Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.     

Experimental and clinical applications of fibrin glue

A 2-year experience with laboratory and clinical applications of fibrin glue is presented. An autologous technique, which eliminates the danger of multidonor preparations, has been developed in our blood bank. While one can obtain different fibrinogen concentrations from the same amount of a patient's blood, in vitro mechanical testing demonstrated that at higher fibrinogen concentrations there is an increase in shear adhesive strength. Evaluation of skin-graft take in 16 Sprague-Dawley rats did not demonstrate significant differences in healing when adhesive use was compared with suture technique. In a clinical study, four different groups of patients (facial burns, hand burns, difficult graft sites, and miscellaneous surgical applications) benefited from autologous or single-donor fibrin glue for a total of 82 cases. There are several distinct advantages to the use of fibrin adhesive: The autologous technique eliminates the risk of transmissible viral diseases (AIDS, hepatitis); it can be used as a sealant in the treatment of seromas, dural leaks, and lymphoceles; and it improves hemostasis and early graft adherence. Face and hands are resurfaced with sheet grafts in a single procedure, obtaining a better aesthetic result with complete graft take and immediate start of physical therapy. Neither sutures nor pressure dressings are required. The minimal postoperative care associated with early return to normal activities seems to increase the satisfaction of patients and nurse personnel.     

Molecular evidence for the rapid propagation of mouse t haplotypes from a single, recent, ancestral chromosome

Mouse t haplotypes are variant forms of chromosome 17 that exist at high frequencies in worldwide populations of two species of commensal mice. To determine both the relationship of t haplotypes to each other and the species within which they exist, 35 representative t haplotypes were analyzed by means of 10 independent molecular probes, including five DNA clones and five polypeptide spots identified by means of two- dimensional gel electrophoresis. All of the tested haplotypes were found to share restriction fragments and polypeptide spots that are absent in mice carrying wild-type forms of chromosome 17. This observation provides the first direct evidence that all of the known t haplotypes are descendents of a single ancestral chromosome. The absence of variation among t haplotypes could mean that this ancestral chromosome existed relatively recently, in which case it would be necessary to postulate introgressions of t haplotypes across species lines to explain their presence in both Mus domesticus and M. musculus. Alternatively, it is possible that the ancestral chromosome existed prior to the split between M. domesticus and M. musculus and that, by chance, our probes fail to detect polymorphisms that exist among the t haplotypes. A further result of our analysis is the characterization of a partial t haplotype in a wild population of Israeli mice.      

Interaction of the peptide CF3-Leu-Ala-NH-C6H4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A

The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.     

Robustness of activity of Calvin cycle enzymes after high light and low temperature conditions in Antarctic vascular plants

Only two vascular plants have naturally colonized the Maritime Antarctic: Colobanthus quitensis and Deschampsia antarctica. We propose that one of the reasons of their success in this environment is the robustness of their CO₂ assimilation machinery. In order to understand the mechanisms involved in the positive photosynthetic rates under stressful conditions, we analyzed changes in the activity of two key Calvin cycle enzymes: Ribulose bisphosphate carboxylase oxygenase (Rubisco) and stromal Fructose-1,6-bisphosphatase after high light and low temperature treatments. Our results show that the activity of both enzymes does not decrease after 48 h high light/low temperature treatments, a feature usually observed in plants adapted to harsh environments. The activation state of both enzymes remained high throughout the treatments. This feature has been related to the redox state of the chloroplast, suggesting that both plants maintain their redox balance under high light and/or low temperature conditions assayed. Both plants differed in their responsiveness to cold acclimation as observed by gas exchange and enzymatic measurements. We propose that these differences may be related to microclimate adaptations to the environment they naturally develop in the Maritime Antarctic.     

Response of cultured IEC-17 normal rat intestinal epithelial cells to X radiation

The growth parameters and radiosensitivity of normal rat intestinal epithelial cells, IEC-17, were studied. The cells were cultured by standard methods and exposed to an array of doses (1-12 Gy) of 250 kVp X rays. The survival curves generated exhibited no initial shoulder and were bimodal. The Do of the first component was about 0.2 Gy and the second component. 5.0 Gy. The ability of this cell line to repair sublethal lesions was examined by fractionation studies; repair was completed within 60 min after the first dose. When Chinese hamster ovary (CHO) cells were grown under the same conditions used for the IEC-17 cells and then irradiated with single doses, a typical survival curve with a Do of 1.4 Gy was obtained. The survival curves obtained for the IEC-17 cell line are consistent with the response of a morphologically distinct single population containing two functionally separate types of cells.