[Life extension study on high-yield non-myeloablating bone marrow transplantation from young to old mice].

Tissue renewal is the known phenomenon, when the progeny of resident or circulated stem cells (SC) replaces the vanishing cells. The delivery of stem cells via circulation should result in stem cell homing and differentiation into wide variety of tissues and gives promise as therapy for many diseases of tissue failure including aging itself. To test this hypothesis, we created chimeric mice C57BL/6 by bone marrow (BM) transplantation from young 1.5 months old donors to 21.5-months old recipient mice of the same strain C57BL/6. We applied here the recently published new transplantation technique, which allows to get high scores of chimerism due to very high amount of transplanted cells (1.5 x 10(8) per mouse or 25% of its total BM count). In the earlier works only 1% of total BM count (about 5 x 10(6) cells per mouse) was usually transplanted to lethally irradiated mice, what excluded the possibility to apply this method for life extension. As a result of the modified technique implementation, the mean post-transplantation life (starting the 21.5 months old) of treated mice was 4.9 months versus 3.4 months for untreated mice. The difference in 1.5 months counts for 44% extension of mean post-transplantation life.

Induced absorption band of holotransketolase and its interpretation.

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1;-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.

[Cytological and molecular changes in the atypical case of accelerated human aging]. / Tsitologicheskie i molekuliarnye izmeneniia pri netipichnom sluchae uskorennogo stareniia cheloveka.

A complex research of cells of a patient with unusual form of premature ageing was made. The clinical picture is not typical for any of known forms of hereditary premature aging--progerias. Skin fibroblasts of the patient AG has limited proliferation capacity in vitro. It was shown by fluorescent-immunochemical hybridization (FISH-method), that the level of stable chromosome aberrations in AG blood lymphocytes was characteristic of aged 55-65 years, though as he was only 26 years old. Some characteristic peculiarities, typical for progerias, were found in the reaction of skin fibroblasts of AG to growth factors addition. Some clinical and biochemical peculiarities are results rather, than reasons of the disease. The conclusion is that the premature ageing in this case is a manifestation of Werner's syndrome--one of hereditary forms of accelerated senescence.

Cleaving of ketosubstrates by transketolase and the nature of the products formed.

The interaction of transketolase ketosubstrates with the holoenzyme has been studied. On addition of ketosubstrates cleaving both irreversibly (hydroxypyruvate) and reversibly (xylulose 5-phosphate), identical changes in the CD spectrum at 300-360 nm are observed. The changes in this spectral region, as previously shown, are due to the formation of the catalytically active holoenzyme from the apoenzyme and the coenzyme, and the cleavage of ketosubstrates by transketolase. The identity of the changes in transketolase CD spectrum caused by the addition of reversibly or irreversibly cleaving substrates indicates that in the both cases the changes are due to the formation of an intermediate product of the transketolase reaction--a glycolaldehyde residue covalently bound to the coenzyme within the holoenzyme molecule. Usually, in the course of the transferase reaction, the glycolaldehyde residue is transferred to an aldose (acceptor substrate), resulting in the recycling of the holoenzyme free of the glycolaldehyde residue. The removal of the glycolaldehyde residue from the holoenzyme appears to proceed even in the absence of an aldose. However, the glycolaldehyde cannot be found the free state because it condenses with another glycolaldehyde residue formed in the course of the cleavage of another ketosubstrate molecule yielding erythrulose.

One-substrate transketolase-catalyzed reaction.

Apart from catalyzing the common two-substrate reaction with ketose as donor substrate and aldose as acceptor substrate, transketolase is also able to catalyze a one-substrate reaction utilizing only ketose (xylulose 5-phosphate) as substrate. The products of this one-substrate reaction were glyceraldehyde 3-phosphate and erythrulose. No free glycolaldehyde (a product of xylulose 5-phosphate splitting in the transketolase reaction) was revealed.

Acceptor substrate inhibits transketolase competitively with respect to donor substrate.

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.

Influence of transketolase substrates on its conformation.

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate-i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose-differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.

Cooperativity and flexibility of active sites in homodimeric transketolase.

Here we summarize evidence for non-equivalence of two structurally similar active sites in transketolase and other thiamine-dependent enzymes. This non-equivalence takes place when the enzymes interact with various ligands (inhibitors, cations, coenzyme and substrates). Data on different strains in the structure of the holotransketolase subunits are also given. The above results are discussed within the framework of a concept of permanent alternative site oscillation of the transketolase molecule in the presence and in the absence of substrate as a manifestation of a 'flip-flop' mechanism.

Kinetic investigation of cooperativity in coenzyme binding by transketolase active sites.

The two-step mechanism of coenzyme (thiamine diphosphate, ThDP) binding with two initially identical active sites of apotransketolase has been examined with a kinetic model. Cooperativity between sites in the primary ThDP binding and in the following conformational transition has been analyzed. The only reliable difference between sites is shown to be the tenfold difference in the backward rate constants of the conformational transition; this means that the cooperative interaction between sites takes place only after termination of both steps of ThDP binding in both sites.

Kinetic mechanism of active site non-equivalence in transketolase.

The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.

Determination of constants of substrate primary binding with baker's yeast transketolase by kinetic modelling.

A kinetic model of bisubstrate reaction catalyzed by baker's yeast transketolase is proposed. The model considers individual stages of substrates reversible primary binding. The model corresponds to the observed kinetics of product accumulation within a wide range of initial substrate concentrations. Kinetic parameters for the best simulation of the experimental data are defined. The equilibrium constants of the primary binding of both the initial and produced ketose and also the initial aldose were unequivocally determined by varying the initial substrate concentrations. The dissociation constants of the primary enzyme-substrate complex for the initial ketose (xylulose 5-phosphate) and the reaction product (sedoheptulose 7-phosphate) were found to differ by more than by two orders of magnitude. The result is discussed in the context of the hypothesis of flip-flop functioning of the transketolase active sites.

Inhibition of transketolase by N-acetylimidazole.

Transketolase from baker's yeast is rapidly inactivated in the presence of N-acetylimidazole. According to kinetic data, acetylation of one amino acid residue of the protein per active site is sufficient for TK* inactivation. The holoenzyme is inhibited more slowly than is apotransketolase. The presence of a tyrosine residue in the enzyme's active site, essential for activity, is suggested.