Supplementary Figure 1 from Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis
Abstract The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product wsa sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow λgt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5′ and 3′ untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon‐inducible genes: 204 and interferon regulatory factor 2. In addition, a 12‐base sequence matching the interferon‐stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5′ untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon α. The MNDA mRNA was not induced by interferon α in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell‐specific response to interferon.
We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage.
A newly designed Falling Weight Deflectometer (FWD) has been developed that significantly speeds up the testing process and overall productivity of the device. The new device is currently being referred to as the Fast FWD or FFWD for short. The speed of the device, and the fact that it is capable of imparting full scale truck wheel loads to the pavement surface suggested that it might be a viable tool for conducting accelerated pavement testing experiments. Two pavement test sections were tested to failure with the FFWD. Preliminary tests show that the FFWD can be used as an intermediate tool, between the small scale laboratory tests (bending beam, shear, and triaxial tests) and full scale accelerated pavement tests.
Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins. In addition, a Mr 62,000 protein reacted only with B-SWGA in the nuclear matrix fractions from normal rat liver and the induced hepatoma. A polypeptide band (approximate molecular weight, 213,000) in the Walker 256 NRF reacted with concanavalin A and biotinylated ricinus communis agglutinin. One polypeptide band (approximate molecular weight, 182,000) reacted with concanavalin A in all three tissues, with biotinylated ricinus communis agglutinin and B-SWGA in the Walker NRF, and with B-SWGA in the hepatoma NRF. Another polypeptide band (approximate molecular weight, 138,000), reactive with all three lectins, was present in all three tissues. Our findings are consistent with previous reports of lectin binding proteins in the eukaryotic cell nucleus and indicate that certain glycoproteins isolated in nuclear preparations are found specifically in 3'-MeDAB-induced hepatoma and Walker 256 transplantable carcinosarcoma.
