Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Relative importance of size versus territorial residency in intraspecific aggression by symbiotic male water mites (Acari: Unionicolidae)

Body size plays an important role in the outcome of aggressive encounters between males of the symbiotic freshwater miteUnionicola formosa. Since intruding males displace resident males as often as resident males successfully defend their territory (mantle cavity of a mussel), resident males do not hold a competitive advantage during male-male aggressive encounters. Larger male size probably enhances a male's success when competing for receptive females.     

Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of X-rays

The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.     

Isolation and identification of seven metabolites of 25-hydroxydihydrotachysterol3 formed in the isolated perfused rat kidney: a model for the study of side-chain metabolism of vitamin D

The in vivo metabolism of dihydrotachysterol3, an analogue of vitamin D3 and a potent calcemic factor, has been studied in the rat. This in vivo metabolism is compared to the in vitro metabolism of 25-hydroxydihydrotachysterol3 in the perfused rat kidney. Using mass spectrometry and ultraviolet spectroscopy, we have identified seven novel metabolites derived from 25-hydroxydihydrotachysterol3. The seven compounds represent intermediates on two renal pathways (24-oxidation and 26,23-lactone formation) also observed for 25-hydroxyvitamin D3. No evidence was found for the renal synthesis of a 1-hydroxylated metabolite of 25-hydroxydihydrotachysterol3 analogous to the hormone 1,25-dihydroxyvitamin D3. Two of the compounds formed in vitro, 24,25-dihydroxydihydrotachysterol3 and 25-hydroxydihydrotachysterol 26,23-lactone, were also formed in vivo. In vivo studies also revealed the formation of two other unidentified metabolites which are presumed to be formed nonrenally and may be calcemic factors. This work shows that dihydrotachysterol3 metabolism is complex and probably utilizes the same side-chain enzymes as vitamin D3. In addition, our work also confirms that intermediates postulated to lie on pathways to 26,23-lactone in the vitamin D3 series are also formed for the side chain in dihydrotachysterol3.     

Developmental modulation of a glial cell-associated glycoprotein, 5B12, in an insect, Acheta domesticus

The expression of an insect (Acheta domesticus) adult glial cell-specific antigen, 5B12 undergoes major changes during development. The 5B12 antigen is detected as early as 20-25% of embryonic development, when immunoreactivity is distributed throughout the periphery, present at the luminal surface of epithelial cells which compose developing limb buds, sensory appendages, and the body cavity. The antigen is also localized on the cell surface of neural elements within commissural tracts in the embryonic CNS. 5B12 is secreted extracellularly in the periphery, where it is associated with the embryonic basal lamina in developing cercal sensory appendages. Luminal surface expression is transient, and disappears by 95% of embryonic development. As development proceeds, 5B12 distribution becomes more restricted, so that in the adult the antigen is predominantly associated with specific glial elements within the nervous system where it occurs as a specialized component of the extracellular matrix. The 5B12 antigen is also associated with discrete central and peripheral fiber tracts. Antigen 5B12 is present in whole embryos and in the adult CNS as a Mr 185-kDa glycoprotein. Distinct carbohydrate moieties with chondroitin sulfate-like properties are situated on the 5B12 epitope. Thus the glia-associated 5B12 macromolecule has the characteristics of a small proteoglycan. Based upon features of its distribution, pattern of spatiotemporal expression, and biochemical properties, it is speculated that 5B12 participates in events related sequentially to the development and the function of the insect nervous system.     

Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.     

The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.