Influence of substituent ribose on transition state affinity in reactions catalyzed by adenosine deaminase

Adenosine deaminase from calf intestine hydrolyzes adenine at a limiting rate four orders of magnitude lower than that for adenosine, while Km values for these substrates are about the same (Wolfenden, R., et al. (1969), Biochemistry 8, 2412-2415). Reactivity of 6-substituents, toward nucleophilic displacement, is found to be affected only slightly by removal of ribose as a 9-substituent, in model reactions. Substituent ribose thus appears to stabilize, selectively, the transition state for enzymatic deamination. In contrast with the small influence of substituent ribose on the apparent binding affinity of substrates, removal of substituent ribose from a potential transition state analogue, 1,6-dihydro-6-hydroxy-methylpurine ribonucleoside, results in a lowering of its affinity for the enzyme by several orders of magnitude. The synthesis of the analogue and related compounds is described, and their properties compared with those of other photoadducts and of the naturally occurring inhibitors covidarabine and coformycin. Binding of these inhibitors is found to result in the appearance of ultraviolet-absorbing bands in the neighborhood of 323 nm.     

Bombesin-like immunoreactivity in developing human lung

Bombesin-like immunoreactivity (BLI) was detected by a specific radioimmunoassay in extracts of 15 human lungs from fetuses, neonates, infants and children. A higher concentration of lung BLI was found in the fetal/neonatal group (2053.2 ± 316.3 ng bombesin/g protein, n=6) compared to the infant/children group (416.3 ± 64.3 ng/g, n=9). The peaking of BLI in lungs during the fetal/neonatal period suggests an important function for this peptide in intrauterine life and neonatal adaptation.     

The inside and outside of gap-junction membranes visualized by deep etching

We have viewed the membrane specializations that occur at gap junctions from the inside and outside of cells in replicas of quick-frozen and deep-etched samples. Gap junctions were split to expose the normally apposed outside surfaces of their membranes, which displayed uniform 8-9 nm protrusions with central pores. Such pores were also observed in the protoplasmic-face particles of freeze-fractured gap junctions, even after the junctions were induced to crystallize by treatment with metabolic inhibitors or by homogenization. Crystallized junctions have been shown to be in the closed, high-resistance state; hence the channel-closing mechanism must not be located in the regions viewed so far. In washed-out broken cells, the inner surfaces of gap junctions possess smooth surfaces with no visible pores. These surfaces are devoid of special undercoatings of cytoskeletal elements, suggesting that crystallization observed during uncoupling is an intramembrane phenomenon. Hypertonicity, in itself, may produce the same sort of hexagonal crystallization of gap-junction components that is usually observed after uncoupling.     

Thermoinduced structural changes in the membranes of animal organs following administration of antioxidants and during growth of malignancies

By means of weak-bound spin probes--stable iminoxyl free radicals differing in the level of hydrophobity there were studied thermoinduced structural transitions in the membranes of cell organells of animal liver after intraperitoneal injection of antioxidants and in the course of malignant growth, and in the nuclear membranes of Ehrlich ascite carcinoma as well. It has been found that during the growth of Ehrlich ascite carcinoma changes in rotationary mobility of probes in cell nuclei isolated from the liver of tumour-carrying animal are similar to the changes observed after antiocidants injection. A different pattern is observed in tumour cells. The membranes of ascite cell nuclei are characterized by a weak dependence of tau c on temperature for both probes. Within the temperature range studied no characteristic structural transitions proceeding in the nuclei of intact animals are observed.     

Fluid transport in a thick layer above an active ciliated surface.

The discrete cilia approach is employed to describe the flow field above an active ciliated surface in a layer of finite depth. The infinite-size cilia surface model predicts a uniform flow above the cilia surface. Modifications as a result of a finitely extending cilia surface inside a finite-size dish predict a backward parabolic profile above an active cilia surface. Experiments are described which demonstrate a good fit between theoretical predictions and observations. The results provide a sound physical basis for the proper interpretation of fluid-flow observations close to an active ciliary field.     

Immobilized organelles and whole cells into protein foam structures: scanning and transmission electron microscope observations

Protein foam structures bearing cells and organelles were produced by using a co-crosslinking method with serum albumin and glutaraldehyde at sub-zero temperature. Morphological observations obtained with scanning and transmission electron microscopy indicate macroporous and homogeneous structures. The glutaraldehyde concentration was varied to reveal its effect on immobilized red cells. It was observed that the structural appearance of preatreated cells or subcellular fractions (thylakoids from lettuce ; spheroplasts and chromatophores from R. capsulata) is preserved during the immobilization process. The morphological features of foam particles are always related to the observed kinetic activities of the specimens.     

Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions.

Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.