Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.     

Purification, characterization and some properties of diacetyl(acetoin) reductase from Enterobacter aerogenes

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem, 18, 116-119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces L-glycols from the corresponding L-alpha-hydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged alpha-dicarbonyls converting them into L-alpha-hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of L-glycol: NAD+ oxidoreductase and the recommended name of L-glycol dehydrogenase(NAD). The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described.     

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.     

An embryo-specific protein of barley (Hordeum vulgare).

An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 microM) or mannitol (9% mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structure.     

High species richness in an Estonian wooded meadow

Abstract. A wooded meadow at Laelatu in western Estonia was found to be very rich in vascular plants. The maximum number of species found was 25 in a 10 cm x 10 cm plot, 42 in a 20 cm x 20 cm plot and 63 in a 1 m² plot. Species richness is related to the management history of the site. The highest richness was found in sites with the most regular long-term mowing. Species density was lower in the case of fertilization or temporary cessation of mowing. The richest community had an LAI of 2.8 and an above-ground dry biomass of 175 g/m². With increasing height of the herb layer the number of species decreases. Small disturbances cause a remarkable decrease in species density.     

A remnant New Zealand carr

Carr vegetation was once extensive in New Zealand. It can be divided into Cordyline australis I Carex secta carr, comprising an open wood with scattered large herbs and an abundance of Carex species, and podocarp carr, dominated by tall conifers. Both have been almost eliminated by agricultural development. We studied a remnant of Cordyline australis I Carex secta carr in South Island, which graded at one end into salt marsh. Eight communities were recognized, including one pure salt marsh and two more with saline influence. Near the stream that provided moisture and alluvium for the area were herbaceous communities. In the poorly-drained area away from the stream were communities dominated by shrubs and small trees. The soil environment is similar to that of many European carrs, though generally less organic. A number of the exotic species are also found in European carrs.     

Vegetation of a coastal sand dune system in southern New Zealand

The introduction of vigorous exotic species has destroyed much of the native sand dune vegetation along the coasts of South Island, New Zealand. However, some dunes built by the native cyperad Desmoschoenus spiralis still remain in the southwest. The dunes at Cole Creek, on the West coast of South Island, were chosen as a relatively pristine study area. 15 transects were laid through semi-fixed dunes into dwarf forest. Environmental measurements were taken at regular intervals through the dunes: pH, soil moisture, organic matter, conductivity, nutrient status and elevation. Classification and ordination of these data demonstrated that two major environmental areas were present - open dune and dwarf forest - with an abrupt ecotone between them. Vegetation analyses revealed a loosely-banded pattern, parallel to the sea. Vegetation types with few but constant species such as Desmoschoenus spiralis and Calystegia soldanella, predominated in the open dune. Forest species were rarely found seaward of the dune/forest boundary, though there was evidence the Spatial Mass Effect was operating. Multiple regression and canonical correlation of the vegetation and environmental factors showed that the main factors affecting vegetation patterns were the environmental complex related to distance from the sea, elevation above mean tide, soil alkalinity and moisture.     

Impacts of annual weather conditions on forest productivity

The response of four northern deciduous tree species to annual climate variation is quantified at two intensively measured sites in northern Michigan, USA. Response to changes in temperature and moisture differ with the species and is dependent on other site conditions. Relationships identified in these field studies indicate that projected climate changes may have dramatic effects on the productivity of at least some commercially important tree species in the northern United States.     

Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production.

The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.