Small-angle X-ray scattering studies of fibrin film: comparisons of fine and coarse films prepared with thrombin and ancrod

Measurements of small-angle x-ray scattering have been made on films prepared from fine and coarse (i.e., formed at high and low, respectively, pH and ionic strength) clots of bovine fibrin by osmotic shrinkage or compression in one dimension. Intensity profiles were obtained with pinhole geometry on films stretched up to a stretch ratio of 1.43. In unstretched coarse films, repeat spacings were seen at about 245, 120, and 77-80 A. These peaks can probably be identified with the first, second, and third orders of the well-known fibrin repeat of 225 A. In unstretched fine films, only the 77-80 A spacing was seen. In this case, the first two orders may be weak because the half-staggered arrangement of monomer units giving rise to the 225 A reflection is not reinforced by lateral aggregation of protofibrils; the third order may be strong since the molecular subdomains appear to divide the repeat roughly into thirds. After stretching, the 77-80 A spacing persisted in the meridional direction but almost disappeared in the equatorial. Experiments on unstretched films prepared with ancrod substituted for thrombin gave similar results.     

N-iodoacetyltyramine: preparation and use in 125I labeling by alkylation of sulfhydryl groups

Preparation and use of N-iodoacetyltyramine in generation of 125I-labeled compounds is described. The kinetics of alkylation of N-acetylcysteine by N-iodoacetyltyramine (k2 = 3.0 M-1 s-1) and N-chloroacetyltyramine (k2 = 0.12 M-1 s-1) indicate that N-iodoacetyltyramine is more useful for labeling sulfhydryl-containing compounds to high specific activity with 125I. Conditions for preparation of carrier-free 125I-labeled N-iodoacetyl-3-monoiodotyramine in 50% yield based on starting iodide are described. The high degree of group specificity of N-iodoacetyl-3-monoiodotyramine reaction with sulfhydryl groups is demonstrated by the high reactivity toward sulfhydryl-containing bovine serum albumin and low reactivity toward N-ethylmaleimide-blocked bovine serum albumin and IgG. 125I-labeled N-iodoacetyl-3-monoiodotyramine was also used to prepare an 125I-labeled ACTH derivative that retains full biological activity, further demonstrating the selectivity toward reactions with sulfhydryl groups.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Studies of blocking mechanisms of carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes by neuroactive drugs

1. The incorporation of 32Pi into 4 phospholipids of rat cortical synaptosomes was altered in the presence of carbachol (1 mM), viz. a decrease of phosphatidylinositol-4,5-bisphosphate and phosphatidyl-4-phosphate by 34 and 21%, and an increase of phosphatidylinositol and phosphatidic acid by 52 and 96% of basal controls respectively. 2. The IC30 values calculated from the dose-response curves for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and [3H]QNB binding to the cortical synaptosomes were similar for the typical antimuscarinic agents (i.e. atropine, pirenzepine and trihexyphenidyl), and tricyclic antidepressants (i.e. amitriptyline, doxepin and imipramine) studied. 3. The IC30 values obtained for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and high potassium-induced 45Ca2+-uptake by this preparation were similar for neuroselective calcium channel blockers (i.e. cinnarizine and flunarizine) studied. 4. Our results suggest that the neuroactive drugs studied can either act at, or beyond the receptor level, perhaps on the availability of calcium ion, to block carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes.     

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.     

The insulin A and B chains contain structural information for the formation of the native molecule. Studies with protein disulphide-isomerase.

It has been shown previously [Tang, Wang & Tsou (1988) Biochem. J. 255, 451-455] that, under appropriate conditions, native insulin can be obtained from scrambled insulin or the S-sulphonates of the chains with a yield of 25-30%, together with reaction products containing the separated A and B chains. The native hormone is by far the predominant product among the isomers containing both chains. It is now shown that the presence of added C peptide has no appreciable effect on the yield of native insulin. At higher temperatures the content of the native hormone decreases whereas those of the separated chains increase, and in no case was scrambled insulin containing both chains the predominant product in the absence of denaturants. Both the scrambling and the unscrambling reactions give similar h.p.l.c. profiles for the products. Under similar conditions cross-linked insulin with native disulphide linkages can be obtained from the scrambled molecule or from the S-sulphonate derivative with yields of 50% and 75% respectively at 4 degrees C, and with a dilute solution of the hexa-S-sulphonate yields better than 90% can be obtained. The regenerated product is shown to have the native disulphide bridges by treatment with CNBr to give insulin and by the identity of the h.p.l.c. profile of its peptic hydrolysate with that for cross-linked insulin. It appears that the insulin A and B chains contain sufficient information for the formation of the native molecule and that the role of the connecting C peptide is to bring and to keep the two chains together.     

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: Effects on catalysis, activation by sulfate, and thermal stability

A hinge-bending domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheK _m values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the AlaPro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withT _m near 54°C, two transitions are evident for the mutant enzyme withT _m values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.     

原生质体来源的大白菜悬浮细胞系的超低温保存

原生质体来源的大白菜 Brasstca campessris var.pekinsis 悬浮细胞系在二甲亚砜的保护下,能在液氮中(-196℃)长期冻存。加入山梨醇能增强保护作用;而加入甘露糖则降低保护作用。培养基对冻存也有明显的影响。在液氮中存放的时间长短对细胞存活率没有多大影响。冻后相对活性最高可达75.4％,恢复生长快,化冻后重新悬浮培养6天,生长量可达300-500％。遮光比不遮光对恢复更有利。冻存后恢复生长的悬浮细胞,能与未经冰冻的对照一样进行原生质体分离和培养。