Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Lipoprotein receptor ‘CK’-dependent signalling in human platelets

The study addressed to understand whether or not lipoproteins at low concentrations could modulate Receptor-C dependent platelet signalling revealed that LDL, like exogneous cholesterol, had the capacity to initiate PLD-dependent platelet signalling in a dose dependent fashion and this effect was inhibited in presence of HDL; cAMP; DTT; Zn⁺⁺ and butanol whereas cGMP had no effect upon this PLD-dependent signalling. Further Receptor C from platelet in the purified-form displayed LDL-or cholesterol-dependent autophosphorylation at the tyrosine residues and this Receptor-C tyrosine kinase (Receptor-C_k) activity contributed to the observed LDL-or cholesterol-dependent PLD activity in human platelets. Based upon these results coupled with earlier results, an attempt was made to define the lipoprotein-dependent platelet signalling pathway.     

Thorotrast kinetics and radiation dose

Summary As a presupposition for estimating the mean tissue dose from intravascularly injected Thorotrast results of investigations on tissue distribution and steady state activity ratios of²³²Th and daughters in Thorotrast patients were compiled and are presented as best estimates. Special emphasis has been given to the non-uniformity of Thorotrast distribution on the organ 2and cellular level on the basis of results from animal experiments. Moreover, the variation widths of the mean tissue doses were calculated from the individual standard errors of the mean Thorotrast tissue distribution and activity ratios.According to the results of Thorotrast tissue distribution analyses about 97% of intravascularly injected colloidal ThO₂ are retained by the organs of the reticulo-endothelial-system (RES) of the average Thorotrast patient (liver: 59%; spleen: 29%; bone marrow: 9%). Only 0.7 and 0.1% are distributed within the lungs and the kidneys, respectively. The fractional retention of²³²Th in the marrow-free skeleton proved to be 2% on the average. Considering in addition the results on the steady state activity ratios between²³²Th and its daughters and self-absorption of-energy in Thorotrast agglomerates the mean annual tissue doses to the liver, spleen, red bone marrow, lungs (respiratory zone), and cells on bone surface, e.g., from 30 ml intravascularly injected Thorotrast are about 30 (10–70), 80 (30–200), 10 (4–27), 4.5 (1.8–11.3), and 15 (6–38) rad. The variation widths of the mean tissue doses given in brackets are based upon an average individual standard error of the mean Thorotrast tissue distribution and activity ratios of 150%. The data on mean tissue doses, however, do not include variations of the dose due to macroscopic inhomogeneities of Thorotrast distribution on the organ level, which in the liver may go up to a factor of 50. Contrary to the mean tissue dose the local annual dose, i.e., the dose to cells adjacent to the surface of 0.1–50 µm Thorotrast aggregates is between 40 and 40,000 rad.Paper presented on the occasion of a WHO meeting of a Scientific Group on the Long-Term Effects of Radium and Thorium in Man. Geneva, Sept. 12–16, 1977Dedicated to Prof. Dr. med. F. Sommer, Homburg/Saar, on the occasion of his 65th birthday     

Mutagenic effectiveness and efficiency of EMS,DES and gamma-rays in rice

Summary Data on chlorophyll mutation frequency after treatment with EMS, DES and gamma-rays and sequential administration of gamma-rays and the two alkylating agents in three varieties of rice have been used to work out quantitatively the effectiveness and efficiency of each mutagen and combination treatment. For effectiveness, the order is EMS > DES and for efficiency it is EMS > DES > gamma-rays. In some sequential treatments (Gamma-rays + DES in IR8 and Basmati; DES + gamma-rays in IR8 and Jhona; Gamma-rays + EMS in IR8 and Basmati; and EMS + gamma-rays in IR8, Jhona and Basmati) mutation frequency is more than additive (synergistic) but these treatments are decisively less efficient because of their relatively high injurious effects in the M₁. generation. EMS induces more albinas than gamma-rays do. The mutational spectrum patterns induced by gamma-rays and DES are alike. In general, combination treatments tend to increase the frequency of albinas over other types of chlorophyll mutants.     

Changes in phosphatases and lipids following scrotal gamma irradiation in adult rat testis.

Effects of localized low (2.5 Gy) and high (10 Gy) levels of gamma irradiation on the testis of albino rats were studied. A marked increase in the testicular total lipid, phospholipid and cholesterol content was observed at all post-treatment intervals except at 16 weeks where the contents decreased. A significant decrease in the activity of acid phosphatase/g of testis was seen at both the doses, the minimum value being at 4 weeks. The decrease in acid phosphatase activity is correlated with the state of germ cell population in seminiferous tubule which is found to be depleted at 4 week interval. The alkaline phosphatase activity/g testis however, showed a significant increase, the maximum being at 4 weeks post-treatment. Thereafter, the values of the enzyme activity showed a slight recovery at 16 weeks post-irradiation. ATPase activity increased initially followed by a significant decrease at all post-treatment intervals.     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

Emergence of deciduous teeth in Punjabi children, north India.

Data on deciduous tooth emergence of 312 children aged 4 to 31 months of Punjabi parentage are presented. Probit analysis was used to derive the median age of tooth emergence. Female children are found to be advanced with respect to tooth emergence than their male counterparts. While comparing the present data with those from other populations it is found that, in general, the mean number of emerged teeth in Punjabi children is more at most ages, with lower median age of eruption for most teeth. Magnitude of interage variability in the eruption times is noticed to be maximum in the 16-17 and 20-21 months age groups. The findings of the study suggest that number of teeth can be used as a parameter for the estimation of age.