Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein.

Increasingly, enriched populations of hematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C-terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosylated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an Ig fusion protein and used as an immunogen to generate a rabbit polyclonal antiserum. The antiserum reacted against the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony-forming units-granulocyte-macrophage as compared to unfractionated marrow mononuclear cells. These findings suggest that the canine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation.

Effects of rhIL-11 on normal dogs and after sublethal radiation.

The effects of recombinant human interleukin-11 (rhIL-11) were studied in normal dogs and dogs given otherwise sublethal total-body irradiation (TBI) without marrow transplantation. Ten normal dogs were given rhIL-11 subcutaneously, twice daily for 14 days at varying doses, two dogs at 30 micrograms/kg/day, four dogs at 60 micrograms/kg/day, two dogs at 120 micrograms/kg/day, and two dogs at 240 micrograms/kg/day. Peripheral blood platelet counts increased in all dogs. The increase in platelet counts ranged from 1.4 to 3.1 times the pre-treatment level. The greater increases of platelets were associated with higher doses (p = 0.01). No change in platelet size was evident except at the dose of 240 micrograms/kg/day. There were no changes in the total white blood cell (WBC) count or differential. A higher proportion of megakaryocytes with a DNA content of 32N/64N was observed in dogs treated with rhIL-11 at day 7 (n = 6) than for control dogs that did not receive rhIL-11 (n = 7; p = 0.01). In both peripheral blood and marrow, significantly increased hematopoietic progenitors (i.e, colony-forming unit granulocyte/macrophage [CFU-GM]) were present 7 and 14 days after the start of treatment. Concentrations of serum fibrinogen increased by a median of 155 mg/dL at day 7 of rhIL-11 (p < 0.01). Cholesterol also increased by a median of 52 mg/dL at day 14 (p < 0.01). There was a single death of a non-irradiated dog from pneumonitis on day 15 after the start of rhIL-11 administration at a dose of 120 micrograms/kg/day. All other non-irradiated dogs tolerated rhIL-11 without any significant adverse effects. Five dogs were given 200 cGy TBI without marrow grafting, followed by 240 micrograms/kg/day rhIL-11 subcutaneously in two divided doses for 28 days starting within 2 hours of TBI. The results in this group were compared with 10 dogs that had previously or concurrently been given 200 cGy without marrow grafting or hematopoietic growth factors. Two of the five treatment dogs died of pneumonitis on day 13 compared to one death among 10 control dogs on day 24. Among dogs that survived to hematologic recovery, the rhIL-11 dogs had decreased platelet counts (< 150,000) for a median of 24 days (range = 24 to 41) compared to a median of 28 days (range = 21-40) for the control group. Treatment with rhIL-11 increased platelet counts, platelet size, ploidy number of megakaryocytes, and marrow and peripheral blood CFU-GM in normal dogs.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber.

The Ca2+ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca2+-activated tension and relaxation. Pretreatment of the skinned fibers with AT-P gamma S and high Ca2+ (10(-5)M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca2+ insensitive tension. In contrast, pretreatment with low Ca2+ (10(-8)M) and ATP gamma S results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca2+-sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca2+-activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP-dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca2+-activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.

Regulation of Ca2+-activated tension in limulus striated muscle.

Skinned tail and leg muscle fibers of the limulus were used to study the mechanism of Ca2+ regulation of contraction. Although a Ca2+-sensitive 31,000 dalton protein phosphorylation could be observed in the presence of [gamma-32P] ATP no such phosphorylation occurred in the presence of [gamma-32P] ITP. Ca2+-activated tension occurred equally as well in ATP and ITP. For this reason we eliminated the possibility that a Ca2+-sensitive myosin light chain kinase/phosphatase system is the mechanism responsible for the Ca2+-activated tension. Other agents known to affect a myosin light chain kinase/phosphatase system showed negative results (ATP gamma S, trifluoperazine, catalytic subunit of the cyclic adenosine 3',5'-monophosphate dependent protein kinase and calmodulin). Troponin I reversibly inhibits Ca2+-activated tension. These results are consistent with thin filament regulation being responsible for Ca2+-activated tension in skinned fibers.

Calcium-regulatory mechanisms. Functional classification using skinned fibers.

The primary purpose of this study was to determine whether various agents (adenosine 3-thiotriphosphate [ATP gamma S], trifluoperazine [TFP], troponin I, the catalytic subunit of the cyclic adenosine 3',5'-monophosphate dependent protein kinase [C-subunit], and calmodulin [CaM]) could be used to classify skinned fiber types, and then to determine whether the proposed mechanisms for Ca2+ regulation were consistent with the results. Agents (ATP gamma S, TFP, C-subunit, CaM) expected to alter a light chain kinase-phosphatase system strongly affect the Ca2+-activated tension in skinned gizzard smooth muscle fibers, whereas these agents have no effect on skinned mammalian striated and scallop adductor fibers. Troponin I, which is known to bind strongly to troponin C and CaM, inhibits Ca2+ activation of skinned mammalian striated and gizzard fibers but not scallop adductor muscle. The results in different types of skinned fibers are consistent with proposed mechanisms for Ca2+ regulation.