The ontogeny of soleus muscles in mdx and wild type mice.

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.

The genes encoding subunits of ATP synthase are conserved in the reduced plastid genome of the heterotrophic alga Prototheca wickerhamii.

The heterotrophic unicellular alga Prototheca wickerhamii is closely related to the photoautotrophic Chlorella vulgaris but has a 54,100-bp plastid DNA (ptDNA) that is much smaller than the chloroplast DNA of C. vulgaris (150,613 bp). The nucleotide sequence of 28,093 bp of the Prototheca ptDNA has been determined. No genes for photosynthetic functions have been found, except for sequences encoding six subunits of the ATP synthase ( atpA, atpB, atpE, atpF, atpH, and atpI). Transcripts of these atp genes have also been detected. Whether the leucoplasts of Prototheca contain a functional ATP synthase has still to be elucidated. Identified genes further include tufA, minD, cysT, and genes coding for three rRNAs, 22 tRNAs, and 12 ribosomal proteins. The results support the idea that, in the reduced plastid genome of Prototheca, genes coding for components of the plastid translational apparatus have been preferentially retained, and might be needed for the expression of the atp genes and some unassigned ORFs.

New processing of lupin protein isolates and functional properties.

A growing demand for functional plant proteins could be identified, which properties are customized for specific applications and formulations as food ingredients. Native lupin proteins (alpha, beta, gamma) conglutin have a good solubility at appropriately chosen conditions. A novel procedure has been proposed to maintain the native protein properties. Lupin proteins are extracted from hexane deoiled lupin. The protein product type E comprises high molecular weight proteins (alpha, beta-conglutin), which are separated using alkaline extraction and acid precipitation procedures. The protein product type F is enriched in the gamma-conglutin fraction and is separated from the acid pre-extract applying cross flow filtration at pH 7-8. For the zirconium oxide membrane the filtration rate can be increased by appropriately chosen pH conditions up to 70 l/m2h. Lupin protein fraction (type E and F) are highly soluble protein isolates with outstanding emulsification, salt tolerance and foaming properties. These new lupin proteins (type E and F) offer extremely interesting properties for application in food systems and are available from pilot plant fractionation.

A model system for studying postnatal myogenesis with tetracycline-responsive, genetically engineered clonal myoblasts in vitro and in vivo.

The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 microg ml(-1)) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.

Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2.

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Analysis of the role of Hsp25 phosphorylation reveals the importance of the oligomerization state of this small heat shock protein in its protective function against TNFalpha- and hydrogen peroxide-induced cell death.

The role of murine Hsp25 phosphorylation in the protection mediated by this protein against TNFalpha- or H2O2-mediated cytotoxicity was investigated in L929 cell lines expressing wild type (wt-) or nonphosphorylatable (mt-) Hsp25. We show that mt-Hsp25, in which the phosphorylation sites, serines 15 and 86, were replaced by alanines, is still efficient in decreasing intracellular reactive oxygen species levels and in raising glutathione cellular content, leading the protective activity of mt-Hsp25 against oxidative stress to be identical to that of wt-Hsp25. To independently investigate the role of Hsp25 phosphorylation, we blocked TNFalpha-induced phosphorylation of wt-Hsp25 using SB203580, a specific inhibitor of the P38 MAP kinase. This treatment did not abolish the protective activity of Hsp25 against TNFalpha. The pattern of Hsp25 oligomerization was also analyzed, showing mt-Hsp25 to constitutively display large native sizes, as does wt-Hsp25 after TNFalpha treatment in the presence of SB203580. Our results, therefore, are consistent with the possibility that the hyperaggregated form of Hsp25 is responsible for the protective activity against oxidative stress and that the phosphorylation of serines 15 and/or 86 by interfering with this structural reorganization, may lead to the inactivation of Hsp25 protective activity.

Repression of human heat shock factor 1 activity at control temperature by phosphorylation.

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.

The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress.

Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1.

Alpha A-crystallin confers cellular thermoresistance.

The bovine eye lens protein alpha A-crystallin has been overexpressed both by stable transfection of HeLa cells and by transient transfection of NIH 3T3 cells. In both experimental systems alpha A-crystallin overexpression results in an increased cellular thermoresistance as judged by different clonal survival assays. In contrast, similar overexpression of another stable lens protein, beta B2-crystallin, does not confer thermoresistance. These results indicate that the structural relationship of alpha A-crystallin to the small heat shock proteins HSP25/27 and to alpha B-crystallin is sufficient for the shared thermoprotective function of all of these molecules and strongly suggests that the chaperone-like properties that they have in common are responsible for the conferred cellular thermoresistance.

Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.

Small heat shock proteins (sHsps) show a very rapid stress- and mitogen-dependent phosphorylation by MAPKAP kinase 2. Based on this observation, phosphorylation of sHsps was thought to play a key role in mediating thermoresistance immediately after heat shock, before the increased synthesis of heat shock proteins becomes relevant. We have analysed the phosphorylation dependence of the chaperone and thermoresistance-mediating properties of the small heat shock protein Hsp25. Surprisingly, overexpression of Hsp25 mutants, which are not phosphorylated in the transfected cells, confers the same thermoresistant phenotype as overexpression of wild type Hsp25, which is either mono- or bis-phosphorylated at serine residues 15 and 86 within the cells. Furthermore, in vitro phosphorylated Hsp25 shows the same oligomerization properties and the same chaperone activity as the nonphosphorylated protein. No differences between phosphorylated and nonphosphorylated Hsp25 are detected in preventing thermal aggregation of unfolding proteins and assisting refolding of denatured proteins. The results suggest that chaperone properties of the small heat shock proteins contribute to the increased cellular thermoresistance in a phosphorylation-independent manner.

Development and tissue-specific distribution of mouse small heat shock protein hsp25.

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13-20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related alpha, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism.

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells.

hsp25 is a small, growth-related, mammalian stress protein which is highly accumulated in the stationary phase of Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat-shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT II6 and EAT II8) exhibit constitutive over-expression of the small heat-shock protein, hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT II6 and EAT II8 is significantly decreased depending on the degree of cadmium-regulated over-expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over-expressing cells for primary inoculation could be demonstrated.

Generation of antibodies against human hsp27 and murine hsp25 by immunization with a chimeric small heat shock protein.

A hybrid protein containing the N-terminal part of the murine stress protein hsp25 (amino acids 1 to 110) and the C-terminal part of the human stress protein hsp27 (amino acids 111 to 208) was expressed in E. coli using a T7 polymerase/promoter system. The recombinant hybrid protein was purified and used for immunization of rabbits. In contrast to immunization experiments using hsp25 and hsp27 alone, immunization with the hybrid protein hsp25/27 leads to antibodies which can be used for detection of both hsp25 and hsp27.

[The effect of LPH on the behavior of parameters of the kallikrein-kinin system (KKS) in the early phase of experimental endotoxic shock in the rat]. / Einfluss von LPH auf das Verhalten von Parametern des Kallikrein-Kinin-Systems (KKS) in der Frühphase des experimentellen Endotoxinschocks der Ratte.

The effect of a new shock-protective agent LPH on parameters of the kallikrein-kinin-system was studied in a standardized endotoxin shock model. Activation of KKS in endotoxin shock was not significantly influenced by LPH. On the other hand, LPH by itself caused activation of KKS. Decrease of kininogen level by endotoxin, however, was prevented by the pretreatment with LPH.

Treatment of relapsed acute lymphocytic leukemia in adults.

Thirty-three patients with ALL/AUL in first relapse were treated with an induction of prednisone, vindesine, daunorubicin, Erwinia asparaginase, i.t. MTX (phase I), high-dose cytarabine, and etoposide (phase II). Twenty-one (64%) achieved a complete remission, one a partial remission. Side effects of induction-phase I were predominantly hematological with subsequent infections and gastrointestinal toxicity. In phase II some patients had additional cutaneous, ocular, and hepatic toxicity. The treatment efficiently induced remissions with tolerable toxicity in relapsed ALL. The disease-free survival, however, needs to be improved.