The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose.

Exposure during early development to chemicals with hormonal action may be associated with weight gain during adulthood because of altered body homeostasis. It is known that organotins affect adipose mass when exposure occurs during fetal development, although no knowledge of effects are available for exposures after birth. Here we show that the environmental organotin tributyltin chloride (TBT) exerts adipogenic action when peripubertal and sexually mature mice are exposed to the chemical. The duration and extent of these effects depend on the sex and on the dose of the compound, and the effects are relevant at doses close to the estimated human intake (0.5µg/kg). At higher doses (50-500µg/kg), TBT also activated estrogen receptors (ERs) in adipose cells in vitro and in vivo, based on results from acute and longitudinal studies in ERE/luciferase reporter mice. In 3T3-L1 cells (which have no ERs), transiently transfected with the ERE-dependent reporter plus or minus ERα or ERß, TBT (in a dose range of 1-100nM) directly targets each ER subtype in a receptor-specific manner through a direct mechanism mediated by ERα in undifferentiated preadipocytic cells and by ERß in differentiating adipocytes. The ER antagonist ICI-182,780 inhibits this effect. In summary, the results of this work suggest that TBT is adipogenic at all ages and in both sexes and that it might be an ER activator in fat cells. These findings might help to resolve the apparent paradox of an adipogenic chemical being also an estrogen receptor activator by showing that the two apparently opposite actions are separated by the different doses to which the organism is exposed.

Alternatives to animal experimentation for hormonal compounds research.

Alternatives to animal testing and the identification of reliable methods that may decrease the need for animals are currently the subject of intense investigation worldwide. Alternative testing procedures are particularly important for synthetic and natural chemicals that exert their biological actions through binding nuclear receptors, called nuclear receptors-interacting compounds (NR-ICs), for which research is increasingly emphasizing the limits of several models in the accurate estimation of the physiological consequences of exposure to these compounds. In particular, estrogen receptor interacting compounds (ER-ICs) have a great impact on human health from the therapeutic, nutritional, and toxicological point of view due to the highly permissive nature of the estrogen receptors towards a large number of natural and synthetic compounds. Similar to in vitro systems, recently generated animal models (e.g., animal models generated for the study of estrogen receptor ligands) may fulfill the 3R principles: refine, reduce, and replace. If used correctly, NR-regulated models, such as reporter mice, xenopus, or zebrafish, and models obtained by somatic gene transfer in reporter systems, combined with imaging technologies, may contribute to strongly decreasing the overall number of animals required for NR-IC testing and research. With these models, flexible and highly standardized parameters and reporter marker quantification can be obtained. Here, we highlight the need for the substitution of currently used testing models with more appropriate ones that can reproduce the features and reactivity of specific mammalian target tissue/organs. We consider the promotion of this advancement a research priority bearing scientific, economic, social, and ethical relevance.

Estrogen receptor-mediated transcriptional activity of genistein in the mouse testis.

Here we show that genistein, through an estrogen receptor-mediated action, modulates gene expression in the mouse testis throughout development. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in the testis of the pups. Testis are already responsive to genistein as well as to estradiol at day 14.5 of fetal development. Activation of luciferase correlates with an activation of cell proliferation. In conclusion, our results show that genistein affects reproductive organs of male mice at all developmental ages.

Non-Hodgkin's lymphomas of the large bowel-clinical characteristics, prognostic factors and survival.

The aims of this study were to review the clinical presentation of non-Hodgkin's lymphomas of the large bowel, to analyze the prognostic factors using univariate and multivariate methods, as well as the overall survival. We identified 24 cases at our clinic between 1991 and 2005, based on pathohistological analysis and standard diagnostic criteria established by Dawson et al. They accounted for 1.2% of all cases of the large bowel malignancies (24/2021) during this period. The following clinical information such as age, gender, symptoms, tumor localization, operation performed, histology grade, stage of disease, and adjuvant chemotherapy was obtained. Survival function was expressed by Kaplan-Meier curve and Log-rank test was performed for the difference in survival between two patient groups. Multivariate analysis was carried out using the Cox proportional hazard model. Overall mean survival time was 41.91 months. According to the univariete analysis, the factors influencing overall survival rate was operation type (elective and emergent). Tumor stage and operation type were independent prognostic factors for survival, as determined by multivariate analysis. Our results showed that tumor stage and operation type should be considered as the most important prognostic factors in patients with primary non-Hodgkin's lymphomas of the large bowel.

[Use of fibrin glue in the prevention of leakage in pancreatico-jejunal anastomoses]. / Primena fibrinskog lepka u prevenciji popustanja pankreasno-jejunalne anastomoze.

The aim of this study was to test the protective effects of fibrin sealing on the pancreatico-jejunostomy (PJA), the high-risk anastomosis following pancreas head resection. Experimental study was performed on the mongrel dogs, divided in two groups (20 animals each): Experimental group-with end to end "dunking" PJA, protected by temporary occlusion of the pancreatic duct with fibrin sealant/Tissucol/Immuno Ag/, while control group was without any protective procedure. The animals were followed 5 months in order to study: protective effects of such procedure on the PJA quantified with the percent of anastomotic leakage, effects of the exocrine secretion and effects the endocrine function Results: PJA leakage occurred in 13.33% in control group. No leakage was registered in experimental group. Biochemical, histological and electron microscopic study showed slight transitory elevation of amylase levels. Fibrin glue plug was dissolved and pancreatic juice output was reestablished 12th days postoperatively. Long term follow-up showed no damages of the endocrine and exocrine pancreas. Pancreatic duct occlusion with fibrin glue appeared to be an useful method in the prevention of pancreatico-jejunostomy leakage, without negative effects on the exocrine and endocrine pancreas.

Fractal analysis of STM images of photochemical polymer of coniferyl alcohol.

Fractal analysis was applied to images of photochemical lignin polymer obtained using scanning tunneling microscope. We studied the polymer obtained in vitro by ionic mechanism through UV radiation--induced polymerization. The analysis showed the regularity of the lignin-like polymer at different levels of organization. At the 95% confidence level, there was no significant difference in the fractal dimension between images representing different organizational levels of photochemical lignin. That means that lignin produced in in vitro conditions by photochemical mechanism of synthesis, has a fractal structural organization. The obtained values of the fractal dimension are in good agreement with the theoretically predicted value for the polyaddition and polycondensation mechanism of polymerization, known as the bulk model.

Topographical characterization and surface force spectroscopy of the photochemical lignin model compound.

By combining the results from atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM), herein we investigate properties of photochemical lignin model compounds. We provide evidence that photochemical lignin forms random, probably non-functional structures. The topography of such structures is explored using ESEM. Non-functionality of such structures is proved by AFM and atomic force spectroscopy experiments wherein the photochemical lignin functionalized tip is approached to the substrate covered with photochemical lignin. There was no evidence of existence of any kind of host-guest interaction during the approach/retraction experiments. These results provide evidence for our previously stated hypothesis that photochemical lignin polymerization may be one of the degrading effects of UV radiation to the plant cell.

A Comparative Study of Enzymatically and Photochemically Polymerized Artificial Lignin Supramolecular Structures Using Environmental Scanning Electron Microscopy.

Environmental scanning electron microscopy images of the self-assembled structures of enzymatically (DHP) and photochemically polymerized (PCP) artificial lignin are herein presented. Differences in the structural organization between DHP and PCP polymer at the supramolecular level were reported. Based on topological information, we proposed a hypothesis about possible new physiological roles of lignin in live plant cells and the ecological significance of possible in-vivo photochemical lignin polymerization. Copyright 2000 Academic Press.

Visualization of artificial lignin supramolecular structures

In this paper we are presenting the results of our environmental scanning electron microscopy (ESEM) investigation of the lignin model compound--enzymatically polymerized coniferyl alcohol, also known as dehydrogenate polymer (DHP). The goals of this study were to visualize the supramolecular organization of DHP polymer on various substrates, namely graphite, mica, and glass, and to explore the influence of substrate surface properties and associated collective phenomena on the lignin self-assembled supramolecular structure. Based on results obtained with ESEM, combined with previously published results based on scanning tunneling microscopy (STM) and electron spin resonance (ESR) technique, we looked at lignin structure ranging from a monomer on a fraction of nanometer scale to a large aggregate on a fraction of millimeter scale, therefore using six orders of magnitude range of size. Herein, we are presenting evidence that there are at least four different levels of the supramolecular structure of lignin, and that its supramolecular organization is well dependent on the substrate surface characteristics, such as hydrophobicity, delocalized orbitals, and surface-free energy.

[The study of microviscosity of plasma membranes of Nitella cells during rest and excitation]. / Isuchenie mikroviazkosti plazmaticheskoi membrany kletki Nitella v sostoianiiakh pokoia i vozbuzhdeniia.

Changes in the microviscosity of excitable membranes was investigated using resonance Raman spectroscopy of carotenoids. The Raman resonance spectra of carotenoids in Nitella cells were excited by 514.5 nm line of an argon ion laser. The bands at 1525 cm-1, 1160 cm-1 and 1008 cm-1 were observed and they were assigned to C=C, C-C and C-CH vibrations, respectively. The rhythmic excitation of cell reduced the intensity and increased the ratios of intensity of major carotenoid bands with no noticeable shift in the position of peaks. The Arrhenius plot of relative intensity ratios of 1525 cm-1 and 1160 cm-1 bands versus reciprocal temperature showed a change of the slope in the range of 13-18 degrees C. This indicates a membrane phase transitions in which a reorientation of carotenoids species takes place. The interpretation was supported by parallel microcalorimetric and EPR measurements. The decrease of microviscosity with increasing temperature is probably caused by changes in polyene chain conformation. It is suggested that membrane microviscosity during NH4(+)-stimulated rhythmic excitation of algal cells increases, and membrane-associated carotenoids act as microviscosity-sensitive "potential sensor" for the channel.

Fractal analysis of STM images of lignin polymer obtained by in vitro synthesis.

Lignin, the structural polymer of the plant cell walls, is produced by free radical polymerization of phenolic alcohols, catalyzed by different peroxidases. The mechanism and the structural organization of lignin in the cell have not been completely understood. In this study we applied fractal analysis to images of lignin polymer obtained using scanning tunneling microscope. The analysis showed the regularity of the polymer at different levels of organization. According to the results obtained, at the 95% confidence level, there is no significant difference in the fractal dimension between images representing different organizational levels of lignin. In other words, lignin produced in in vitro conditions has fractal structural organization and, consequently the polymer can be expected to be regular in in vivo conditions. The value of the fractal dimension 1.929 +/- 0.021 is in good agreement with the theoretically predicted value for polyaddition and polycondensation mechanism of polymerization. The mechanism of in vivo lignin synthesis is discussed in terms of various experimental and theoretical evidences. In this paper, we could show that fractal analysis of the lignin polymer is a useful complementary approach to the experimental data collection in structural and phenomenological studies.

Temperature dependence of delayed fluorescence induction curve transients.

The temperature dependence of delayed millisecond fluorescence (DF) induction curve transients was investigated. The transients were obtained by keeping intact maize leaf segments in the dark for 30 to 300 s before illumination. The temperature dependence of DF induction revealed abrupt changes in activation energy of the recombination process. Those changes highly overcome the lowest energy barriers for certain types of chemical reactions, implying significant possible alterations in the DF mechanism itself. On the other hand, the particular transients responses expressed some specificities. Both the electrochemical gradient controlled changes, and the temperature induced changes contribute to it.

Spontaneous ultraweak bioluminescence in plants: origin, mechanisms and properties.

An analysis of theoretical knowledge and experimental results of ultraweak luminescence (UWL) is provided. The role of excited state of molecules and free radicals, formed in various biochemical reactions, in UWL is discussed. UWL of model reactions and in vivo systems are compared. The hypothesis of coherent electromagnetic field as a source of UWL is also discussed. Spectral, kinetic and temporal properties of UWL are summarised, as well as their connection with its origin and role in the organism. Attention is paid to recent progress in experimental methods of low-light detection. The possible use of UWL in environmental studies, selection and other applications is discussed.

[Prevention of thromboembolic disease in gynecologic surgery]. / Prevencija tromboembolijske bolesti kod ginekoloskih operacija.

We presented two groups of patients gynecologically operated and examined in a five year period; the first group of 11536 patients was under thromboembolic protection, whereas the second group of 8532 patients was without thromboembolic protection. Protective measures concerning thromboembolic disease were carried out by applying elastic stockings 24 hours before operation and by early post-operative getting up from bed. Low-molecular dextran was applied before operation as well as during the operation in the amount of 500-1000 ml. In risky patients with varicosities, recidive thrombophlebitis and cardiovascular diseases, we applied small doses of heparin subcutaneously two hours before the operation and after the operation every eight hours five days long. With such prevention of thromboembolic disease in gynecologic surgery, we achieved very favorable effects in reducing mortality to 0.05% concerning the operated, while it amounts to 0.3% in those who were without this kind of protection.

A study of lignin formation at the molecular level by scanning tunneling microscopy.

A scanning tunneling microscope (STM) was used to observe the temporal formation and organization of dehydrogenative polymer (DHP) synthesized from coniferyl alcohol. The images obtained elucidate this structure for the first time. The structure of DHP, as seen from STM images, shows long-range order. It appears that DHP consists of building units or modules assembled into larger assemblies called supermodules. Supermodules are interconnected into the overall lattice-like polymer structure with or without spherical regions. One module consists of about 20 monomers, while the supermodule contains about 500 monomers. Calculated molecular weights for modules and supermodules agree with DHP molecular weight distribution peaks. Samples prepared at two different pH values, 6.4 and 7.6, have the same characteristics. The results presented demonstrate that the process of lignification, even in in vitro conditions, is highly ordered, and as such contribute to our understanding of the structure of lignin, a significant constitutive and functional element of cell walls.

Irreversible structural changes in thylakoid membranes at high temperatures. Detection by luminescence and EPR.

The character of structural changes in thylakoid membranes caused by temperature variation was investigated. Experiments were performed on maize leaf segments in vivo and in a closed temperature cycle 24-50-24 degrees C. Two biophysical methods were used for detection: luminescence and EPR. Arrhenius plots of delayed fluorescence (DF) versus reciprocal temperature revealed two break points, T1 and T2. The MeFASL (10.3) spin probe monitoring properties close to membrane surface detected only T2 transition temperature. The results were interpreted in terms of a fluidity change which starts in a membrane centre at T1 and gradually displaces toward the surface at T2. The T1 and T2 transition temperatures are sensitive to pretreatment history of plants indicating that high temperature and drought-induced membrane alterations are irreversible. Activation energies E1, E2 and E3 were determined for temperature regions below T1 between T1 and T2, and above T2, respectively. The E1 and E3 activation energies showed greater sensitivity to stress than did E2. There are some indications that the DF method could be used to screen temperature sensitive and temperature resistant genotypes.

Effect of propagators and inhibitors on the ultraweak luminescence from maize roots.

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide-radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.

Functional and antigenic antithrombin III values in plasma and serum from 115 regular blood donors and antithrombin III in prediction of postoperative thrombosis.

Antithrombin III (AT-III) was determined functionally using chromogenic substrate (S-2238) and immunologically using radial immunodiffusion (RID) in plasma and serum from 115 blood donors. There was a decrease in functional activity by 43.5% and in antigen concentration by 18.4% during in vitro coagulation when corresponding serum and plasma samples were compared. A positive correlation was found between the two methods in plasma (r = 0.784) as well as in serum ( r = 0.658). RID in serum correlated well with RID in plasma (r = 0.811), but the correlation for S-2238 in plasma and serum was poor (r = 0.411). Functional AT-III decrease during coagulation was uncorrelated with age and fibrinogen, and was not statistically affected by sex, smoking or blood groups. Preoperative functional AT-III activity was measured in 25 patients undergoing elective hip replacement, 60% of whom developed postoperative thrombosis. The functional activity in serum discriminated well between the thrombotic and nonthrombotic group of patients (p less than 0.025), whereas the activity in plasma showed only a minor difference.