[Report on notifications according to section 8d of the German Transplantation Act (TPG) for the years 2009-2011]. / Bericht zur Meldung nach § 8d Transplantationsgesetz (TPG) für die Jahre 2009 bis 2011.

In Germany, the Tissue Act came into effect on 1 August 2007. Since then, every tissue establishment is legally obligated to keep a record of its activities according to section 8d subsection 3 of the Transplantation Act (TPG). An annual report must be submitted to the Paul Ehrlich Institute once a year up to 1 March of the subsequent year. The report should include the types and quantities of tissues procured, conditioned, processed, stored, distributed or otherwise disposed of, imported, and exported. The report should be made on a TPG-based notification form published on the Internet by the Paul Ehrlich Institute. The present report according to section 8d subsection 3 of the TPG is based on data of the reporting years 2009-2011. Six years after implementation of the TPG's reporting obligation for tissue establishments, the number of tissue establishments known by the Paul Ehrlich Institute has increased from 349 in 2007 to 949 in 2011. In the course of continuous optimization of the notification forms, including tissue-specific glossaries, the reported data of most of the tissues and tissue preparations have become more conclusive.

[Report on notifications according to Section 8d of the German Transplantation Act (TPG) for the year 2008]. / Bericht zur Meldung nach § 8d Transplantationsgesetz (TPG) für das Jahr 2008.

In Germany, the tissue law came into effect on 1 August 2007. The law implemented the requirements of EC directives on quality and safety of human tissues and cells in the German Transplantation Act ("Transplantationsgesetz," TPG) and in the German Medicinal Products Act. Accordingly, tissue establishments are obligated to keep a record of their activities and to submit an annual report to the Paul-Ehrlich-Institut (PEI). The report shall include the types and quantities of tissues procured, conditioned, processed, stored, and distributed, or otherwise disposed of, imported and exported. For this purpose, the PEI published TPG-based notification forms in the Bundesanzeiger and in the Internet. The data provided by tissue establishments have been anonymized and compiled in a general report. The analysis revealed inconclusive data, which can be due to a number of different causes. To achieve better consistency of data provided in the future, the explanations for completing the notification forms will be amended. Thus far, compiled data are not appropriate to draw conclusions on the availability of tissues and tissue preparations in Germany, but the data can serve as reference points.

During Drosophila embryogenesis the beta 1 tubulin gene is specifically expressed in the nervous system and the apodemes.

We determined the in vivo distribution of the beta 1 tubulin from D. melanogaster using isotype specific antibodies. Maternally expressed beta 1 tubulin is incorporated into mitotic spindles. Later in development a strong expression in the CNS is observed. Furthermore, all chordotonal organs and the apodemes are marked by beta 1 tubulin. Nuclear run-on assays and stage specific in vitro transcription showed a zygotic expression of the beta 1 tubulin gene from the extended germ-band stage onwards. Using the P-element system, we identified several elements; upstream between -2.2 kb and the transcription initiation site, elements for low level expression in the CNS are present. In the intron between +0.44 kb and +2.5 kb enhancer elements are located that drive the expression in the chordotonal organs and the apodemes. Between the start site and +0.44 kb (273 bp) and +2.5 kb and the second exon (315 bp), maternal and CNS enhancers result in full level expression of a lacZ-beta 1 reporter gene. We show, that the beta 1 tubulin gene is very early effector gene starting its expression shortly after the commitment of neuroblast cell fate. This gene offers an excellent model system for the identification of neural and apodeme specific transcription factors.

During Drosophila spermatogenesis beta 1, beta 2 and beta 3 tubulin isotypes are cell-type specifically expressed but have the potential to coassemble into the axoneme of transgenic flies.

alpha and beta Tubulins exist in a number of different isotypes with distinct expression patterns during development. We have shown by immunofluorescent staining that beta 1, beta 2 and beta 3 tubulins are distributed very specifically in the testes of Drosophila. beta 3 Tubulin is present exclusively in cytoplasmic microtubules of cells somatic in origin, while the beta 1 isotype is localized in the somatic cells and in early germ cells of both the microtubules of the cytoskeleton as well as in the mitotic spindle. In contrast, beta 2 tubulin is present in all microtubular arrays (cytoskeleton, meiotic spindles, axoneme) of germ cells from meiotic prophase onward, though not detectable in somatic cells. Thus, a switch of beta tubulin isotypes from beta 1 to beta 2 occurs during male germ cell differentiation. This switch is also observed in the distantly related species Drosophila hydei. By fusing beta 1 or beta 3 amino acid coding regions to the control region of the beta 2 tubulin gene and performing germ line transformation experiments, we have examined the copolymerization properties of the different tubulin isotypes. Neither beta 1 nor beta 3 are detectable in the axoneme in the wild-type situation. Analysis of transgenic flies carrying beta 2-beta 1 fusion genes or beta 2-beta 3 fusion genes revealed that both beta 1 and beta 3 tubulin isotypes have the potential to co-incorporate with beta 2 tubulin into microtubules of the sperm axoneme. Male flies homozygous for the fusion genes (beta 2-beta 1 or beta 2-beta 3) remain fertile, despite the mixture of beta tubulin isotypes in the axoneme.

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.

Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs.

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

The expression of beta 1 and beta 3 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis.

In Drosophila beta tubulins are encoded by a small gene family and the four members of this family are differentially expressed. mRNAs transcribed from two of these genes, namely the beta 1 and beta 3 tubulin genes, are abundant during embryogenesis. While the beta 1 tubulin gene is constitutively expressed during development, beta 3 mRNA is restricted to two distinct phases: mid embryogenesis and metamorphosis. The transcription initiation sites are identical in both these stages and comparison of presumptive promoter regions reveals no extensive homologies between the genes. In situ localization shows beta 1 tubulin mRNA to be maternally expressed in the nurse cells of the egg chambers and evenly distributed during early embryogenesis. In contrast, during later stages of embryogenesis beta 1 tubulin transcripts are predominantly expressed in neural derivatives. The beta 3 tubulin gene expression is also spatially regulated, beta 3 mRNA being restricted to the mesoderm.

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.