Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.      

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Cyclic AMP as an intracellular messenger of gonadotrophin-induced steroidogenesis during the development of the embryonic chick ovary

(1) Ovine luteinizing hormone (LH) stimulates cyclic AMP (cAMP) and progesterone production (P) throughout late ontogeny of the chick ovary and cAMP mimicks LH in stimulating P secretion but: (2) P/cAMP ratios are lower at the earliest stages than at hatching, LH enhancing this tendency. (3) Immediately before hatching, on day 19 time-courses of LH stimulations of cAMP and P are different. (4) cAMP and P respond differently to increasing doses of LH but similarly to increasing doses of forskolin. (5) 1.5 mM dibutyryl cAMP (I) and 100 ng LH (II) increase P maximally, 2.5- and 3.3-fold respectively, but a mixture (I + II) increases P 7.5-fold.     

Azospirillum sp. Promotes Root Hair Development in Tomato Plants through a Mechanism that Involves Ethylene

Tomato seeds were inoculated with the plant growth–promoting rhizobacteria Azospirillum brasilense FT326, and changes in parameters associated with plant growth were evaluated 15 days after inoculation. Azospirilla were localized on roots and within xylematic tissue. An increase in shoot and root fresh weight, main root hair length, and root surface indicated that inoculation with A. brasilense FT 326 resulted in plant growth improvement. The levels of indole-3-acetic acid (IAA) and ethylene, two of the phytohormones related to plant growth, were higher in inoculated plants. Exogenously supplied ethylene mimicked the effect of inoculation, and the addition of an inhibitor of its synthesis or of its physiological activity completely blocked A. brasilense growth promotion. Based on our results, we propose that the process of growth promotion triggered by A. brasilense inoculation involves a signaling pathway that has ethylene as a central, positive regulator.     

Effect of salt stress on growth parameters, enzymatic antioxidant system, and lipid peroxidation in wild chicory (Cichorium intybus L.)

Efficient utilization of saline land for food cultivation can increase agricultural productivity and rural income. To obtain information on the salt tolerance/susceptibility of wild chicory (Cichorium intybus L.), the influence of salinity (0–260 mM NaCl) on chicory seed germination and that of two salinity levels of irrigation water (100 and 200 mM NaCl) on plant growth, antioxidative enzyme activity, and accumulation of proline and malondialdehyde (MDA) were investigated. The trials were performed outdoors, in pots placed under a protective glass covering, for two consecutive years. Seeds showed a high capacity to germinate in saline conditions. The use of 100 mM NaCl solution resulted in 81 % germination, whereas seed germinability decreased below 40 % using salt concentrations above 200 mM NaCl. Wild chicory showed tolerance to medium salinity (100 mM NaCl), whereas a drastic reduction in biomass was observed when 200 mM NaCl solution was used for irrigation. MDA, present in higher amounts in leaves than in roots, decreased in both tissues under increasing salinity. Proline content increased remarkably with the level of salt stress, more so in roots than in leaves. In salt stress conditions, the activity of antioxidant enzymes (APX, CAT, POD, SOD) was enhanced. The electrophoretic patterns of the studied enzymes showed that the salinity of irrigation water affected only the intensity of bands, but did not activate new isoforms. Our results suggest that wild chicory is able to grow in soil with moderate salinity by activating antioxidative responses both in roots and leaves.