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.

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.

[Comparative assessment of myelocariocytes cell cycle in patient with hemodepressions]. / Sravnitel'naia otsenka kletochnogo tsikla mielokariotsitov bol'nykh gemodepressiiami.

The proliferative activity of bone marrow cells was studied by flow cytofluorometry in 8 patients with aplastic anemia (AA), 6 with myelodyspastic syndrome (MDS), and 26 normal subjects aged 21-40 years. The myelokaryocyte proliferation was inhibited at the G0-1 stages of cell cycle, the number of cells in the S phase was decreased, and the coefficient of variations cV of DNA cytofluorometry values were increased in AA patients in comparison with the norm. By contrast, in MDS patients the number of cells in the G0-1 phase was decreased and that of cells in the S phase increased in comparison with the norm and with AA patients; cV was increased. These differences in the cell cycle characteristics are suggested to be used as a differential diagnostic criterion in AA and MDS.

[Activation of lymphocyte nucleoli in culture adding the putative allergen to culture media: a tool for allergy diagnosis]. / Aktivatsiia iadryshek limfotsitov v kul'turakh s dobavleniem predpolagaemogo allergena v sredu kak sposob diagnostiki allergii.

A method for diagnosis of allergy from activation of the peripheral blood lymphocyte nucleoli in short-lived cultures of peripheral blood leukocytes of 16 patients with clinical manifestations of drug allergy is described. The tests were positive in 75% cases.

[Results of cytological studies of smears of the buccal mucosa in elderly subjects]. / Rezul'taty tsitologicheskogo issledovaniia otpechatkov so slizistoi polosti rta u lits pozhilogo vozrasta.

Cytological studies of smear impressions from different sites of the buccal mucosa were carried out in 25 subjects (22 women and 3 men) aged 61-85 years. A group of healthy subjects aged 16-33 years were controls. Cells of different degree of differentiation are present in normal mucosa with different epithelial status: intermediate cells, mature cells with pyknotic nuclei, or surface cells without nuclei. In elderly subjects, the percentage of anuclear cells in the last stage of differentiation is increased in almost all sites of the mucosa, with the main characteristics of cytograms of young subjects retained.

[Dental care for patients with hematological diseases]. / Stomatologicheskaia pomoshch' bol'nym s gematologicheskimi zabolevaniiami.

Clinical and cytological studies in 41 hematological patients aged 19-70 years showed appreciable shifts in the immune status and nonspecific reactivity virtually in all examinees. Layers of cellular epithelium of late (V-VI) and early (III-IV) stages were more often detected in smears impressions than individual cells. Cells of stages I-II were scarce and never formed layers. In patients with leukemia, signs of dissociation of epitheliocyte nucleus and cytoplasm maturation were observed ("mature" cytoplasm and "young" nuclei). These specific shifts should be borne in mind when rendering dental care to hematological patients.

[An estimation of the level of epithelial cell differentiation in impressions from different sites of the oral mucosa in healthy persons]. / Otsenka urovnia differentsirovki kletok épiteliia v otpechatkakh s raznykh uchastkov slizistoi obolochki polosti rta zdorovykh liudei.

Impressions from various sites of the buccal mucosa of 20 normal subjects aged 16 to 33 years were examined. Mature epitheliocytes of the V differentiation stage predominate in the cytological impressions of the lingual and buccal mucosa. Cells of the V stage of differentiation predominate in the lower lip mucosa, and there are just few cells of the IV (transitional) stage. In impressions of the palatal mucosa mainly cells without nuclei (stage IV) were found. These data will help more objectively assess the cytological findings in different diseases involving the buccal mucosa.

[Changes in the oral mucosa in chronic secondary disease]. / Izmeneniia slizistoi obolochki polosti rta pri khronicheskoi vtorichnoi bolezni.

Four patients with acute leukemia suffering from a chronic secondary illness (CSI) which developed 1.5 to 34 months after allogenous transplantation of bone marrow are described. One patient (female) was followed up over time: 14, 15, and 20 months after CSI was diagnosed, the rest patients were examined once, 15.5, 23, and 29 months, respectively, after the disease was diagnosed. Besides visual characterization of the buccal mucosa, results of cytologic examination of smear impressions are presented. Specific features of mucosa involvement at the peak of clinical manifestation of CSI and later, when the rest symptoms of secondary disease to a certain measure regressed, are described. The authors emphasize a similarity between the detected changes in the buccal mucosa and those observed in diseases which might be caused by immunity disorders.

[Results of screening hematological and cytochemical blood studies of 906 children living in Bryansk Province at places with different intensities of soil contamination with cesium-137 and strontium-90]. / Rezul'taty skriningovykh gematologicheskikh i tsitokhimicheskikh issledovanii krovi 906 detei, prozhivaiushchikh v Brianskoi oblasti v mestakh s raznoi plotnost'iu zagriazneniia pochvy tseziem-137 i strontsiem-90.

Populations of children living in the Bryansk territory (radionuclide contamination 0.2-63.9 Cu/km2) are characterized by heterogeneous blood counts, though relevant mean values are close to control. Mean cytochemical indices indicated a significant reduction in activity of point nonspecific esterase (NSEP), a marker of mature T-cells, in children from all the contaminated districts. Shifts in cytochemical blood lymphocytogram by NSEP test evidencing rejuvenascence of T-lymphocyte pool were recorded in 12-33% of children from different villages. A 10% decrease in NSEP suggested poor adaptation and feasibility of immunodeficiency. In one-third of children with low NSEP the number of lymphocytes with large-granular PAS reaction may reflect uneffective B-lymphopoiesis in these children. In two villages significantly contaminated with 137Cs and 90Sr half of the children had blood hemoglobin above 150 milligrams. Children from three villages exhibited a sharp rise in the number of lymphocytes with intensive-granular PAS reaction. These changes may be related to thyroid abnormalities. The number of children at risk of health deterioration grows with growing environmental contamination with 137Cs.

[T-activin in the treatment of patients with recurrent aphthous stomatitis]. / T-aktivin v lechenii bol'nykh retsidiviruiushchim aftoznym stomatitom.

The authors sum up the experience gained in tactivin therapy of patients with recurrent aphthous stomatitis and analyze the effects of this drug on the patients' cellular and humoral immunity. Thirty patients with various forms of the condition were subcutaneously injected tactivin in a dose of 40 micrograms for 10 days. Biochemical parameters were virtually unchanged after therapy, whereas the immunity parameters evidenced essential shifts in humoral and cellular immunity. Clinical and laboratory data indicate a sufficiently high immunocorrective effect of tactivin in therapy of patients with recurrent aphthous stomatitis.

[Kemantan treatment of patients with recurrent aphthous stomatitis]. / Lechenie kemantanom bol'nykh retsidiviruiushchim aftoznym stomatitom.

The authors assess the therapeutic efficacy of kemantan, an immunomodulator, used in the treatment of 30 patients with severe stubborn recurrent aphthous stomatitis (RAS). The blood immunological and biochemical characteristics were checked up over the course of treatment. Clinical and laboratory studies have shown that this immunocorrective agent is sufficiently effective for fibrous and cicatrizing RAS running a permanent course: the periods of the pathologic elements epithelialization are shortened, the permanent course of the condition is interrupted and the remissions are prolonged; clinical cure has been achieved in a number of cases. Kemantan is indicated for RAS patients only in cases with immunologic insufficiency, with immunologic responses monitored over the course of the treatment.