were 29 percent of growth retarded liveborn fetuses. Perinatal mortality was 167/1000. Renal insufficiency was noted in 12 of 37 (32.4%) pregnancies, 54% of pregnant women had hypertension, 8.1% hyperkalemia, 78.4% were anemic, 27% had significant bacteriuria and 21.6% overt pyelonephritis. There was one case of acute deterioration in renal function that required hemodialysis, and one case of preeclampsia. Renal insufficiency or hypertension reduce drastically the chances for a successful outcome of pregnancy in gravidas with kidney disorders. However renal insufficiency in the presence of hypertension, carries even poorer prognosis, with perinatal mortality of 428/1000.

[Electromyography of the uterus in monitoring pregnancies with symptoms of premature labor]. / Pouzdanost elektromiografije uterusa u pracenju trudnoca sa simptomima prijevremenog porodaja.

Characteristics of uterine muscle action potentials in 100 gravidas with symptoms of preterm labor or imminent abortion (presence of contractions with cervical changes) from 18th till the 32nd weeks of pregnancy are analysed. Twenty four gravidas with uncomplicated pregnancy were monitored electromyographically from the 20th weeks until term, once a week. There was low electrical activity in uncomplicated pregnancies up to 7 days before labor. A similar trend was observed in pregnancies that were finished before term. But, in symptomatic pregnancies finished preterm, the index of uterine muscle electrical activity (expressed in the percentage of voltage plus frequency) was continuously, although not significantly, above the values in symptomatic and asymptomatic term pregnancies. In only 2 out of 18 (11.1%) before term terminated pregnancies and in 45 out of 82 (54.9%) in term accomplished pregnancies, was electrical silence was registered during clinical symptoms (p less than 0.01). In preterm accomplished pregnancies, higher voltages and more frequent occurrence of biphasic and polyphasic spikes were registered (p less than 0.05), although the single parameter was of no prognostic significance. With a successful tocolytic treatment, the index of uterine muscle electrical activity was significantly reduced (from 57.16 to 20.86, t = 2.52, p less than 0.02) and was similar to that registered in symptomatic term pregnancies subjected to no tocolytic treatment. Although intravenous tocolysis was shown diminish voltage and frequency, in preterm delivered pregnancies no reduction in electrical activity was observed. Uterine electromyography could be used as a predictor of successful or unsuccessful tocolytic treatment.

[Peroral and intracervical administration of prostaglandin E2 for stimulation of term labor in premature rupture of fetal membranes]. / Peroralna i intracervikalna primjena prostaglandina E2 u stimulaciji rocnog poroda kod prijevremenog prsnuca vodenjaka.

The outcome of stimulation of labour in 219 primiparous patients with more than 6 hours following a premature rupture of the membranes, and with an ripe cervix, without the established labour, was analysed according to the mode of treatment. One hundred and thirty eight (138) women received PGE2 peroral tablets, 14 intracervical PGE2 gel, and 67 oxytocin intravenously. There were 73.2% vaginal deliveries in the group that received PGE2 per os, 77.5% in the group that received oxytocin, and 92.9% in the group that was given intracervical gel. The differences were significant. The cervix remained unripe in 9.4% patients that received PGE2 perorally, in 14.9% of those that received oxytocin, while in the gel group it became favourable in all parturients. Uterine polisistoly was encountered in 10.9% labours after peroral stimulation, in 7.1% in the gel group and in 1.5% in the oxytocin group, and perinatal asphyxia in 16.7%, 7.1% and 13.4%, respectively. When too frequent, especially if combined with oxytocin and given to patients with a moderately favourable cervix, peroral stimulation predisposes to uterine polisitoly. Intracervical application of PGE2 gel is the method of choice in primigravid patients with a premature rupture of the membranes and the unripe cervix (Bishop score O). In gravidas with a more favourable cervix (Bishop score 4-6) the administration of oxytocin is acceptable with less complications. In those with a moderately favourable cervix (Bishop score 1-4) the gel application proved to be preferable, although stimulation could be carried out as well.