Azobenzene photocontrol of peptides and proteins.

The last few years have witnessed significant advances in the use of light as a stimulus to control biomolecular interactions. Great efforts have been devoted to the development of genetically encoded optobiological and small photochromic switches. Newly discovered small molecules now allow researchers to build molecular systems that are sensitive to a wider range of wavelengths of light than ever before with improved switching fidelities and increased lifetimes of the photoactivated states. Because these molecules are relatively small and adopt predictable conformations they are well suited as tools to interrogate cellular function in a spatially and temporally contolled fashion and for applications in photopharmacology.

Specific UV photodissociation of tyrosyl-containing peptides in multistage mass spectrometry.

UV photodissociation (UVPD) at 262 nm has been carried out on protonated tyrosyl-containing peptides formed by trypsin digestion of apo-transferrin. Under UVPD, the main event is the fragmentation of the C(alpha)-C(beta) bond of the tyrosyl residues leading to a radical ion 107 Da below the precursor ion. The dissociation rate of this specific cleavage appears to be strongly dependent on the peptide sequence and is more prominent on the singly protonated species than on the doubly protonated state. The fragmentation spectra resulting from collisional activation of the protonated even-electron native peptides and of the odd-electron radical species prepared by UVPD are dominated by y-type backbone cleavages. A comparison of their respective y-ion pattern shows complementarities since the combination of both increases the sequence coverage of the peptide sequence. The specific detection of the neutral loss of 107 Da from peptides witnesses the content of at least one tyrosyl residue and, though preliminary, is proposed as a potential new filtering strategy during protein database searching.

Effect of K, MG aspartate on some biological parameters of aging rats.

The effect of K and Mg salts of aspartic acid (Cardilan) on the serum concentration of selected proteins and phagocytic activity in aging male Wistar rats was investigated. Cardilan was administered in tap water for 7 days a month for 3 months before the last observed interval (12, 18 and 24 month). In a part of animals, the aging process was accelerated by sublethally gamma-irradiation. The administration of Cardilan slowed down the changes in the concentration of prealbumin, albumin, haptoglobin, haemopexin, C3 complement in non-irradiated rats (DC). This effect was extended to the changes in transferrin level in irradiated rats (IDC). The phagocytic activity in both DC, IDC rats was lower compared with controls drinking water (DW, IDW), but not significantly. The effect of Cardilan administration appears to be the greatest in 24-month-old rats, when the treated animals survived better by 25% in IDC group and by 26% better in DC rats, compared with those of the same age controls. Potassium and magnesium salts of aspartates are suitable compounds for life prolongation in the experimental conditions.

Different onsets of oxidative damage to DNA and lipids in bone marrow and liver in rats given total body irradiation.

We examined time-dependent changes in antioxidant vitamins and oxidative damage to DNA and lipids in the bone marrow, liver, and plasma of rats given total body irradiation (TBI) with X-rays at 3 Gy. The oxidative damage to DNA and lipids was evaluated by measuring increases of 8-hydroxydeoxyguanosine (8OHdG) in DNA and 4-hydroxy-2-nonenal (HNE), respectively. After the TBI, marked increases in 8OHdG and HNE were detected at 3 to 5 h in the bone marrow, while gradual increases in these parameters were detected after a few days in the liver. These changes in 8OHdG and HNE were well correlated within each tissue. In the bone marrow, levels of both vitamin C and vitamin E were decreased by the TBI; however, the changes in vitamin C were earlier and greater than those in vitamin E. In the liver, the level of vitamin C did not decrease, but that of vitamin E decreased due to the TBI. Changes in HNE, vitamin C, and vitamin E in the plasma were similar to those in the liver. Within each tissue, the time of decrease in antioxidants was almost the same as that of the increase in oxidative damage. An increase in total iron due to the TBI was also detected in these tissues. In particular, the total iron in the bone marrow was markedly increased at a few hours after the TBI, with a slight increase in transferrin and no increase in ferritin. Exposure studies performed on cells or isolated DNA showed that an increase in 8OHdG was detected immediately after irradiation at more than 100 Gy in bone marrow cells and at less than 10 Gy in isolated DNA, suggesting that an increase in 8OHdG is undetectable even in bone marrow immediately after the TBI at 3 Gy. These results indicate that the onset of oxidative damage to DNA and lipids was delayed after TBI at 3 Gy, that it was quite different in the bone marrow and the liver, and that an increase in iron and decrease in antioxidant vitamins were involved in the mechanism of oxidative damage.

Testicular toxicity of the transferrin binding radionuclide 114mIn in adult and neonatal rats.

Adult (70 d) and neonatal (7 d) male rats were dosed (i.p.) with 37 MBq/kg (1 mCi/kg; approximately 1 microgram elemental indium/kg) 114mIn, a transferrin-binding radionuclide. In adults, approximately 0.25% of the injected activity localised within the testis by 48 h postinjection and remained constant for up to 63 d. In neonates, 0.06% of the activity was in the testis by 48 h, and this declined such that by 63 d only 0.03% remained. At 63 d, treated rats had reduced sperm head counts and abnormal testicular histology that was more marked in animals dosed as adults than as neonates. In vitro, uptake of 114mIn into seminiferous tubules isolated from 7-, 20-, or 70-d-old rats was compared with that of 125I. Both radionuclides were readily accumulated by the tubules. Whilst 114In uptake into 20- and 70-d tubules was inhibited by excess transferrin, uptake into 7-d tubules was unchanged. 125I uptake was not affected by excess transferrin. These data support the contention that some radionuclides may cross the blood-testis barrier by utilisation of the physiologic iron-transferrin pathway, which may lead to greater testicular damage in adult compared to neonatal animals.

Alteraçöes nutricionais e do metabolismo energético na irradiaçäo abdominal: estudo experimental / Nutritional and energy metabolism changes in abdominal irradiation: experimental study

Estudaram-se os efeitos da irradiaçäo sobre o estado nutricional e o metabolismo energético do hospedeiro. Utilizaram-se 48 ratos adultos machos Wistar, mantidos em gaiolas metabólicas, e divididos em grupos controle (C) e irradiado (R). No período 1, pré-irradiaçäo, os ratos foram submetidos a calorimetria indireta no dia 4 (calorimetria 1) que forneceu as variáveis gasto energético (GE), quociente respiratório (QR), substrato total oxidado (STO) e proteínas (PO), glicides (GO) e lípides oxidadas (LO). A seguir transcorreu o período 2, de irradiaçäo, quando os ratos R receberam 300 cGy/ diários de irradiaçäo abdominal, por cinco dias, sob restriçäo em molde acrílico. Cada rato do grupo C passou a receber alimentaçäo pareada e foi submetido a irradiaçäo simulada. Calorimetrias foram realizadas durante esse período (II e III) e após o seu final (IV). No sacrifício (dia 14), dosou-se hemoglobina, hermatócrito, albumina e transferrina. A análise estatística dos resultados foi feita com significância de 0,05 e 0,01. No período 1, näo houve diferenças entre os grupos em relaçäo às variáveis analisadas. No período 2 ocorreu reduçäo da ingestäo de dieta e perda percentual de peso corpóreo nos dois grupos. Apesar da ingestäo semelhante, a reduçäo ponderal foi maior no grupo R que no C. A incorporaçäo de nitrogênio decresceu significantemente no período 2. Apenas a média de albumina sérica apresentou-se menor no grupo R. O QR reduziu-se na avaliaçäo calorimétrica III, mantendo-se mais baixo em IV no grupo R, sem retornar aos níveis iniciais. Houve reduçäo significante do GE apenas no grupo R, em III. No grupo R o STO diminuiu com a irradiaçäo. Em ambos houve reduçäo da PO e GO, que no grupo R se manteve reduzida em IV, mantendo menor PO. Nas condiçöes do presente estudo, a irradiaçäo abdominal determinaou reduçäo de ingestäo alimentar, perda ponderal corporal, hipoalbuminemia, reduçäo de incorporaçäo de nitrogênio e promoveu queda do gasto energético e do quociente respiratório, levando ainda a uma alteraçäo do perfil de consumo de substratos. Conclui-se que a irradiaçäo pode conduzir a desnutriçäo protéico-calórico, näo decorrente exclusivamente da anorexia a ela associada

[Nutritional and energy metabolism changes in abdominal irradiation--experimental study]. / Alterações nutricionais e do metabolismo anergético na irradiação abdominal-estudo experimental.

In this study the effects on nutritional status and energetic metabolism due to abdominal irradiation were analysed. Adult male Wistar rats (48), were divided in two groups Control (C) and Radiated (R). The rats were maintained all time in metabolic cages. The study was done in two periods: Period 1 begun at 0 day, where rats adapted to cages and oral diet, had food and water "ad libitum". At the day 4 indirect calorimetric measurements were performed (calorimetry I). At Period 2, group R rats abdominal radiation at a 300cGy/day rate, for 5 consecutive days, and group C started a pair feeding process linked individually to R rats and suffered application of simulated-radiation. Two other calorimetric measurements (II,III) were performed during Period 2. After radiation the last calorimetry was performed (IV). At sacrifice (day 14) blood was collected for determination of hemoglobin, haematocrit, albumin and transferrin. There were no statistical differences among groups C and R during Period 1 (p < 0.05). Great reduction in food intake and weight variation were found in Period 2, but weight loss was significantly higher in R rats. Nitrogen balance decreased in Period 2, but without difference among the groups (p < 0.05). Serum albumin was significantly lower in R rats. Respiratory quotient decreased in both groups during Period 2, but R rats kept it lower (p < 0.05). The energy expenditure level decreased after radiation in Group R. During Period 2 total substrate oxidation decreased in R rats. Radiation decreased glucose and protein oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron paramagnetic resonance and difference ultraviolet studies of Mn2+ binding to serum transferrin.

Serum transferrin is the mammalian protein whose normal function is to transport ferric ions through the blood among sites of absorption, storage, and utilization. It has two specific metal-binding sites that bind a variety of metal ions in addition to ferric ion. The macroscopic equilibrium constant for the binding of the first equivalent of Mn2+ to apotransferrin has been determined by electron paramagnetic resonance spectroscopy (EPR) to be logKM1 = 4.06 +/- 0.13 at pH 7.4 in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (Hepes). An equilibrium constant for nonspecific binding of Mn2+ to apotransferrin of logKns = 2.93 +/- 0.13 has also been obtained by using EPR. Binding of Mn2+ to apotransferrin and to both C- and N-terminal nonferric transferrin has also been studied by difference UV spectroscopy. The second stepwise macroscopic equilibrium constant for the formation of Mn2Tf is logKM2 = 2.96 +/- 0.13. The site-specific microconstants for Mn2+ binding are logkN = 3.13 +/- 0.09 for the N-terminal site and logkC = 3.80 +/- 0.09 for the C-terminal site. There does not appear to be any significant cooperativity between the two sites with respect to metal binding. An equilibrium model for the speciation of Mn2+ in serum has been developed which estimates that almost 90% of Mn2+ is bound to serum proteins, but only approximately 1% is bound to transferrin. The weak binding of Mn2+ to apotransferrin and the obvious inability of transferrin to compete with albumin indicates that the appearance of Mn-transferrin as a major serum species in vivo must involve oxidation of the metal to form the much more stable Mn(3+)-transferrin complex. The computer model confirms that albumin has a sufficient binding affinity to complex most of the Mn(II) in serum in competition with the common low molecular weight ligands in serum. However, there is insufficient data to rule out the possibility that some other protein, such as alpha 2-macroglobulin, may compete with albumin for Mn(II).

[The morphofunctional indices of the erythrocytic link in hemopoiesis in persons constantly working in an area of intensified radioecological control]. / Morfofunktsional'nye pokazateli éritrotsitarnogo zvena gemopoéza u lits, postoianno rabotaiushchikh v zone usilennogo radioékologicheskogo kontrolia.

A study of the peripheral blood indicates that persons constantly working in the zone of rigid radiation control showed a statistically valid reduction of the hemoglobin concentration, appearance of spinous and spheric erythrocyte forms, reduction of the mechanical resistance. Simultaneously 18% of males and 26% of females revealed moderate hypochromic anemia with a normal level of serum iron. The intensity of Fe-transferrin electron paramagnetic resonance was statistically reduced, that may be related to reduction of trivalent iron to bivalent and formation of a pool of free bivalent iron--a potent inductor of lipid peroxidation. Activation of oxidative processes in blood is confirmed by significant fluctuations of the blood ceruloplasmin level.

Influence of radiation treatment on serum transferrin and tumor necrosis factor-alpha.

This study deals with the effect of radiation treatment (RT) on serum transferrin and tumor necrosis factor-alpha (TNF-alpha) in patients with malignant tumors. In 21 patients who received 36-60 Gy in 20 to 30 sessions of RT, serum transferrin and TNF-alpha were determined pre-RT, after 10 to 15 sessions (middle of RT) and after 20 to 30 sessions (end of RT). The values of serum transferrin pre-RT were significantly higher than those in the middle and at the end of RT (p < 0.001). The values of TNF-alpha were increased by RT and were significantly higher at the end of RT as compared to the pre-RT values (p < 0.05). The values of serum transferrin and TNF-alpha show a tendency to negative correlation, either as a whole or separately pre- and under-RT. However, no correlation was statistically significant.

[The dose dependence of the development of compensatory-restorative body reactions to irradiation. The EPR method]. / Dozovaia zavisimost' razvitiia kompensatorno-vosstanovitel'nykh reaktsii organizma pri obluchenii. Metod EPR.

The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase.

[EPR study of the blood of animals during an acute radiation lesion]. / Issledovanie metodom EPR krovi zhivotnykh v khode ostrogo luchevogo porazheniia.

The intensity of the ESR signal of methemoglobin, Fe3+-transferrin, and Cu2+-ceruloplasmin in blood and spleen of mice exposed to 6 Gy radiation was shown to undergo considerable changes at early times of acute radiation sickness.

Immunoelectrophoretic study of the effect of radiation on protein mixtures.

This paper demonstrates the suitability of immunoelectrophoretic methods in studies of radiation effects on protein mixtures. Single serum proteins (IgG, albumin, transferrin), albumin-transferrin mixtures and human serum were irradiated with doses of 3-52 kGy. The irradiated proteins were analysed using rocket-, crossed-, crossed-with-intermediate-gel, and fused-rocket immunoelectrophoretic methods. Using crossed immunoelectrophoresis with intermediate gel, complex formation between albumin and transferrin was observed. Gel filtration monitored by fused-rocket immunoelectrophoresis was demonstrated to be a most promising method for studying protein degradation and hybrid formation.

[Diploid radiation gynogenesis in carp. III. Analysis of gynogenetic progeny by biochemical markers]. / Issledovaniia po diploidnomi radiatsionnomu ginogenezu u karpa. Soobshchenie III. Analiz ginogeneticheskikh potomstv po biokhimicheskim markeram.

Studies have been carried out on carp gynogenetic offspring by four autosome genes: Tf (transferrin), Est(s) (muscle esterases, slow), Est(f) (muscle esterases, fast), My (myogenes). Homozygosity and phenotypical homogeneity by all the loci studied are established for carps of the second generation of the induced gynogenesis. In the first generation obtained from heterozygous females, the correlation of homozygous offspring of two classes corresponding to the theoretically expected one (1:1). Heterozygous offspring makes (%): 5.0 (Tf), 9.1 (Est (s)), 28.4 (Est (f)). Three loci are mapped in relation to the centromere by heterozygote frequency. Genetic inactivation of the radiated sperm has been confirmed using biochemical markers. Due to the codominant nature of inheritance and high polymorphism, protein loci are considered to be convenuent genetic markers.

Effect of magnitude of daily continuous radiation dose on serum proteins and survival in rats.

Changes in serum prealbumin, A1-macroglobulin, transferrin, and complement levels were studied in rats irradiated with daily doses of 100R (0.0258 C/kg), 200R (0.0516 C/kg) and 400R (0.1032 C/kg) until death. The daily dose of 100R applied for 39 days produced no changes in prealbumin and transferrin and a rise in the levels of A1-macroglobulin and complement. Shortly before death, i.e., between 39 and 44 days of irradiation, the levels of all four patients dropped. Daily doses of 200 and 400R caused a reduction in the levels of all four proteins since the very beginning. The LD 50 was 4 270R (1.1017 C/kg) at 100R/day, 4 060 R (1.0475 C/kg) at 200R/day, and 4 520 R (1.1342 C/kg) at 400R/day.

Ethoxyformylation and photooxidation of histidines in transferrins.

The chemical reactivity of histidines in ovotransferrin and human serum transferrin was studied utilizing two different reactions. Upon dye-sensitized photooxidation of ovotransferrin and ethoxyformylation of human serum transferrin and ovotransferrin, losses in histidine and iron-binding activity were observed. All of the histidines in both apoproteins could be ethoxyformylated by the use of 170 to 400 molar excesses of reagent resulting in complete loss in activity. The histidines of human serum transferrin showed a greater reactivity toward the reagent than did those of ovotransferrin. The binding of each iron protected two histidines from ethoxyformylation, and in both cases the proteins remained completely active. First-order losses in histidine and iron-binding activity were observed when ovotransferrin was irradiated in the presence of methylene blue. Comparison of the first-order rates indicates the loss of two histidines per binding site accounts for the inactivation of the protein. However, iron binding did not protect ovotransferrin from photoinactivation as expected. Evidence from both modification technqiues indicates: (1) Histidines are essential for iron-binding activity. (2) There are two essential histidines in each binding site. The advantages of using two modification reactions, ethoxyformylation and photooxidation, in the study of the functional role of histidines in proteins are demonstrated in this work.