Assembly of amino-terminally deleted desmin in vimentin-free cells.

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells.

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.

[Severe accidental hypothermia : Treatment using an intravascular temperature management catheter]. / Schwere akzidentelle Hypothermie : Behandlung mithilfe eines intravaskulären Katheters zum Temperaturmanagement.

Different techniques have been reported for the treatment of severe accidental hypothermia. In this case, we successfully used an intravascular catheter temperature management system which has been developed to induce reversible therapeutic hypothermia in patients following resuscitation. In our patient, the initial core temperature was 26.7 °C, and the temperature management system allowed for successful rewarming without complications with a maximum rate of about 1 °C/h.

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation.

The 'high ammonia pathway' enzyme glutamate dehydrogenase (NADP+) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. Purified glutamate dehydrogenase (NADP+) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP+) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP+) inactivation is caused or followed by rapid proteolysis.

Expression of cDNA-encoded human acid alpha-glucosidase in milk of transgenic mice.

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid alpha-glucosidase cDNA was placed under control of the alpha S1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 micrograms/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid alpha-glucosidase meets the criteria for therapeutic application.

Abnormal incisor-tooth differentiation in transgenic mice expressing the muscle-specific desmin gene.

Immunocytochemistry and electron microscopic observations on the incisor-tooth organ of transgenic mice expressing the muscle-specific desmin gene under the direction of the vimentin promoter, reveal that the expression of the hybrid transgene occurs both in mesenchymal cells and differentiating odontoblasts. The muscle-specific desmin, as estimated by fluorescence intensity, is more expressed in immature mesenchymal cells than in postmitotic differentiated odontoblasts. The expression of the transgene generates alteration of the odontoblast-intermediate filament network and interferes with the secretory activity of both odontoblasts and ameloblasts. Our results are consistent with the hypothesis that odontoblasts have inductive properties on the differentiation of ameloblasts and that intermediate filaments among other factors play the role of cell and tissue organizer.

Muscle-specific expression of a dominant negative desmin mutant in transgenic mice.

To extend our knowledge of the functions of desmin and vimentin intermediate filaments in the developing organism, a construct encoding a truncated desmin subunit driven by the desmin promoter (pDDV), was introduced into the murine germ line. The resulting mutant desmin subunit was assembly-incompetent and capable of disrupting both preexisting desmin and vimentin filaments in a dominant negative fashion in transfected C2C12 muscle cells and in transgenic mouse muscle tissue. Expression of the pDDV was tissue-specific in transgenic mice. High level expression of pDDV occurred in a small percentage of desmin-containing muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse desmin pattern instead of the dots and clumps into which mutant desmin typically accumulates in undifferentiated C2C12 muscle cells in tissue culture. Disruption of the endogenous desmin filaments in Sartorius muscle results in ultrastructural abnormalities.

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin.

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.

Disruption of vimentin intermediate filaments in transgenic mice by expression of a dominant negative mutant desmin subunit.

To investigate putative functions of vimentin intermediate filaments in the context of intact tissues and the developing organism, a construct (pVDV), driven by the vimentin promoter and encoding a truncated desmin subunit, was introduced into the murine germ line. The mutant desmin was assembly-incompetent and capable of disrupting preexisting vimentin filaments in a dominant negative fashion, both in transgenic mouse tissues and in fibroblast cultures derived from these mice. Mutant desmin expression strongly enhanced vimentin turnover. In tissues of some transgenic mouse lines, high level expression of pVDV occurred in 10 to 40% of vimentin-containing cells and, surprisingly, in 1 to 10% of the skeletal and tongue muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse staining pattern instead of the punctated aggregates into which mutant desmin typically accumulates in other cell types. The overexpression of pVDV and the concomitant disruption of the endogenous vimentin filament network and enhanced vimentin turnover in a significant percentage of cells did not cause detectable developmental abnormalities.

Identification of two silencers flanking an AP-1 enhancer in the vimentin promoter.

We have studied the 5' upstream sequences required for the transcriptional regulation of the hamster gene encoding the intermediate filament protein, vimentin. Although vimentin is regarded as the intermediate filament protein of mesothelial tissue, it is also produced in most cultured cells. The human mammary carcinoma cell line, MCF-7, belongs to the exceptions. It contains no vimentin, and the complete upstream promoter region is inactive in this particular cell line. By using transient transfection of chimeric constructs into MCF-7 and HeLa cells, and subsequent chloramphenicol acetyltransferase assays, we were able to show the presence of two negative control regions flanking a double AP-1 enhancer element. Our data indicate that these elements exert their effect irrespective of orientation and position, suggesting that they are silencers. In vitro footprinting assays, gel mobility assays and Southwestern (protein-DNA) blotting revealed the presence of trans-acting factors interacting with both silencer elements. The silencing effect was particularly pronounced in MCF-7 cells, although DNA-binding proteins are present in HeLa cells as well.

Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk.

At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost-effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development.

[Surgical therapy of cancer of the hepatic duct bifurcation]. / Zur Problematik der chirurgischen Therapie des Hepaticusgabel-Carcinoms.

We report about the diagnostic and therapeutic approaches in 62 cases of Klatskin tumors operated during the last 5 years. In most patients the definite treatment consisted either of primary endoscopic drainage of the bile ducts or of a secondary endoscopic drainage after explorative laparotomy. Only one third of the patients could be operated in curative intention. Operative procedures included local tumor resections as well as a central bile duct resection combined with hemihepatectomy. The main problem in these operations is to avoid a biliodigestive anastomosis with tumor infiltrated bile ducts, because these patients would have been better treated endoscopically. After extended preoperative diagnostic procedures including CT-scan, ultrasound, ERCP and angiography in most cases the criteria for irresectability can be defined and unnecessary operative interventions can be avoided.

Generation of transgenic dairy cattle using 'in vitro' embryo production.

We have combined gene transfer, by microinjection, with 'in vitro' embryo production technology, enabling us to carry out non-surgical transfer, to recipient cows, of microinjected embryos that have been cultured from immature oocytes. Using this approach, we have established 21 pregnancies from which 19 calves were born. Southern blot analysis proved that in two cases the microinjected DNA had been integrated in the host genome.

Guinea-pig atrial cardioballs.

Myocytes from atria of adult guinea-pigs were isolated by means of a previously described enzyme perfusion with some modifications. The problem of Ca-intolerance of the dispersed cells was circumvented by (i) avoiding cooling of the cells below 25 degrees C and (ii) increasing the Ca concentration slowly already during the enzyme perfusion. Isolated atrial myocytes were taken in long-term cell culture. Under this condition they become spherical within about 2 days. The rounded stage (cardioballs), which is found in the cultures for a period of ca. 10 days is highly suited for electrophysiological studies using the different recording configurations of the patch clamp technique, including 'tight-seal whole-cell recording' with simultaneous cell dialysis.

Formation of 7 alpha- and 7 beta-hydroxylated bile acid precursors from 27-hydroxycholesterol in human liver microsomes and mitochondria.

In a search for enzymes involved in the formation of bile acids from 27-hydroxycholesterol in humans, the metabolism of this and other side-chain oxygenated steroids was studied in human liver microsomes and mitochondria. The microsomal fraction contained enzyme(s) catalyzing 7 alpha-hydroxylation of 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, whereas the 7 alpha-hydroxylation of cholesterol and 3 beta-hydroxy-5-cholenoic acid was low. Only small amounts of 7 beta-hydroxylated products were formed. Purification and subfractionation of microsomal protein yielded a fraction of cytochrome P-450, which required NADPH and NADPH-cytochrome P-450 reductase and catalyzed 7 alpha-hydroxylation of the side-chain oxygenated 3 beta-hydroxy-delta 5-C27-steroids but was inactive toward cholesterol. Added cholesterol did not inhibit the observed enzymatic activity. The results provide evidence that this enzyme is different from cholesterol 7 alpha-hydroxylase. The mitochondrial fraction contained enzyme(s) that catalyzed an isocitrate-dependent 7 alpha-hydroxylation of 3 beta-hydroxy-5-cholestenoic acid. The activity was much lower with 27-hydroxycholesterol. The mitochondrial fraction also catalyzed the oxidation of the 27-hydroxy group and contained a 3 beta-hydroxy-delta 5-steroid dehydrogenase active on 7 alpha-hydroxylated C27-steroids. The metabolic end product of the reactions catalyzed by these enzymes was 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. A considerable fraction of the 7 alpha-hydroxy-delta 5 intermediates was also converted to the corresponding 7 beta-hydroxysteroids, probably by way of the 7-oxosteroids, suggesting the presence of an epimerizing enzyme in the mitochondrial fraction.

Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

Structure and expression of the mouse casein gene locus.

The analysis of yeast artificial chromosomes (YACs) containing the complete mouse casein gene locus revealed the presence of five casein genes, alpha-, beta-, gamma-, delta-, and kappa-casein, in this order, in the locus. The alpha- and beta-casein genes are only 10 kb apart and have convergent transcriptional orientations. The distance between the beta-casein gene and the alpha s2-like gamma-casein gene is about 70 kb, and these genes have divergent transcriptional orientations. The gamma- and delta-casein genes, both encoding a alpha s2-like casein, are linked within 60 kb and convergently transcribed. The kappa-casein gene is located about 100 kb from the delta-gene. Except for the presence of the delta-casein gene, the organization of the mouse casein locus resembles that of the bovine locus, including the transcriptional orientation of the genes. In contrast to the other casein genes, which are strongly induced at mid-lactation, expression of the delta-casein gene is abruptly induced upon parturition. Comparative analysis of alpha s2-like sequences from various species suggests that the ancestral alpha s2-like gene duplicated around the time of radiation of the rodent and artiodactylid ancestors.

Baby hamster kidney (BHK-21/C13) cells can express striated muscle type proteins.

When baby hamster kidney (BHK-21/C13) cell lines are subjected to low-serum medium, cell morphology changes from polygonal to elongated and occasionally fusion of cells is also observed. BHK-21 cells initially growing in Eagle's modified minimum essential medium (EMEM) containing 10% newborn bovine serum were induced to differentiate by changing the culture medium after the cells had grown to confluency. After this point the cells were grown in a low-serum medium (EMEM with 2% normal horse serum), for at least 4 days. The expression of different muscle-specific proteins (desmin, titin and skeletal muscle myosin) and of tropomyosins was studied in both polygonal and elongated BHK-21 cells using the indirect-immunofluorescence assay, two-dimensional (2D)-gel electrophoresis and immunoblotting. Filamentous staining was found with the desmin antisera in the polygonal cells and at all stages of BHK-cell elongation. While no reaction was seen with the titin and myosin antibodies in the polygonal cells, a punctate staining reaction for titin was detected 2 days after medium-change, although the cells had not yet elongated. After 4 days titin was found in a striated pattern. Filamentous staining was seen with the skeletal-muscle-specific myosin antibody at this stage. Confirmatory results were obtained from immunoblotting assays and 2D-gel electrophoresis of cytoskeletal preparations from undifferentiated and differentiated BHK cells. These latter experiments showed the initiation of tropomyosin expression only in the differentiated cells. The positive staining with antibodies to skeletal muscle myosin and titin indicates a striated-muscle nature of the (elongated) BHK-21/C13 cells.