Outcome of neurological early rehabilitation patients carrying multi-drug resistant bacteria: results from a German multi-center study.

BACKGROUND: Colonization or infection with multi-drug resistant (MDR) bacteria is considered detrimental to the outcome of neurological and neurosurgical early rehabilitation patients. METHODS: In a German multi-center study, 754 neurological early rehabilitation patients were enrolled and and reviewed in respect to MDR status, length of stay (LOS) and the following outcome variables: Barthel Index (BI), Early Rehabilitation Index (ERI), Glasgow Outcome Score Extended (GOSE), Coma Remission Scale (CRS), Functional Ambulation Categories (FAC). RESULTS: The mean age of the study population was 68.0 ± 14.8 years. Upon admission, the following prevalence for MDRs was observed: MRSA (methicillin resistant staphylococcus aureus) 7.0% (53/754), ESBL- (extended spectrum beta-lactamase) producing bacteria strains 12.6% (95/754), VRE (vancomycin resistant enterococci) 2.8% (21/754). Patients colonized or infected with MDR bacteria (MDR+) were significantly more frequently diagnosed with a critical illness polyneuropathy - CIP - than non-colonized (MDR-) patients: 29.0% vs. 14.8%. In addition, they were more frequently mechanically ventilated (MDR+: 55/138, 39.9%; MDR- 137/616, 22.2%). MDR+ patients were referred to rehabilitation earlier, had a longer LOS in early rehabilitation, lower BI on admission and at discharge, lower ERI on admission and lower CRS at discharge than MDR- patients. There was a highly significant correlation of the BI upon admission with the BI at discharge (rs = 0.492, p < 0.001). GOSE at discharge differed significantly between both groups (χ 2-test, p < 0.01). Perhaps of greatest importance, mortality among MDR+ was higher in comparison to MDR- (18.1% vs. 7.6%). CONCLUSIONS: The outcome of neurological early rehabilitation patients colonized or infected with MDR bacteria including MRSA or ESBL producing strains is significantly poorer than by non-colonized patients. There is some evidence that the poor outcome could be related to the higher morbidity and lower functional status upon admission.

[5-Year-Follow-up of the MEmbeR Multicenter Study on Medical-Occupational Rehabilitation]. / 5-Jahres-Follow-Up der multizentrischen Evaluationsstudie zur medizinisch-beruflichen Rehabilitation (MEmbeR).

In Germany, medical-occupational rehabilitation represents an essential link between rehabilitation programs focusing either on medical or occupational rehabilitation. Its main objective is return to work. The current study presents the vocational integration 5 years after medical-occupational rehabilitation and determines possible prognostic factors for long-term occupational integration. To evaluate the effectiveness of medical-occupational rehabilitation, a 5-year-follow-up interview was conducted with participants (n=105) of the multicenter study on medical-occupational rehabilitation (MEmbeR). As a main result, 76% of the participants were still employed 5 years after medical-occupational rehabilitation and the return to work rate was 57%. Prognostic factors for long-term occupational integration could not be identified. However, a low degree of disability, an unrestricted capacity for teamwork as well as an unrestricted ability to judge might be beneficial factors for a successful reintegration. The high amount of participants who returned to work 5 years after medical-occupational rehabilitation, supports the concept of medical-occupational rehabilitation. However, more studies are needed to identify further factors influencing the outcome.

[Course of rehabilitation in early neurological/neurosurgical rehabilitation. Results of a 2014 multi-center evaluation in Germany]. / Rehabilitationsverlauf von Patienten in der neurologisch-neurochirurgischen Frührehabilitation.

BACKGROUND: In Germany, neurological-neurosurgical early rehabilitation is well established in the treatment of severe neurological diseases. To develop quality standards, knowledge of the current rehabilitation course is required. PATIENTS AND METHODS: A retrospective analysis was performed on the course of rehabilitation from patients in an early neurological/neurosurgical rehabilitation program in 16 centers from 10 German states. The odds for a good or poor outcome were investigated using a multivariate logistic regression model. RESULTS: Seven hundred and fifty-four patients were included in the study. The average age of the patients was 68 ± 15 years. Of the patients studied, 26 % were on mechanical ventilation commencing their neurological rehabilitation. The average duration of stay was 56 ± 51 days. Weaning rate from mechanical ventilation was 65 % and the rate of weaning from tracheal cannula was 54 %. Mean improvement in the Barthel Index of 17 points, significant reduction of dysphagia (from 62 to 30 %) and depended walking (from 99 to 82 %), and the achievement of phase C (the next stage of rehabilitation) in 38 % can still be counted as signs of successful rehabilitation. During their course of stay, near 10 % of the patients died. Of these, 67 % received solely palliative care. In the multivariate logistic models, the absence of the factor "necessity for mechanical ventilation on admission" (odds ratio 0.61; 95 % confidence interval (CI): 0.42 … 0.89) increased the chance for good outcome and the presence of this factor the risk of dying with an odds ratio of 8.07 (95 % CI: 4.54-14.34). DISCUSSION: In spite of the severity of neurological deficits, significant functional progress has been made. These results could be interpret as positive proof of the efficacy of neurological/neurosurgical early rehabilitation programs.

[The MEmbeR Multicenter Study on medical-occupational rehabilitation]. / Multizentrische Evaluationsstudie zur medizinisch-beruflichen Rehabilitation (MEmbeR).

INTRODUCTION: MEmbeR is a prospective multi-center study on medical-occupational rehabilitation in Germany. METHODS: 196 neurological, psychiatric, orthopaedic, and internal medicine patients from 21 rehabilitation centres all across Germany have been enrolled and followed-up for 2 years after discharge. Primary outcome parameter was defined as return to work. Further, the SF-12 and a Mini-ICF-Rating have been used. RESULTS: Mean age was 34.1 (9.9) years, length of stay 150.0 (223.5) days. Prior to occupational rehabilitation, 69.9% were unable to work, 2 years after discharge only 5.6%. Rate of participants seeking a job was reduced from 19.7% to 3.1%. In summary, 78.1% returned to work. Employed participants were younger (32.8 [9.7] vs. 38.5 [9.4] years, p=0.001) and less disabled (Degree of Disablement [GdB]: 20.0 [31.2] vs. 36.1 [33.7], p<0.05). CONCLUSION: The multicenter cohort study MEmbeR provides further knowledge about the outcome of medical-occupational rehabilitation in Germany.

Mutations in the gene encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome.

The dystonias are a common clinically and genetically heterogeneous group of movement disorders. More than ten loci for inherited forms of dystonia have been mapped, but only three mutated genes have been identified so far. These are DYT1, encoding torsin A and mutant in the early-onset generalized form, GCH1 (formerly known as DYT5), encoding GTP-cyclohydrolase I and mutant in dominant dopa-responsive dystonia, and TH, encoding tyrosine hydroxylase and mutant in the recessive form of the disease. Myoclonus-dystonia syndrome (MDS; DYT11) is an autosomal dominant disorder characterized by bilateral, alcohol-sensitive myoclonic jerks involving mainly the arms and axial muscles. Dystonia, usually torticollis and/or writer's cramp, occurs in most but not all affected patients and may occasionally be the only symptom of the disease. In addition, patients often show prominent psychiatric abnormalities, including panic attacks and obsessive-compulsive behavior. In most MDS families, the disease is linked to a locus on chromosome 7q21 (refs. 11-13). Using a positional cloning approach, we have identified five different heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE), which we mapped to a refined critical region of about 3.2 Mb. SGCE is expressed in all brain regions examined. Pedigree analysis shows a marked difference in penetrance depending on the parental origin of the disease allele. This is indicative of a maternal imprinting mechanism, which has been demonstrated in the mouse epsilon-sarcoglycan gene.

[Long-term course of patients in neurological rehabilitation Phase B. Results of the 6-year follow-up in a multicenter study]. / Langzeitverlauf von Patienten der neurologischen Rehabilitation Phase B. Ergebnisse der 6-Jahres-Nachuntersuchung einer Multicenterstudie.

BACKGROUND: After conclusion of emergency care for severe neurological diseases patients in Germany are admitted at an early stage to so-called Phase B rehabilitation. No studies have been carried out on the long-term course of these patients. PATIENTS AND METHODS: In a prospective study in 2002 patients in Phase B from 9 centers were included and follow-up investigations were carried out after 5 and 6 years. Assessment instruments used were the Barthel index, the Rankin scale and the EQ-5D. Factors for the risk of a poor outcome and the chances for a good outcome were evaluated using multivariate logistic regression. RESULTS: A total of 1,280 patients were included in the study. A high age increased the risk of dying with a hazard quotient (HQ) of 1.05 (95% CI: 1.04-1.06) and high point counts in the coma remission scale (HQ 0.93; 95% CI: 0.92-0.96) and Barthel index (HQ 0.97; 95% CI: 0.97-0.98) on discharge reduced the risk of dying after 5 years. The factors swallowing impairment (OR 3.1; 95% CI: 1.7-5.5) and obligatory surveillance at the end of rehabilitation (OR 3.2; 95% CI: 1.2-8.6) increased the risk of a poor result in the Rankin scale 2-4 and the factors communication disorder (OR 5.0; 95% CI: 2.0-12.8) and PEG (percutaneous endoscopic gastrostomy) (OR 19.7; 95% CI: 2.7-144.4) on discharge increased the risk of a reduced health-related quality of life (defined as EQ-5D VAS <70) after 6 years. CONCLUSIONS: If support for bodily functions can be successfully reduced during Phase B rehabilitation, the patients will have a good outcome with respect to 5-year survival. If this is not successful the outcome is unfavorable with respect to survival and with respect to achieving self-sufficiency and health-related quality of life after 6 years.

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.

Increased oncogenicity of subclones of SV40 large T-induced neuroectodermal tumor cell lines after loss of large T expression and concomitant mutation in p53.

A model for medulloblastoma-like primitive neuroectodermal tumors was established in rat using retrovirally transduced SV40 large T antigen (LT) as an inducing agent (O. D. Wiestler et al., Brain Pathol., 2: 47-59, 1992). A cell line isolated from such a tumor and clonal derivatives thereof were biologically and molecularly characterized. In the parental tumor cell line, TZ870, which had been selected for G418 resistance, virtually all cells expressed LT and wild-type p53, which were complexed to each other. When plated in soft agar, these cells grew relatively slowly and formed disperse colonies. However, when grown without selection pressure, these cells reproducibly gave rise to LT-negative and G418-sensitive derivatives, LT-0 cells. Surprisingly, these latter cells exhibited a higher degree of malignancy both in vitro, growing readily to large colonies in soft agar, and in vivo, where they gave rise to a rapidly growing malignant tumor. Clonal selection from TZ870 cells revealed two types of clones: in one type, LT expression was stably maintained, even without selection pressure, whereas the other type lost the LT coding sequences. All LT-negative clones exhibited the same phenotype as the LT-0 cells. Reexpression of LT had no effect. However, LT no longer formed complexes with p53, and p53 was metabolically stable, suggesting that it had been mutated. Sequence analyses and diagnostic restriction digests of the p53 gene revealed that (a) both the parental LT-transformed cells and their derivatives contained only one complete p53 allele and (b) all LT-positive clones expressed wild-type p53, whereas all LT-negative clones expressed a mutant allele with a common mutation at Cys-174-->Tyr, indicating their clonal origin. We assume that the loss of LT coding sequences is the consequence of the p53 mutation, perhaps by inducing genomic instability, and that both the p53 mutation and additional genetic alterations that accompany the loss of LT coding sequences might contribute to enhanced malignancy.

Criterion validity and sensitivity to change of the Early Rehabilitation Index (ERI): results from a German multi-center study.

BACKGROUND: Evaluation of functional status is difficult in neurological and neurosurgical early rehabilitation patients. The Early Rehabilitation Index (ERI) was introduced in Germany over 20 years ago, but since then validation studies are lacking. The ERI (range -325 to 0 points) includes highly relevant items including the necessity of intermittent mechanical ventilation or tracheostomy. METHODS: The present paper analyzed data from a German multi-center study, enrolling 754 neurological early rehabilitation patients. Together with ERI, Barthel Index (BI), Glasgow Coma Scale (GCS), Glasgow Outcome Score Extended, Coma Remission Scale (CRS), Functional Ambulation Categories and length of stay were obtained. RESULTS: ERI showed significant improvements from admission to discharge (p < 0.001). In addition, there were significant correlations of the ERI upon admission and at discharge with BI, CRS and GCS. CONCLUSIONS: Evaluation of our study data suggest that the ERI may be used as a valid assessment instrument for neurological and neurosurgical early rehabilitation patients.

Differential effects of phosphorylation of rat p53 on transactivation of promoters derived from different p53 responsive genes.

The tumor suppressor protein p53 is phosphorylated at multiple sites in the amino-terminal transactivation domain and at several sites in the carboxy-terminal region. Phosphorylation appears to modulate its DNA binding activity. Here we demonstrate that phosphorylation of p53 also modulates its transcriptional activity. Okadaic acid treatment of cells resulted in enhanced phosphorylation of p53 and concomitantly in enhanced transactivation of an mdm2 promoter-linked luciferase reporter gene. This effect was cell type specific, however, since transactivation was enhanced in rat and mouse fibroblasts but reduced in the human Saos-2 cell line. Moreover, the effect was dependent on the promoter. In rat cells transcription from the mdm2, waf1 (cip1) and bax gene promoters, and the artificial PG13 promoter was enhanced by okadaic acid treatment whereas that from the cyclin G promoter was reduced. When various phosphorylation site mutants of p53 were tested for transactivation of these promoters, they behaved differently. Amino-terminal mutants exhibited reduced transcriptional activities on mdm2, waf1 and cyclin G promoters but enhanced activities with bax and PG13 promoters. On the other hand, a mutant at the cdk phosphorylation site, A313, showed reduced activity with mdm2 and waf1 promoters but enhanced activity with the cyclin G promoter, and finally, mutant A390 exhibited enhanced activity on waf1 and bax promoters, but reduced activity on the cyclin G promoter. These results suggest that phosphorylation of p53 may have different effects on its transcriptional activity, depending on the cellular environment and the particular response element. Moreover, both, amino- and carboxy-terminal phosphorylation sites seem to be involved in modulating the DNA-binding and the transactivation activities.

Purification and characterization of a protein kinase which is activated by SV40 large T-antigen and phosphorylates the tumor suppressor protein p53.

In SV40-transformed or infected rat cells phosphorylation of the tumor suppressor protein p53 is enhanced due to activation of kinases. At least three different kinases can be co-precipitated with p53-large T (LT) immune complexes, casein kinase II representing the major activity, a cyclin dependent kinase (Cdk), and a kinase which appears to be specifically activated by LT (E Müller, B Boldyreff and KH Scheidtmann, Oncogene 8: 2193-2205, 1993). In this paper we describe the purification and identification of the LT-activated kinase that phosphorylates a site adjacent to the Cdk site in rat p53. To monitor the activity a synthetic peptide was used containing glutamic acid at the position of Ser-313, thus mimicking a phosphorylated Cdk site. With a combination of Mono Q chromatography and subsequent affinity chromatographies with p13suc1 and a p53-fragment as ligands a 42 kDa protein kinase was purified to near homogeneity from SV40-transformed rat cells. This kinase phosphorylated both the peptide substrate and the native rat p53. Interestingly, phosphorylation of the specific site seemed to depend on prior phosphorylation of the Cdk site. On the other hand, the kinase seemed to be activated by LT, as the activity towards the peptide substrate was significantly higher in extracts from wild-type LT-transformed cells than from normal or mutant LT-transformed cells. This activation was not restricted to rat cells but occurred in SV40-transformed mouse and infected monkey cells as well. Phosphorylation of the specific site by LT-activated kinase was not dependent on the presence of LT in vitro suggesting that activation of the LT-activated kinase is probably indirect rather than through direct interaction with LT. Cell cycle studies revealed that the LT-activated kinase is cell cycle regulated, since its activity was not detectable in M phase but increased during G1 phase after which it remained relatively constant.

Characterization of protein kinase activities associated with p53-large-T immune complexes from SV40-transformed rat cells.

Interaction of viral oncoproteins, such as SV40 large T, with cellular growth suppressor proteins Rb and p53 is presumed to inactive or modulate their growth suppression functions, thereby leading to transformation. An additional transformation-related activity of LT leads to hyperphosphorylation of p53. To search for kinases that might be responsible for this effect, p53-LT complexes were immunopurified from different SV40-transformed rat cell lines and assayed for associated kinase activities, in vitro. Protein kinase activity was readily observed in p53-LT immunecomplexes from wild-type transformed cells but was low or undetectable in p53 from mutant-transformed or normal cells. Optimal activity required the presence of Mn++. p53 was phosphorylated at all sites found in vivo. In contrast, LT was phosphorylated only at a subset of formerly identified sites and at additional sites not seen in vivo. The p53-LT-kinase complex was assayed for the presence of casein kinases, cdk like kinases, or DNA-activated kinase, using specific effectors, antibodies, or purified enzymes as tools. DNA-activated kinase or cdc2/cdk2 were not detectable, although the purified enzymes phosphorylated p53 in vitro. Casein kinase 2 represented the major activity, which on p53 phosphorylated not only the C-terminal Ser390 but also several sites in the N-terminal region. One additional activity, not identified so far, may represent an LT-induced or activated kinase. This kinase seems to enhance overall phosphorylation of p53 and, perhaps other substrates, and may thereby contribute to transformation.

Complex regulation of the DNA-binding activity of p53 by phosphorylation: differential effects of individual phosphorylation sites on the interaction with different binding motifs.

The tumor suppressor protein p53 exists in different phosphorylation states depending on the cellular environment and perhaps the stage of the cell cycle. These different phosphorylation states can be mimicked in the baculovirus expression system by employing the phosphatase inhibitor okadaic acid. Hyperphosphorylation of p53, particularly of Ser313 and/or Ser309, stimulated its DNA binding activity (Fuchs, Hecker and Scheidtmann, Eur. J. Biochem. 228, 625, 1995). Here we show that hyperphosphorylation of p53 has different effects on its DNA-binding activity, depending on the phosphorylation sites and the binding motif: (i) Phosphorylation of amino-terminal sites appeared to reduce binding to the RGC consensus motif, whereas additional phosphorylation of both, Ser313 and Ser309 led to enhanced binding. (ii) Upon hyperphosphorylation, binding to the RGC motif was enhanced whereas binding to the p53 response element of the bax1 gene promoter was diminished. (iii) DNA binding was also greatly enhanced by antibodies Pab 122 and 421 directed against the carboxyl terminus, but this latter effect was superimposed by the phosphorylation state of p53. Thus, the DNA binding activity of p53 appears to be regulated in a complex way in that (i) binding to a given sequence motif may be regulated by differential phosphorylation and/or by interaction with other factors; (ii) binding to different motifs may be modulated in opposite ways. Thus, the different genes that are regulated by p53 may be differently affected by these parameters.

Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phosphorylates core histones.

We cloned a cDNA coding for a novel serine/threonine kinase, Dlk, a protein of 448 amino acids with a predicted molecular weight of 51.3 kDa. The kinase domain shows 81% amino acid sequence identity to the recently identified DAP kinase (death associated protein kinase) (Deiss et al., Genes & Dev., 9, 15-30, 1995), therefore, the new kinase was called Dlk, for DAP like kinase. Northern analyses revealed a single mRNA species of 1.7 kb which was ubiquitously expressed. However, expression levels varied considerably in different cell lines and tissues. Moreover, expression was downregulated upon UV irradiation. Dlk exhibited autophosphorylation activity, predominantly towards threonine residues and phosphorylated the regulatory subunit of myosin light chain, but in this case exclusively at serine residues. Dlk seems to be tightly associated with insoluble nuclear structures, presumably chromatin, since it was resistant to various rigorous extraction procedures but it was partially released upon DNase I digestion of nuclei. Consistent with this, purified Dlk phosphorylated core histones H3, H2A and H4 as exogenous substrates and endogenous histone H3 in kinase assays with nuclear extracts. Expression as GFP-fusion protein revealed a diffuse as well as a speckled nuclear staining suggesting an association with replication or transcription centers.

p53 phosphorylation mutants retain transcription activity.

To investigate the effect of phosphorylation on the transcription activity of p53, ten phosphorylation mutants were constructed covering all the identified phosphorylation sites of rat p53. These included mutants of two casein kinase I sites (Ser6 and Ser9), two DNA-PK sites (Ser15 and Ser39), a p34cdc2 site (Ser313), the adjacent Ser312 and a casein kinase II site (Ser390). Two double phosphorylation mutants (Ser4, 6 and Ser15, 390) and one triple phosphorylation mutant (Ser4, 6 and 15) were also constructed. The transcription activity of all the p53 phosphorylation mutants was tested by transfection into two different types of cells, Saos-2 cells and p53(-/-) fibroblasts derived from p53 knock out mice, which both lack endogenouse p53. Surprisingly, all the p53 phosphorylation mutants retain transcription activity and the seven mutants tested can also suppress cell growth.

C-terminal truncation of Dlk/ZIP kinase leads to abrogation of nuclear transport and high apoptotic activity.

Dlk (also termed ZIP kinase) is a novel serine/threonine kinase with a unique C-terminal domain that is rich in arginine and contains three putative NLS motifs and a functional lecuine zipper. Dlk is indeed localized in the nucleus where it shows a speckled distribution. To elucidate the biological functions of Dlk, we wanted to identify the signals relevant for nuclear transport and further the nuclear structures which Dlk binds to. Expression of various deletion and point mutations of Dlk as GFP fusion proteins revealed that the leucine zipper is required for association with speckles and the most C-terminal NLS is necessary and sufficient for nuclear transport. Interestingly, a C-terminal deletion mutant defective for nuclear transport exhibited a pronounced colocalization with actin filaments and, even more strikingly, was a very potent inducer of apoptosis. This apoptotic activity was abrogated, however, when this mutant was retargeted to the nucleus via a heterologous NLS from large T, indicating that Dlk only exerts an apoptotic activity in the cytoplasm. To identify the speckle like structures to which Dlk binds we performed immunofluorescence analyses with antibodies directed against representative marker proteins of replication, transcription, or splicing centers. None of these marker proteins revealed a colocalization with Dlk. Instead, we found a partial colocalization with PML bodies which seem to play a key role in regulation of apoptosis. Taken together, these data strongly suggest a functional role for Dlk in control of cell survival which is dependent on its subcellular localization.

Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis.

Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk. This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro. Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase. We employed the yeast two-hybrid system to identify interaction partners of Dlk that might serve as regulators or targets. Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis. Complex formation between Dlk and Par-4 was confirmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo. The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 - 417, rather than to the leucine zipper. Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin filaments. These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis.

Identification of amplified genes from SV40 large T antigen-induced rat PNET cell lines by subtractive cDNA analysis and radiation hybrid mapping.

Primitive neuroectodermal tumors (PNETs) such as human medulloblastomas are genetically heterogeneous and therefore poorly understood. In a rat model the SV40 large T antigen was used to induce neoplasms with characteristic features of PNETs. Tumor development requires a latency period of 8-11 months implicating secondary genetic alterations. To identify such secondary alterations we performed comparative analyses of two phenotypically identical PNET-derived cell lines. Indeed, these cell lines displayed distinct high-level amplification sites. Using a combination of subtractive cDNA analysis and radiation hybrid mapping we have now identified genes in the amplicon regions of the two cell lines. Interestingly, one of these genes encodes the rat homolog of a cytosolic branched chain aminotransferase (BCAT(C)) previously shown to be amplified in a mouse teratocarcinoma cell line. We propose that this simple cloning strategy may serve as a powerful tool for the isolation of genes implicated in known chromosomal aberrations in primary tumors and tumor cell lines.

Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells.

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.

Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells.

Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.