Application of the small-angle x-ray scattering technique for the study of two-step equilibrium enzyme-substrate interactions

The small-angle x-ray scattering (SAXS) technique is used for the investigation of two-stage equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analyzing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium, and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of double-stranded oligonucleotide with Eco dam methyltransferase (MTase) have been determined and demonstrate a high cooperativity of MTase binding when the ternary complex containing the dimeric enzyme is formed. The structural parameters (R_g, R_c, semiaxes) have been determined for free enzyme and polynucleotides and of enzyme-substrate complexes, indicating structural rearrangements of the enzyme in the interaction with substrates. © 1996 John Wiley & Sons, Inc.     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Components of early maternal environment affecting the predisposition to catalepsy

Reciprocal pup substitution (cross-fostering) in cataleptic GC (designated so by the initials of words "genetic" and "catalepsy") and control Wistar females resulted in attenuation of cataleptic predisposition in GC rats fostered by Wistar foster-mothers. The latter demonstrate a more intense maternal care than GC females. There was a significant negative correlation between the frequency of mother staying in nest and the duration of pinch-induced catalepsy in pups fostered by her. In the home-cage retrieval test, the females of the strains compared showed a significant dependence of the latencies of approach to, and retrieval of, pups on their own and the pups' genotype.     

Involvement of adenine nucleotide carrier in the mechanism of the apolipoprotein C induced decrease of membrane potential in rat liver mitochondria

Apolipoprotein C (apo C) was shown to decrease the Ca2+ capacity and membrane potential of mitochondria isolated from rat liver. The specific ligands of adenine nucleotide carrier, ADP and carboxyatractyloside (CAT), inhibited the effect of apo C on the mitochondrial membrane potential. The effect of ADP and CAT was revealed in the absence of Ca2+. We conclude that in the presence of apo C, adenine nucleosides carrier transforms into a pore, and this causes the decrease in the membrane potential of the mitochondria. ADP and CAT support the primary conformation of the carrier and therefore inhibit the effect of apolipoprotein C.     

Effects of change of maternal environment during early postnatal development on behaviour in cataleptic rats

Pinch-induced catalepsy was compared at an age of 2 weeks and at weaning in cataleptic GC and control Wistar rats reared by their biological mothers or subjected to reciprocal in-or cross-fostering. Besides, some open-field parameters were studied in the same groups of rats at an age of 2 months. Significant interstrain differences in all the behavioural parameters studied were found. Reciprocal cross-fostering tended to diminish interstrain differences in most parameters. It brought about a decrease of duration of pinch-induced catalepsy at 2 weeks and at weaning in GC rats, and an increase of duration of catalepsy at weaning in Wistar females. Besides, cross-fostering decreased the duration of freezing in the open-field test in GC rats at 2 months.     

The effect of anabiosis autoinducers on the bacterial genome

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d1 factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 micrograms/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C1-AHB) and hexylresorcinol (C6-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C6-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C1-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C6-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes (the phenotypic dissociation of microbial populations and the formation of resting microbial forms) is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.     

Correlation between cataleptic freezing and prestimulation inhibition of the startle reflex in rats

In rats of GC strain bred for predisdposition to cataleptic freezing, a significant negative correlation between the duration of freezing and the level of prepulse inhibition (PPI) of the startle reflex, has been found. Besides, in a group of GC rats specific by their "nervousness" and jumpiness, there was also a negative correlation between the duration of freezing and the habituation to the startle reflex. None of this correlation have been found in Wistar rats. Since impairment of the PPI and habituation of the startle reflex is considered to be characteristic of schizophrenia, it is believed than cataleptic freezing in the GC rats may be used as a model of schizophrenic psychopathology.     

Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.