Adipose tissue-derived stromal cells acquire endothelial-like features upon reprogramming with SOX18.

Adipose tissue-derived stromal cells (ASC) form a rich source of autologous cells for use in regenerative medicine. In vitro induction of an endothelial phenotype may improve performance of ASCs in cardiovascular repair. Here, we report on an in vitro strategy using direct reprogramming of ASCs by means of ectopic expression of the endothelial-specific transcription factor SRY (sex determining region Y)-box18 (SOX18). SOX18 induces ASCs to express a set of genes involved in vascular patterning: MMP7, KDR, EFNB2, SEMA3G and CXCR4. Accordingly, SOX18 transduced ASCs reorganize under conditions of shear stress, display VEGF-induced chemotaxis and form tubular structures in 3D matrices in an MMP7-dependent manner. These in vitro findings provide insight into molecular and cellular processes downstream of SOX18 and show that reprogramming using SOX18 is sufficient to induce several endothelial-like features in ASCs.

The human kinesin-like protein RB6K is under tight cell cycle control and is essential for cytokinesis.

Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.

Vascular endothelial genes that are responsive to tumor necrosis factor-alpha in vitro are expressed in atherosclerotic lesions, including inhibitor of apoptosis protein-1, stannin, and two novel genes.

Activation and dysfunction of endothelial cells play a prominent role in patho-physiological processes such as atherosclerosis. We describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by monocyte conditioned medium or tumor necrosis factor-alpha. A minority of the fragments (22/106) represent known genes involved in various processes, including leukocyte trafficking, vesicular transport, cell cycle control, apoptosis, and cellular protection against oxidative stress. Full-length cDNA clones were obtained for five novel transcripts that were induced or repressed more than 10-fold in vitro. These novel human cDNAs CA2_1, CG12_1, GG10_2, AG8_1, and GG2_1 encode inhibitor of apoptosis protein-1 (hIAP-1), homologues of apolipoprotein-L, mouse rabkinesin-6, rat stannin, and a novel 188 amino acid protein, respectively. Expression of 4 novel transcripts is shown by in situ hybridization on healthy and atherosclerotic vascular tissue, using monocyte chemotactic protein-1 as a marker for inflammation. CA2_1 (hIAP-1) and AG8_1 are expressed by endothelial cells and macrophage foam cells of the inflamed vascular wall. CG12_1 (apolipoprotein-L like) was specifically expressed in endothelial cells lining the normal and atherosclerotic iliac artery and aorta. These results substantiate the complex change in the gene expression pattern of vascular endothelial cells, which accompanies the inflammatory reaction of atherosclerotic lesions.

On the anomalous temperature behaviour of the EPR signal of monovalent nickel in hydrogenase.

The dependence on temperature in the range between 4.2 K and 20 K was measured for the EPR signal of monovalent nickel in H2-reduced hydrogenase from Chromatium vinosum and from Methanobacterium thermoautotrophicum. In accordance with measurements on the hydrogenase from Desulfovibrio gigas [Teixeira, M., Moura, I., Xavier, A. V., Huynh, B. H., DerVartanian, D. V., Peck, H. D., Jr, LeGall, J. and Moura, J. J. G. (1985) J. Biol. Chem. 260, 8942-8950; and Cammack, R., Patil, D. S. and Fernandez, V. M. (1985) Biochem. Soc. Trans. 13, 572-578], the enzyme from C. vinosum showed a distinct transformation of the EPR signal of nickel in this temperature region. The light sensitivity did not change. EPR spectra recorded at 9 GHz and at 35 GHz showed that the transformation of the spectrum at 4.2 K is caused by spin coupling to an unknown paramagnet. No coupling was apparent at temperatures above 20 K. At 4.2 K, additional, very broad signals in the region g= 1.2-3, as well as a signal around g = 5, were detected In the enzyme from C. Vinosum, both in the H2-reduced state and in the Ar-reoxidised state. The possible origin of the paramagnetic species responsible for these signals is discussed. The EPR signal of monovalent nickel in the enzyme from M. thermoautotrophicum showed no significant changes in line shape between 4.2 K and 70 K, nor were any additional signals detected. This suggests that in the reduced form of this enzyme similar paramagnetic species might be absent or not reduced.

Monovalent nickel in hydrogenase from Chromatium vinosum. Light sensitivity and evidence for direct interaction with hydrogen.

Redox titrations with hydrogenase from Chromatium vinosum show that its nickel ion can exist in 3, possibly 4, different redox states: the 3+, 2+, 1+ and possibly a zero valent state. The 1+ state is unstable: oxidation to Ni(II) occurs unless H2 gas is present. The Ni(I) coordination, but not that of Ni(III), is highly light sensitive. A photoreaction occurs on illumination. It is irreversible below 77 K, but reversible at 200 K. The rate of this photodissociation reaction in 2H2O is nearly 6-times slower than in H2O, indicating the breakage of a nickel-hydrogen bond. This forms the first evidence for an H atom in the direct coordination sphere of Ni in hydrogenase and for the involvement of this metal in the reaction with hydrogen.

Evolution of yeast ribosomal DNA: molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae.

We have studied the evolution of the yeast ribosomal DNA unit to search for regions outside the rRNA genes that exhibit evolutionary constraints and therefore might be involved in control of ribosome biosynthesis. We have cloned one complete rDNA unit of Kluyveromyces lactis and Hansenula wingei and established the physical and genetic organisation of both units. Both species belong to the subfamily of the Saccharomycetoideae. The lengths of the rDNA units of K. lactis and H. wingei are 8.6 and 11.1 kb respectively, and both comprise the 5S rRNA gene in addition to the large rRNA operon. Sequence conservation was monitored by restriction enzyme mapping as well as heteroduplex analysis of the two cloned rDNA units with S. carlsbergensis rDNA. These analyses showed that, phylogenetically, K. lactis is closer to S. carlsbergensis than H. wingei. The non-transcribed spacers (NTS) of both K. lactis and H. wingei have diverged completely from S. carlsbergensis; moreover in H. wingei the NTS are about double the length of these in the other two species. The transcribed spacers of both K. lactis and H. wingei contain conserved tracts. A homologous sequence of about 60 bp was found in the middle of the external transcribed spacer of H. wingei upon heteroduplexing with S. carlsbergensis rDNA, whereas the sequence at the transcription initiation site itself was insufficiently homologous to form a duplex. The sequence of the homologous region was determined both in H. wingei and K. lactis and compared with that of S. carlsbergensis. The function of this conserved element within the external transcribed spacer is discussed.

A conserved sequence element is present around the transcription initiation site for RNA polymerase A in Saccharomycetoideae.

To identify DNA elements involved in the initiation of rRNA transcription in yeast we located the start site of the rRNA operon of Kluyveromyces lactis and Hansenula wingei, both members of the Saccharomycetoideae, by S1 nuclease analysis and determined the surrounding nucleotide sequences. Comparison of these sequences with those of Saccharomyces carlsbergensis, S. cerevisiae and S. rosei (all belonging to the same yeast subfamily) reveals an identical sequence at the site of transcription initiation from position +1 to +7 which is part of a larger conserved region extending from position -9 to +23; the conserved heptanucleotide sequence is supposed to constitute an important part of the promoter for yeast RNA polymerase A. The non-transcribed spacers (NTS) upstream of position -9 have diverged strongly with the exception of two short elements around positions -75 and -135. The external transcribed spacer (ETS) downstream of position +23 is largely conserved between K. lactis, S. rosei and S. carlsbergensis except for a divergent region around position +75. On the other hand, the ETS of H. wingei has diverged significantly.