Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.     

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA polymerase delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA polymerase delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA polymerase activity. Western blotting of rabbit DNA polymerase delta with polyclonal antibody to calf thymus PCNA-dependent DNA polymerase delta gives the same results as calf thymus delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA polymerase delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA polymerase. The processivity of DNA polymerase delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA polymerase delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA polymerase delta is a single polypeptide of 122 kDa. These features would recategorize the original delta to the epsilon category by recently proposed convention. PCNA-dependent DNA polymerase delta is a relatively minor component of rabbit bone marrow compared to DNA polymerase alpha and PCNA-independent DNA polymerase delta (epsilon), the relative proportions being alpha, 60%; delta, 7%; and epsilon, 30%.     

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit

Different fractions of cellular RNA from erythroid enriched bone marrow cells of the rabbit, extracted by the temperature fractionation method, were investigated by hybridization to globin cDNA. 97.4% of all globin sequences were found in the 4 degrees C franction (cytoplasmic RNA) 0.11% are in the 40 degrees / 50 degrees C fraction and 2.47% in the 65 degrees C and 85 degrees C franctions (pre-mRNA). This shows a substantial purification of the pre-mRNA fractions from cytoplasmic mRNA. 33% of the globin sequences in the 65 degrees C and 85 degrees C fractions are polyadenylated. The poly(A)+-RNA from the 65 degrees C and 85 degrees C fractions separated in a formamide sucrose gradient showed a clear hybridization to globin cDNA in the region between 9S and 28S and around 4S. In a control experiment in which RNA from baby hamster kidney cells (BHK) was mixed with globin mRNA and separated in the same manner hybridization was observed at the 9S position of the gradient only.     

Elevated cyclic GMP concentrations in rabbit bone marrow culture and mouse spleen following erythropoietic stimulation.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.     

Electron microscopy study of the aminoacyl-tRNA synthetase multienzymatic complex purified from rabbit reticulocytes

The morphology of the high molecular weight complex of aminoacyl-tRNA synthetases purified from rabbit reticulocytes has been investigated by electron microscopy. To stabilize it against dissociation, the complex was also studied after chemical crosslinking. Freeze fracture, drying shadowing and negative staining were used. The reticulocyte complex appears as a moderately elongated object with no simple compact shape. Upon rapid drying, the native complex dissociates and shows the presence of approximately 8 globular components, the individual size of which is 80–100 Å. The surface of the cross-linked complex shows several distinct globules which appear to extend out of a central core. The irregularly shaped crosslinked complex has a maximal dimension of 350±50 Å. The morphology of the synthetase complex is discussed with respect to some of the properties of this type of multienzymatic system.     

Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts.

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 X 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.     

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.