Effects of inhibitors and activators of protein kinase C on late erythroid progenitor (CFU-e) colony formation in vitro

Protein kinase C (PKC) plays a central role in external signal transduction for many cell types. To examine the involvement of PKC in the control of erythropoiesis, we tested the effects of PKC inhibitors on in vitro colony formation by late erythroid progenitors (CFU-e) from normal and Friend virus-infected mice. Inhibitors of PKC and other kinases (H-7 and H-8) inhibited CFU-e at concentrations which inhibit PKC. HA1004, an inhibitor of the cyclic nucleotide-dependent kinases and a weak inhibitor of PKC, had little effect on CFU-e. In the absence of erythropoietin, a combination of phorbol ester and Ca++ ionophore significantly increased normal CFU-e. These results suggest PKC plays a role in the transduction of regulatory signals for the growth of CFU-e.     

Effect of gallium nitrate in vitro and in normal rats

Gallium nitrate (GN) is an inhibitor of bone resorption and thereby may result in a change in coupled bone formation. In the present investigation the effects of GN on bone formation were studied in the rat osteosarcoma (ROS) 17/2.8 cell line and normal diploid rat osteoblasts (ROB) in vitro and the femur of rats treated in vivo, measuring mRNA levels for two osteoblast parameters, type I collagen, a marker of matrix formation, and osteocalcin, a bone specific protein and also histone H₄, a marker of cell proliferation. GN, at 50 μM for 3 h, increased type I collagen mRNA levels by 132% in ROS 17/2.8 cells and by 122% in proliferating ROB cells. Osteocalcin (OC) mRNA levels were decreased by 61% in ROS 17/2.8 cells and by 97% in differentiated ROB cells. These changes occurred in the absence of any effects on cell proliferation. Seventy-day-old female rats were then treated with GN, 0.5 mg/kg/day, for 3 weeks. As previously reported, GN decreased serum calcium levels, but had no effect on lumbar or femoral bone density. In contrast to the in vitro effects, GN had no effect on type I collagen steady-state mRNA levels in the femur; however, it decreased OC steady-state mRNA levels in the femur by 58%. These results suggest that GN has similar in vitro effects in transformed and normal osteoblasts, while the collagen-stimulatory effects observed in vitro cannot be extrapolated to in vivo models. The consistent inhibition of osteocalcin in vitro and in vivo suggests a more specific target for GN that may relate to its effects in inhibiting bone resorption in normal rats.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

Isolation and characterization of antigen-Ia complexes involved in T cell recognition

Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is herein demonstrated that the complexes, once formed, are very stable (kd approximately equal to 3 X 10(-6) s-1), but the rate of complex formation is very slow (ka approximately 1 M-1 s-1 explaining the overall low equilibrium constant of approximately 2 X 10(-6) M. Treating the complexes with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance of these complexes in antigen recognition.     

1α,25-Dihydroxyvitamin D3-induced changes in intracellular pH in osteoblast-like cells modulate gene expression

1α,25-Dihydroxyvitamin D₃ exerts rapid nongenomic effects on rat osteoblast-like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1α,25-dihydroxyvitamin D₃ on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1α,25-Dihydroxyvitamin D₃ increased intracellular pH within 10 min in rat osteoblast-like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1β,25-dihydroxyvitamin D₃. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1α,25-dihydroxyvitamin D₃-induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium-free medium. The results indicate that 1α,25-dihydroxyvitamin D₃-induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady-state mRNA levels of genes induced by vitamin D.     

鞑靼滨藜挥发性化学成分分析

对鞑靼滨藜全草的挥发性化学成分进行分析。采用水蒸气蒸馏法及索氏提取法提取鞑靼滨藜全草中的挥发性化学成分,用GC-MS联用技术分析其化学组成,计算其相对百分含量,并对两种提取方法进行比较研究。结果表明:水蒸气蒸馏法提取物的成分比索氏提取法较多。从水蒸气蒸馏法分离出的53个色谱峰中鉴定了51种成分,含量最高的是茴香脑(45.84%),其次为正二十七烷(9.28%),羟基吲哚(6.81%),α-羟基-2-甲基丙基苯乙酸酯(4.03%)。从索氏提取法分离出的26个色谱峰中鉴定了22种成分,含量最高的是正二十九烷(22.63%),其次为硬酯酸(16.69%),十六醛(6.89%),棕榈酸(5.43%)。     

鞑靼滨藜地上部分化学成分的研究

从鞑靼滨藜（Atriplex tatarica）首次分离得到6个化合物,通过理化及波谱分析,它们分别确定为苜素（tricin,1）,苜蓿素-7-O-β-D-吡喃葡萄糖苷（tricin 7-O-β-D-glucopyranoside,2）,异鼠李素（isorhanmetin,3）,异鼠李素．3-芸香糖甙（Isorhamnetin-3-rutinoside,4）,豆甾醇（stigmasteml,5）,β-谷甾醇（β-sitosterol,6）。     

Lectin receptor proximity on HL-60 leukemia cells determined by fluorescence energy transfer using flow cytometry

Fluorescence energy transfer using flow cytometric measurements was utilized to determine the proximity of concanavalin A receptors on the surface of HL-60 promyelocytic leukemia cells before and after induction of differentiation. The HL-60 cells were induced to differentiate into granulocytes using dimethylsulfoxide and into macrophages using 12-O-tetradecanoylphorbol-13-acetate. Concanavalin A was labeled with either fluorescein (donor chromophore) or tetramethylrhodamine (acceptor chromophore), and these species were used to determine lectin proximity. With granulocytic differentiation, the amount of concanavalin A bound remained constant, but a decrease in receptor density was observed. During macrophage differentiation, however, both receptor density and receptor number increased. The increase in concanavalin A binding during differentiation appears to be a result of maturation rather than an initiating event.