Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human ^Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin ^Aγ-globin (βsp/^Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/^Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the ^Aγ-globin gene promoter showed developmental switching and expressed ^Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.     

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes

Summary Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells. Morphologically the cells were 20–30 μm in diameter and exhibited contact inhibition. The cells were not myeloid in origin because they lacked sodium fluoride-inhibitable nonspecific esterase activity, myeloperoxidase activity, and F4/80 antigen. The cell line phenotypes were compared to high endothelial venule (HEV) cells in tissue sections. HEV cells in lymph node tissue sections expressed endoglin, PECAM-1, ICAM-1, VCAM-1, laminin, fibronectin, collagen IV, H2K^d, MECA 79, MECA 325, and vWF. The cell lines expressed endoglin, VCAM-1, fibronectin, and H2K^d. The cell line derived from cervical lymph nodes also expressed laminin and H2D^d. Neither cell line expressed collagen IV, IA^d, ICAM-1, ICAM-2, dendritic cell antigen, or PECAM-1. They also did not express MECA antigens or intracellular vWF, consistent with reports of many cultured endothelial cells. To further substantiate cell line identification, antiserum generated against the cell lines bound specifically to HEV cells in frozen lymph node tissue sections and to both of the lymph node-derived cell lines but not control cell lines. Thus, the lymph node derived-cell lines expressed molecules found on HEV cellsin vivo and most importantly retained the functions of tubule formation, lymphocyte adhesion, and promotion of lymphocyte migration.     

Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.     

Studies of the growth of human bone-derived cells in culture using aqueous two-phase partition

Human bone cells, maintained in culture, have been subjected to partitioning in an aqueous two-phase system on a countercurrent distribution apparatus. A broad cell distribution was obtained indicating cell-surface heterogeneity. Two major cell populations were identified which appeared to be growing at different rates. The fast-growing cells had a less hydrophobic cell surface than the slow-growing cells. Possible relationships of these cell populations with osteoblast differentiation and the potential importance of this technique in studies of osteoblast differentiation are discussed.     

Effect of infusion of uterine purulent exudate into the bovine uterus

Purulent exudate from the uteri of two nonlactating Holstein cows with chronic pyometra was aspirated into a 50 cc syringe via an artificial insemination pipette. The two fractions were mixed, cultured, divided into 14 equal parts and infused into the uteri of 14 other nonlactating cows. All recipient cows had had at least one estrous cycle of normal length and were in the early luteal phase (day 4-8). On days 3, 7, 14 and 21 following infusion, the reproductive tract was examined per rectum, content of the uterus was cultured for bacteria, and endometrial condition was assessed by biopsy and histopathology. The lengths of the estrous cycles of the recipient cows were shortened P < 0.01) from 21 +/- .5 days to 13.3 +/- 1.4 days. Estrus was determined by palpation per rectum and/or observation. Over the experimental period, Pasteurella sp., Corynebacterium pyogenes and other diphtheroids were isolated from 64, 57 and 86% of the cows, respectively. An inflamed endometrium was associated (P<0.10) with the presence of Pasteurella sp. on day 7 following infusion. No other associations were found when the presence of bacteria, vaginal discharge, or short estrous cycles were compared. The infusion of purulent exudate apparently irritated the endometrium causing an early return to estrus rather than causing pyometra. It is probable that postpartum and lactational stress are involved in the development of pyometra.     

Gore-Tex small-vessel angioplasty: A suitable substitute for the use of autogenous saphenous vein grafts

Autogenous saphenous vein has been the material of choice for small-vessel angioplasty and for circulatory access graft reconstruction. In an effort to conserve autogenous saphenous vein, we used expanded polytetrafluoroethylene (PTFE) grafts in 45 patients over a 12-month period. We used Gore-Tex(*) to reconstruct 17 circulatory access grafts, 16 carotid arteries, two brachial arteries, seven femoral arteries, and three popliteal anterior or posterior tibial arteries. The indications for reconstruction were chronic occlusion of the access grafts, trauma to the brachial and anterior tibial arteries, and atherosclerotic disease of the carotid, femoral, and popliteal-tibial arteries. Of the reconstructed circulatory access grafts, one failed immediately because of technical problems in the conduit, and one failed 11 months after reconstruction. All other grafts have functioned well and have produced a marked improvement in flow. Of the 28 patients who underwent reconstruction of arteries measuring 3 mm or less, two had patent arteries but died shortly after operation. The remaining 26 have been followed for one to 43 months. All reconstructed arteries are patent, and there have been no instances of distal embolization or false aneurysm formation. From this brief experience, we conclude that Gore-Tex is a suitable short-term alternative to saphenous vein for small vessel arterioplasty; it also may be the material of choice for reconstructing the outflow tract of occluded access grafts.