Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.     

Role of oxygen in oxidation of lipid and protein during ischemia/reperfusion in isolated perfused rat lung.

Considerable evidence has accumulated that oxygen free radicals play a major role in ischemic injury, particularly when followed by reperfusion. Few reports have demonstrated the occurrence of oxidative damage during the ischemic period, itself. Our laboratory has demonstrated that events occurring during an ischemic period with adequate oxygen supply can mimic the "oxygen paradox," using lipid peroxidation as an index of oxidative stress and lung edema as an index of tissue injury. The present study compares lipid peroxidation and oxidation of soluble (100,000g supernatant) protein during ischemia and reperfusion in isolated rat lung model perfused with artificial medium and ventilated with varying alveolar oxygen tension. Protein oxidation was determined by a modified dinitrophenylhydrazine (DNPH) method using Sephadex G-25 column chromatography to isolate the DNPH bound proteins. Global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. After 1 h ischemia in the isolated rat lung ventilated with 20% oxygen, protein carbonyls and thiobarbituric acid reactive substances (TBARS) increased significantly compared with controls. These changes were more pronounced after 60 min of reperfusion with 95% oxygen in the ventilation gas. With 0% oxygen (95% nitrogen and 5% CO2) content of the ventilating gas during ischemia, TBARS and protein carbonyls remained at the control level. The wet/dry weight ratio showed changes parallel to the indices of tissue oxidation. The presence of 5,8,11,14-eicosatetraynoic, an inhibitor of cyclooxygenase and lipoxygenase pathways, in the perfusate had no effect on the generation of protein carbonyls although inhibition of lipid peroxidation was demonstrated. This implies that the oxidation of soluble protein is not mediated by the eicosanoid metabolic cascade. These data indicate that oxidative processes occur during ischemia and are dependent on the alveolar oxygen concentration. Oxidation of soluble protein can be used as an index of oxidative damage during lung ischemia and reperfusion.     

Promotion of the in vitro renaturation of dodecameric glutamine synthetase from Escherichia coli in the presence of GroEL (chaperonin-60) and ATP.

The folding and assembly of dodecameric glutamine synthetase (GS) from Escherichia coli was examined in the absence and presence of the E. coli heat shock protein, GroEL (chaperonin-60). At nonphysiological temperatures (15-20 degrees C), unfolded GS spontaneously renatured to 80-90% of its original activity in the absence of GroEL. At near-physiological temperatures (37 degrees C), only 20-40% of the original activity returns. Under the latter solution conditions, GroEL and ATP enhance the extent of GS renaturation to 70-80% of the original activity at 37 degrees C. In the absence of ATP, GroEL arrests the renaturation of unfolded GS by forming a stable binary complex. The addition of ATP to this complex resulted in the release of GS subunits and formation of active dodecameric GS. The order of addition of ATP or unfolded GS to GroEL results in differences in the t1/2 values where half-maximal GS activity is attained. At a constant GS concentration, the formation of the GroEL.GS complex followed by ATP addition resulted in approximately a 2-fold increase in the observed t1/2 value compared to that observed when GroEL was preincubated with ATP before the GS renaturation reaction was initiated. These differences in renaturation rates may be related to binding affinity differences between the ATP-free and -bound GroEL conformer for unfolded or partially folded protein substrates [Badcoe, I. G., Smith, C. J., Wood, S., Halsall, D. J., Holbrook, J. J., Lund, P., & Clarke, A. R. (1991) Biochemistry 30, 9195-9200].(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and growth of Virola surinamensis yearlings: Water augmentation in gap and understory

Summary Factors affecting seedling Virola surinamensis (Myristicaceae) survival and growth were investigated on Barro Colorado Island, Panama. Seedlings planted 3 months after germination were monitored in treefall gaps and understory using 2.25 ha irrigated and control plots through the first dry season. During the dry season, irrigated plants in gaps increased total leaf area significantly more than did irrigated plants in the shaded understory. Over the same dry season, control plants in gaps and in the shaded understory lost similar amounts of leaf area. Seedlings in understory were suppressed in stem height and biomass in both irrigated and control plots; these measures were greater in gaps and greatest in irrigated gaps (height). Roots were similar in length in all treatments, but greater in biomass in gaps than understory due to greater proliferation of secondary roots in control and irrigated gaps than in control and irrigated understory. This experiment demonstrates both water and light limitation during the first dry season after germination. V. surinamensis seedlings are capable of survival and modest growth of leaf area in the deep shade of the understory in moist locations; they are severely disadvantaged in shaded understory subject to drought, where most seeds fall and most seedlings establish. The broken canopy of a gap allows shoot and consequently root growth that permits seedlings to survive seasonal drought.     

Expression of the Blast-1 activation/adhesion molecule and its identification as CD48.

We have analyzed the induction and expression of Blast-1 at the mRNA and protein levels and demonstrated its identity with CD48. Blast-1/CD48 is expressed on a wider range of cell types, notably T cells and monocytes, than previously thought, but appears to be restricted to lymphoid and myeloid cells. Resting B and T cells express Blast-1/CD48 molecules at the cell surface; however, they lack the epitope recognized by the 17D6 mAb. Resting B cells express no detectable Blast-1/CD48 mRNA. Induction by EBV infection or stimulation with PMA, IL-4, or PHA results in increased levels of Blast-1/CD48 protein (both 6.28 and 17D6 epitopes) at the cell surface. Detailed analysis of EBV-induced expression revealed that it is due to increased steady-state levels of Blast-1/CD48 mRNA induced by transforming but not nontransforming strains of the virus. Induction by IL-1 beta, ionomycin, or suboptimal levels of PMA plus ionomycin results in increased expression of the 17D6 epitope only. In transfected Cos-7 cells Blast-1/CD48 at the cell surface expresses only the 6.28 epitope, whereas cytoplasmic molecules express both 17D6 and 6.28 epitopes. We suggest that these results are most consistent with the idea that Blast-1/CD48 molecules are complexed at the surface of resting cells and Cos-7 cells, resulting in masking of the 17D6 epitope. Activation causes dissociation of the complex, revealing the 17D6 epitope. The existence of 17D6+6.28- Blast-1/CD48 molecules was demonstrated by immunoprecipitation analysis, which also revealed that, unlike the rest of the molecules, this subset was resistant to digestion with glyosylphosphatidylinositol-specific phospholipase C.     

Identification of Multiple Phosphoinositide-Linked Receptors on Human SK-N-MC Neuroepithelioma Cells

The biochemical and pharmacological characteristics of receptor-stimulated phosphoinositide (PPI) hydrolysis in human SK-N-MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., norepinephrine, oxotremorine-M, endothelin-1, and ATP, each resulted in an increased release (three- to eightfold) of inositol phosphates from [3H]inositol-prelabeled cells. Agonist-stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono- and bisphosphates, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate. Increases in inositol 1,4,5-trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine-M. Norepinephrine-stimulated PPI turnover was potently inhibited by prazosin, WB-4101, and 5-methylurapidil (Ki less than 2.5 nM), but was relatively insensitive to chlorethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an alpha 1A-receptor subtype. The presence of an M1 muscarinic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine-M-stimulated inositol phosphate release (Ki = 35 nM) with a 30-fold greater potency than the M2-selective antagonist, AF-DX 116. Of the three endothelins tested, only the addition of endothelin-1 and endothelin-2 promoted PPI hydrolysis, whereas endothelin-3 was essentially inactive. A P2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK-N-MC cells express at least four PPI-linked receptors. Because the functional coupling of three of these receptors, i.e., alpha 1A-adrenergic, endothelin, and P2 nucleotide, has not been extensively characterized previously in neural tissues, the SK-N-MC cell line may provide a useful model system for studies of these receptors and their regulation.     

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120).

Pneumocystis carinii gp120 can elicit a specific T-cell proliferative response in mice after immunization with a gp120 preparation or with a crude P. carinii homogenate. It can also elicit a proliferative response from SCID mice after recovery from natural infection with P. carinii, implicating this glycoprotein as an important antigen in the host's response to P. carinii infection.     

First World Congress of Cellular and Molecular Biology

Announcement

First World Congress of Cellular and Molecular Biology