Modulators of activity of regulatory proteins acting at ultra low doses

New, previously unstudied bioregulators active at ultra low doses, 10⁸ to 10⁻¹⁷ mg protein/ml, were isolated from vitreoretinal tissue. It was shown that these bioregulators contained regulatory peptides and modulators, represented by proteins with a molecular weight of 15–70 kDa, including blood serum albumin. The nanosize of the bioregulators correlated with their activity at ultra low doses.     

The extremely low frequency electrical properties of plant stems

The electrical properties (variation of capacitance and conductance with frequency) of a plant stem can be conveniently measured in vivo by time domain dielectric spectroscopy. In this technique a voltage step is applied to a stem. The resulting polarization current is sampled by a microprocessor and Fourier-transformed to yield these properties. Spectra were obtained for seven electrode separations along a Poinsettia stem. The inverse capacitance and conductance were plotted vs separation for 50 frequencies from .35 to 350 Hz. Least-square fits yielded the effective dielectric constant and conductivity of the stem over this frequency range. In this way electrode effects were eliminated. A similar procedure was carried out for Coleus. A log-log plot of dielectric constant vs frequency shows a two-stage linear decrease for both plants. The conductivity is primarily DC. The dielectric loss decreases smoothly with frequency for Coleus. These results are compared to those for bone and the inorganic material hollandite. The dielectric properties seem best described by a cooperative, many-body approach.     

Stochastic description of the ligand-receptor interaction of biologically active substances at extremely low doses

Signalling molecules can be effective at extraordinarily low concentrations (down to attomolar levels). To handle such cases, probabilistic methods have been used to describe the formal kinetics of action of biologically active substances in these low doses, although it has been necessary to review what is meant by such a term. The mean numbers of transformed/degraded molecules and their dispersions were calculated for the possible range of ligand-receptor binding schemes. We used both analytical equations and numerical simulations to calculate the coefficients of variation (ratio of standard deviation to mean) and demonstrated that the distribution of the coefficient is highly dependent on the reaction scheme. It may, therefore, be used as an additional factor for discriminating between cooperative and noncooperative models of ligand-receptor interaction over extreme ranges of ligand dilution. The relevance to signalling behaviour is discussed.     

Growth and proliferation stimulating effect of extremely small doses of nitrosomethylurea

The increase to 30-50% of spleen weight of intact and with La leukemia C57Bl/6 mice after 5 days i.p. anticancer drug nitrosomethylurea (10(-3)-10(-8) g/kg) injection was observed. The reaction of intact spleen was more expressed than that of leukemic spleen. The growth-stimulating effect of nitrosomethylurea supersmall doses was accompanied with perturbation in 3H-thymidine cell incorporation, that assume a certain change in cell proliferation.     

Neutron dose effect relationships at low doses

Summary Stimulated by recent observations of non-linearity in the dose effect relationship for the transformation of mammalian cells in vitro by fission neutron irradiation and the reverse dose rate effect in this system, the data for mutation induction in the stamen hairs ofTradescantia occidentalis has been re-examined. The non-linear dose effect relationships suggested in the original reports are confirmed both by the dose effect relationships themselves and by an examination of the statistics of the results. This non-linearity also appears to be present in the more recent observations of other workers. It is suggested that the non-linearity in the system may be due to a sub-population of cells in a particularly sensitive phase of the cell cycle at the time of irradiation. There is a possibility that the data also indicate a qualitative difference in the underlying biophysical actions of neutron and photon radiations.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

The effect of activating ligands on the intrinsic fluorescence of guanine nucleotide-binding regulatory proteins

The intensity of the tryptophan fluorescence of the alpha subunits of guanine nucleotide-binding regulatory proteins increases when they bind guanosine 5'-O-(3-thio)triphosphate (GTY gamma S). The kinetics of the fluorescence enhancement and of the measured binding of [35S]GTP gamma S are well correlated. The addition of Mg2+ to the nucleotide-bound proteins causes a further, rapid increase in the fluorescence intensity. Similar effects result from exposure of the proteins to F- and Mg2+, and the required concentration of F- is reduced by the inclusion of Al3+. It is presumed that the more highly fluorescent state of the G protein alpha subunits represents their active conformation.     

Cytogenetic effects of extremely low doses of plutonium-238 alpha-particle irradiation in CHO K-1 cells

CHO K-1 cells were irradiated during the G1 phase with 0.5-6 rad of alpha particles. There was no appreciable cell killing in this low dose range. Significantly increased frequencies of sister-chromatid exchanges were induced by doses as low as 0.5 rad of alpha-particle irradiation, whereas increased numbers of chromosomal aberrations were observed following exposure to 2 rad. These results suggest that very low doses of alpha radiation may lead to radiation-induced genetic alterations.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

抗冻蛋白与植物低温胁迫反应

植物抗冻蛋白是从许多抗冻植物中分离的、参与植物抵御冻害反应的一类新型蛋白.这类抗冻蛋白具有多个亲水性缚冰域,能直接作用于冰晶,阻止冰晶在细胞间隙形成和再结晶.一些植物抗冻蛋白与致病相关蛋白有序列同源性,具有抗冻和抗病双重活性.植物抗冻蛋白的表达和积累,既受控于发育及转录因子调节,又受到低温、短日照、脱水及乙烯等因素的影响.异源超表达抗冻蛋白基因能赋予敏感宿主植物抗冻能力.文中论述了有关植物抗冻蛋白特性和鉴定,抗冻机制和表达调控,以及遗传转化等方面的研究进展.     

Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties

The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.