Key role of dual specificity kinase TTK in proliferation and survival of pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive human malignancies with an overall 5-year survival rate of <5%. Despite significant advances in treatment of the disease during the past decade, the median survival rate (∼6 months) has hardly improved, warranting the need to identify novel targets for therapeutic approaches. METHODS: Quantitative real time PCR, western blot analyses and immunohistochemical staining of tissue microarrays were used to analyse the expression of TTK gene in primary PDAC tissues and cell lines. To inhibit TTK kinase expression in a variety of pancreatic cancer cell lines, RNA interference was used. Functional roles of this kinase in the context of PDAC were studied using cell proliferation, viability and anchorage-independent growth assays. Western blotting, fluorescence-activated cell sorting analyses and fluorescence microscopy were used to gain mechanistic insight into the functional effects. CONCLUSIONS: We show that the dual specificity kinase TTK (also known as Mps1), is strongly overexpressed in human PDAC. Functionally, cell proliferation was significantly attenuated following TTK knockdown, whereas apoptosis and necrosis rates were significantly increased. In addition, anchorage-independent growth, a hallmark of malignant transformation and metastatic potential, was strongly impaired in the absence of TTK gene function. Interestingly, immortalised normal pancreatic hTERT-HPNE cells were not affected by loss of TTK function. Mechanistically, these effects in cancer cells were associated with increased formation of micronuclei, suggesting that loss of TTK function in pancreatic cancer cells results in chromosomal instability and mitotic catastrophe. Taken together, our data show that TTK function is critical for growth and proliferation of pancreatic cancer cells, thus establishing this kinase as an interesting new target for novel therapeutic approaches in combating this malignancy.

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites.

Neurofascin and NrCAM are two axon-associated transmembrane glycoproteins belonging to the L1 subgroup of the Ig superfamily. In this study, we have analyzed the interaction of both proteins using neurite outgrowth and binding assays. A neurofascin-Fc chimera was found to stimulate the outgrowth of tectal cells when immobilized on an inert surface but not as a soluble form using polylysine as substrate. Antibody blocking experiments demonstrate that neurite extension on immobilized neurofascin is mediated by NrCAM on the axonal surface. Under the reverse experimental conditions where NrCAM induces neurite extension, F11, and not neurofascin, serves as axonal receptor. Binding studies using transfected COS7 cells and immunoprecipitations reveal a direct interaction between neurofascin and NrCAM. This binding activity was mapped to the Ig domains within neurofascin. The neurofascin-NrCAM binding can be modulated by alternative splicing of specific stretches within neurofascin. These studies indicate that heterophilic interactions between Ig-like proteins implicated in axonal extension underlie a regulation by the neuron.

Dissection of complex molecular interactions of neurofascin with axonin-1, F11, and tenascin-R, which promote attachment and neurite formation of tectal cells.

Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin- NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

Structure of the axonal surface recognition molecule neurofascin and its relationship to a neural subgroup of the immunoglobulin superfamily.

The chick axon-associated surface glycoprotein neurofascin is implicated in axonal growth and fasciculation as revealed by antibody perturbation experiments. Here we report the complete cDNA sequence of neurofascin. It is composed of four structural elements: At the NH2 terminus neurofascin contains six Ig-like motifs of the C2 subcategory followed by four fibronectin type III (FNIII)-related repeats. Between the FNIII-like repeats and the plasma membrane spanning region neurofascin contains a domain 75-amino acid residues-long rich in proline, alanine and threonine which might be the target of extensive O-linked glycosylation. A transmembrane segment is followed by a 113-amino acid residues-long cytoplasmic domain. Sequence comparisons indicate that neurofascin is most closely related to chick Nr-CAM and forms with L1 (Ng-CAM) and Nr-CAM a subgroup within the vertebrate Ig superfamily. Sequencing of several overlapping cDNA probes reveals interesting heterogeneities throughout the neurofascin polypeptide. Genomic Southern blots analyzed with neurofascin cDNA clones suggest that neurofascin is encoded by a single gene and its pre-mRNA might be therefore alternatively spliced. Northern blot analysis with domain specific probes showed that neurofascin mRNAs of about 8.5 kb are expressed throughout development in embryonic brain but not in liver. Isolation of neurofascin by immunoaffinity chromatography results in several molecular mass components. To analyze their origin the amino-terminal sequences of several neurofascin components were determined. The NH2-terminal sequences of the 185, 160, and 110-135 kD components are all the same as the NH2 termini predicted by the cDNA sequence, whereas the other neurofascin components start with a sequence found in a putative alternatively spliced segment between the Ig- and FNIII-like part indicating that they are derived by proteolytic cleavage. A combination of enzymatic and chemical deglycosylation procedures and the analysis of peanut lectin binding reveals O- and N-linked carbohydrates on neurofascin components which might generate additional heterogeneity.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.

The role of the GABAA receptor Alpha 1 subunit in the ventral hippocampus in stress resilience.

Pre-pubertal stress increases post-traumatic stress disorder (PTSD) susceptibility. We have previously demonstrated that enriched environment (EE) intervention immediately after pre-pubertal stress protects from the effects of trauma in adulthood. Here, we examined whether exposure to EE would also be beneficial if applied after exposure to trauma in adulthood. We have recently shown that exposure to juvenile stress and under-water trauma (UWT) is associated with increased expression of GABAA receptor subunit α1 in the ventral hippocampus. However, differentiating between affected and unaffected individuals, this increased expression was confined to stress-exposed, behaviorally unaffected individuals, suggesting upregulation of α1 expression as a potential mechanism of resilience. We now examined whether EE-induced resilience renders increased expression of α1 in the ventral hippocampus redundant when facing a trauma later in life. Adult rats were exposed to UWT, with pre-exposure to juvenile stress, and tested in the open field and elevated plus maze paradigms four weeks later. EE exposure during juvenility prevented pre-pubertal stress-induced vulnerability, but not if performed following UWT in adulthood. Furthermore, juvenile EE exposure prevented the trauma-associated increase in α1 expression levels. Our findings emphasize the importance of early interventions in order to reduce the likelihood of developing psychopathologies in adulthood.

Molecular analysis of herpesviral gene products recognized by protective cytolytic T lymphocytes.

The infection of the mouse with murine cytomegalovirus (MCMV) served as a model system to understand the biology of human CMV infection. The contribution of cytolytic T lymphocytes (CTL) to the recovery from infection was studied. Protection against lethal MCMV disease could be conferred on immunodepleted hosts by adoptive transfer of lymphocytes. The antiviral effect was mediated by specifically sensitized T lymphocytes of the CD8+ subset. These cells limited viral spread, prevented tissue destruction by viral cytopathic effects, and protected from lethal disease. Transferred cells have protective therapeutic function even when the virus has already colonized host tissues. CD8+ cells do not require the contribution of CD4+ cells for in vivo function. Selective expression of immediate-early (IE) phase genes in target cells allowed the detection of the immunodominant IE antigen recognized by CTL. The major IE gene ieI encodes a non-structural viral phosphoprotein, pp89, which resides in the nucleus of infected cells where it acts as transcriptional regulator. Expression of gene ieI is under temporal control, and membrane presentation of the protein domain detected by CTL is down-regulated by MCMV early-phase products. A recombinant vaccinia virus expressing gene ieI induced immunity that protected mice against a subsequent challenge with a lethal dose of MCMV. The protective effect was entirely mediated by CD8+ T lymphocytes. Thus, an experimental vaccine expressing a single nonstructural herpesvirus protein can induce a protective cellular immune response.

[Operative treatment of vesico-vaginal fistulae]. / Die operative Behandlung von Blasenscheidenfisteln.

During a period of 16 years, 40 cases of vesico-vaginal fistulae were treated. In 21 of the cases a fistula closing operation could be carried out, 8 vaginally and 13 suprapubically. Out of the 40 cases 19 were cured. In most cases the fistula was approached abdominally and closed by a single-layer method, leaving the vaginal defect open for drainage. This method was suitable for all types of vesico-vaginal fistulae, independent of their size and location but especially suitable for fistulae occurring after irradiation where severe tissue changes had occurred. Urinary diversion is often the only way of helping these patients and the indications for the various types of diversion are discussed.

[Computed tomography as an aid in the differential diagnosis of radiolucent stones and tumors of the upper urinary tract]. / Die Computertomographie als Hilfsmittel bei der Differentialdiagnose nichtschattengebender Konkremente und Tumoren der oberen Harnwege.

The difficult differential diagnosis of urothelial tumors and nonopaque calculi of the upper urinary tract has been considerably improved by the introduction of computed tomography (CT) in urological diagnosis. Nonopaque calculi although urographically not visible can be determined by CT. Usually urothelial tumors can be traced by CT without invasive methods. In case of doubt in addition to the primary scan a so called flow-study, after application of contrast medium, will furnish additional information. The diagnostic facilities provided by CT are illustrated in 18 cases.

[On-line fetal monitoring (meaning for data acquisition) (author's transl)]. / On-line fetal monitoring (Bedeutung für die Datenerfassung)

We demonstrate an enlarged and modernized equipment for the on-line-fetal monitoring. With the aid of illustrations the received informations by help of continuous on-line-fetal monitoring are demonstrated too. They concerned the deposition of electronical and corresponding gasanalytical dates. The importance of registration from all obstetrical parameters and given up drugs by a code-system is put out. Furthermore preferences of the computations are discussed.

The neural cell recognition molecule neurofascin interacts with syntenin-1 but not with syntenin-2, both of which reveal self-associating activity.

Neurofascin belongs to the L1 subgroup of the immunoglobulin superfamily of cell adhesion molecules and is implicated in axonal growth and fasciculation. We used yeast two-hybrid screening to identify proteins that interact with neurofascin intracellularly and therefore might link it to trafficking, spatial targeting, or signaling pathways. Here, we demonstrate that rat syntenin-1, previously published as syntenin, mda-9, or TACIP18 in human, is a neurofascin-binding protein that exhibits a wide-spread tissue expression pattern with a relative maximum in brain. Syntenin-1 was found not to interact with other vertebrate members of the L1 subgroup such as L1 itself or NrCAM. We confirmed the specificity of the neurofascin-syntenin-1 interaction by ligand-overlay assay, surface plasmon resonance analysis, and colocalization of both proteins in heterologous cells. The COOH terminus of neurofascin was mapped to interact with the second PDZ domain of syntenin-1. Furthermore, we isolated syntenin-2 that may be expressed in two isoforms. Despite their high sequence similarity to syntenin-1, syntenin-2alpha, which interacts with neurexin I, and syntenin-2beta do not bind to neurofascin or several other transmembrane proteins that are binding partners of syntenin-1. Finally, we report that syntenin-1 and -2 both form homodimers and can interact with each other.

Organization of the neurofascin gene and analysis of developmentally regulated alternative splicing.

Neurofascin is an axonal member of the L1 subgroup of the immunoglobulin superfamily implicated in neurite extension in the course of embryonic development. Here we have isolated and characterized the gene encoding chicken neurofascin. Comparison of genomic sequences with cDNA sequences provides the structure and localization of intron/exon boundaries and indicates that neurofascin isoforms are generated by alternative splicing of its pre-mRNA. The neurofascin gene is composed of 33 exons distributed over 72 kilobases. Each of the six immunoglobulin- and five fibronectin-type III-like domains is encoded by two exons. While introns between domains are of phase 1, others are of phase 0, 1, or 2. Alternative splicing of neurofascin is developmentally regulated as shown by polymerase chain reaction analysis. Furthermore, plasmid libraries from long range polymerase chain reaction-amplified cDNA of neurofascin were used to examine and quantify the distribution of alternatively spliced exons in individual neurofascin molecules. We found 50 different neurofascin isoforms at different developmental stages and revealed the existence of one major "early" in comparison with multiple "late" neurofascin isoforms.

[Computerized analysis of intrapartum cardiotocograms]. / Untersuchungen zur rechenautomatischen CTG-Analyse sub partu.

307 monitored labors were investigated by means of a discrimination function (DF) in relation to qualitative CTG-parameters. In this connection we could confirm on the one hand well defined qualitative CTG-criteria in their clinical importance by computation corresponding to "normal", "prepathological" (warning-signs) and "pathological" (signs of hypoxia). On the other hand we could demonstrate the exactly recognition of qualitative CTG-parameters in attendance of computation. The boundary ranges of discrimination function resulting from the automatic CTG-analysis respecting fetal condition turned out to be reproduced and clinically reliable. The prediction with regard to neonatal parameters (pHNA, APGAR-score 5 minutes after delivery) is justifying the employment of present soft ware in clinical practice.

[A new form of representation of foetal cardiotocograms (author's transl)]. / Eine neue Form der Darstellung fetaler Kardiotokogramme.

Based on a computerized analysis of CTG's we have developed a new graphical representation of fetal CTG's. The reproduction results from displays over 13 hours labor-progressing. A mathematical model is used. We have applied a discrimination function (DF) which is composed by 10 quantitative electronic parameters. Two fixed periods are compared. For mathematical and statistical evaluation the multivariate analysis of variance in connection with methods of dimension-reduction and discrimination was applied. A nonelementary discrimination function was taken into account. The value of the discrimination function was checked by means of postnatal data (pHNA and APGAR-score 5 minutes postnatal). The predictability "normal" pHNA - related to the computerized CTG- analysis - was exact in 80% (pHNA greater than or equal to 7,20). In 20% we found prepathological pHNA-values (pHNA:7,19-7,11). The predictability "prepathological" pHNA - related to the computerized CTG- analysis - was in 3,5% correlated with pathological pHNA-values (pHNA less than or equal to 7,10). Pathological pHNA - values may be almost ruled out by computerized CTG - analysis. Computerized "normal" CTG's were in 97% correlated with APGAR-scored values (5 minutes postnatal) > 7. Computerized "prepathological" or "pathological" CTG's were in 26% correlated with APGAR-score values (5 minutes postnatal) less than or equal to 7. The proposed computer-program is already used in this form in clinical practice.

[Trendanalysis attended with on-line fetal monitoring (a clinical study) (author's transl)]. / Trendanalytische Darstellungen im Rahmen der on-line fetal monitoring (eine klinische Studie).

Using some clinical examples, we have showed the importance of trend-analysis by application on-line fetal monitoring. The reference to data compression and data diminution is discussed. Trend analysis is at present time still integrated in the system of clinical computing. Valuation in order to clinical evidence is yet questionable.

[Delta pH F changes sub partu and their clinical relevance]. / Delta pHF-Veränderungen sub partu und deren klinische Relevanz.

Investigations of delta pHF-changes in labor led to the opinion, that there exist no agreement between this changes and the fetal or neonatal condition. Nevertheless they represent an important criterion--together with electronic parameters--for estimation of placental function. By achievment of limiting values (delta pHF greater than or equal to 0,10) and persistent pathological fetal heart rate patterns the termination of labor is necessary. A pHF less than 7,25 should no be reached. That ist of great importance for a low acidotic morbidity.--In this connection the determination of pHF to begin of the fetal monitoring is requisite. This is meaningful for the dynamics of pHF-values in labor in accordance with the fetal monitoring.

[A comparison of different methods of measuring bleeding time (author's transl)]. / Vergleichende Untersuchungen zur Bestimmung der Blutungszeit

Two methods of determining bleeding time (Ivy's and Duke's) were compared using a standardized method. Skin cuts were made with either a disposable scalpel or a precision plunger. Ivy's method gave results which were markedly superior to those with Duke's. It was, furthermore, shown that reproducibility of two successive determinations in the same person largely depended on the experience of the investigator. Best results were obtained using the precision plunger and automatic collection of blood. But even when these were used, there were frequent deviations of more than 20% in duplicate samples.

[The "robotron NATALI" microcomputer-assisted birth monitoring system]. / Mikrorechnergestütztes Geburtsüberwachungssystem "robotron NATALI".

The microcomputer system "robotron-NATALI" from VEB Kombinat robotron Dresden, GDR, is applicable for bed side-monitoring and disposed about a new graphical representation of CTG's for fetal condition and uterine activity. A dialogue between physician or nurse and the computer is possible. After parturition all administered data are printed out in form of a partogram, inclusive the graphical representations during labor.

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription.

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection.