Embryonic development of the European smelt Osmerus eperlanus eperlanus (L.) (Neva population)

The embryonic development of the European smelt Osmerus eperlanus eperlanus has been described. The whole period of the development has been subdivided into 6 subperiods. More than 80 stages may be described for all of these periods. The development of the European smelt embryo was studied in the 5 different regimes of a constant temperature in the range 9.5–18.3°C. The duration of different stages under different temperature regimes was expressed in the relative units of τ₀ (the time of a single cleavage) and τ_s (the time of formation of a single pair of somites). It was established that the τ_s value had proportionally changed by embryogenesis. Due to this fact, the duration of the stages was expressed only as τ_s. The dependency of the speed of the European smelt embryogenesis on temperature has been shown. This dependence was expressed using the standard interval τ_s and described using the equation log τ_s(t) = 3.22665?0.13876t + 0.00297t ², where t is the temperature.     

Effect of laser irradiation on microviscosity of aqueous medium in imbibing maize seeds as studied with a spin probe method

A spin probe method was used to study microviscosity of aqueous medium in embryos and endosperm of maize (Zea mays L.) seeds after their irradiation with a Lvov-1 Elektronika laser device. Based on parameters of electron spin resonance (ESR) spectra of nitroxyl radicals (probes) absorbed by imbibing seeds during water uptake, correlation times τ_C were determined for the rotational diffusion of probes in embryos and endosperm of seeds. The τ_C values were found smaller in embryos of irradiated seeds than in untreated seeds; the dependence of τ_C on the duration of seed imbibition was determined. It is concluded that laser irradiation of seeds decreases the microviscosity of aqueous medium in embryo cells and elevates the mobility of the probes. Effect of laser irradiation on τ_C in seed endosperm was less pronounced but also led to the increase in probe mobility.     

Liquid overlaying improves somatic embryogenesis in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. The process of embryogenesis has been categorized into a few distinct stages (induction, proliferation, maturation and germination) in which liquid overlaying at varying levels 0 ml (T₀), 0.25 ml (T₁), 0.5 ml (T₂)_, 0.75 ml (T₃) and 1.0 ml (T₄) was applied on solid medium. It was found that liquid overlaying improved proliferation; maturation, germination of embryos in C. roseus. In proliferation stage, particularly in T₂, torpedo embryo number increased significantly (i.e. 129.6%) as compared to control. Liquid overlaying (T₂, T₃ and T₄) also improved embryo maturation and showed early germination even in maturation medium. It also accelerated normal embryo germination frequency particularly in treatment with T₂ and shortened ‘embryo—plantlet’ recovery time. Biochemical analyses revealed more proline, protein and amino acid with increasing level of liquid overlaying as it improved embryo induction, development and faster germination.     

Dynamic studies of the unwinding of a DNA-like strand

Following Simon and Zimm's recent work, both molecular dynamic and Monte Carlo methods are used to simulate on a computer the unwinding of a DNA-like (N + 1)-unit helical strand. The strand unwinding is assumed to obey (N + 1)-coupled Langevin equations of motion (N = 10–50). The present computer “experiment” was done to elaborate dependence of unwinding behavior of the helix upon (i) configurations of its complementary strand [represented here by a square cylinder (Simon-Zimm model), a circular cylinder, and a fixed helix (double-helical model)]; (ii) interstrand potentials (a hard-cylinder potential, an inverse twelfth-power soft repulsion with or without an inverse third-power attractive tail); (iii) amplitudes of the Brownian motions, and (iv) the molecular weights of the unwinding strand. We found that (i) the Brownian motions strongly couple with the interstrand potentials to produce a shorter “unwinding time” (τ₀) (which characterizes an expotential decay of the unwinding) for a longer renged repulsive potential (without the Brownian motions, no such effect was present); (ii) addition of an attractive tail to the repulsive potentials further reinforces the unwinding, thereby giving a reduced value of τ₀; (iii) τ₀ can be expressed as a product of two factors–unwinding time for a one-dimensional spring-bead model, which mostly accounts for the N ²-dependence in τ₀, and a factor which depends on the Brownian motions and the interstrand potentials; (iv) among the three models described above, under similar situations, the three-dimensional double-helical model has the smallest τ₀; and (v) unless the maximum Brownian displacement exceeds a certain value, the unwinding around a square cylinder takes place in a (unrealistic) stair-step manner whose τ₀ decreases with increase in the Brownian displacements. The N ²-dependence of τ₀ agrees with the Simon and Zimm's machine results as well as Crother's experimental data on T2 DNA; however, it contradicts experimental data of Massie and Zimm, and others. Further possible improvements in connection with the computer simulation are suggested.     

Mo–Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S₂MoS₂CuS₂MoS₂]^3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The ¹H–¹⁵N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.     

On the quantitative relation between dark kinetics of NPQ-induced changes in variable fluorescence and the activation state of the CF<Subscript>0</Subscript>·CF<Subscript>1</Subscript>·ATPase in leaves

The variable fluorescence at the maximum F_m of the fluorescence induction (Kautsky) curve is known to be substantially suppressed shortly after light adaption due to nonphotochemical q_E quenching. The kinetic pattern of the dark decay at F_m consists of three components with rates ~20, ~1, and ~0.1 s^–1, respectively. Light adaptation has no or little effect on these rate constants. It causes a decrease in the ratio between the amplitudes of the slow and fast one with negligible change in the small amplitude of the ultra-slow component. Results add to evidence for the hypothesis that the dark-reversible decrease in variable fluorescence accompanying light adaptation during the P–S phase of the fluorescence induction curve is due to an alteration in nonphotochemical q_E quenching caused by changes in the trans-thylakoid proton motive force in response to changes in the proton conductance g_H+ of the CF₀-channel of the CF₀·CF₁·ATPase.     

Swelling of hydrocolloid dressings

Wound healing is promoted by dressings that maintain a moist environment. Specifically, hydrocolloid dressings allow excess fluid to escape without permitting wound desiccation. However, the fluid handling capacity of hydrocolloid dressings depends on many factors such as the physicochemical properties of the gel formulation, and the design of the dressing. We measured the moisture uptake kinetics of different hydrocolloid dressings by placing the gel side of a sample in contact with water. The time evolution of the thickness was measured by means of a video camera linked to a computer. The theory of Tanaka and Fillmore 1979 was used to predict the kinetics of uniaxial swelling of a cylindrical gel sample. The model allows to associate to an experimental curve a total thickness increase h_f — h₀ (where h_f and h₀ are respectively the final and initial thickness) and a characteristic time τ. The model also relates h_f — h₀ and τ to the physicochemical composition of the dressing, and to the initial thickness h₀. The influence of h₀ is discussed by means of experiments performed on dressings with different initial thickness.     

The rate of development in poikilothermic animals calculated in astronomical and relative time units

• 1.1.|The problem of comparing the rate of development of various species of poikilothermic animals, using the astronomical time units and relative units of development duration (in the unit of τ₀ — the duration of one mitotic cycle in the period of synchronous cleavage divisions) is considered, a number of fish and amphibian species is taken by example.
• 2.2.|A dimensionless criterion of the relative rate of development (CRR) is proposed. In closely related animal species and genera in the early development CCR = 1, and only later on do some species begin to develop faster than the others, as it takes them less time (measured in a lesser number of τ₀) to pass the identical developmental periods (τ_n).
• 3.3.|In more distant animal groups the same name but not identical developmental periods have different relative duration from the early stages of development, due to gastrulation beginning at different stages of the blastulation, i.e. as a result of heterochronies.

     

The effect of different kinds of electrolyte and non‐electrolyte solutions on the survival rate and morphology of zebrafish Danio rerio embryos

The effect of electrolyte and non‐electrolyte solutions on the survival and on the morphology of zebrafish Danio rerio embryos was investigated. Embryos in different ontogenetic stages were incubated in electrolyte (NaCl, KCl, MgCl₂ and CaCl₂) and non‐electrolyte solutions [sucrose and polyvinylalcohol (PVA)] of different concentrations for 5 – 15 min. The embryos were hatched to the long‐pec stage and the effective concentrations which caused a 50% decrease in embryo development (EC₅₀) were determined. The morphometric changes, which were caused by the test solutions, were measured. Ion channel blockers were used to see if active ion transport played a role for embryo survival. Finally, dechorionated embryos were exposed to the test solutions to get indications about the importance of chorion and perivitelline space. For 12 hours post fertilization (hpf) embryos and a 15 min exposure period, EC₅₀ was highest for MgCl₂ (1·60 mol l^?1), followed by sucrose (0·73 mol l^?1), NaCl (0·49 mol l^?1), KCl (0·44 mol l^?1), CaCl₂ (0·43 mol l^?1) and PVA [0·0005 mol l^?1 (2·2%)]. EC₅₀ were lower for early embryonic stages than for advanced stages for all solutions with exception of MgCl₂ and sucrose. At the EC₅₀, MgCl₂ and CaCl₂ solutions did not induce morphometric changes. NaCl and sucrose solutions induced reversible morphometric changes, which were compensated within 10 min. Only the EC₅₀ of KCl and PVA solutions induced permanent morphometric changes, which could not be compensated. Incubation of embryos in electrolyte and non‐electrolyte solutions together with ouabain (blocker of Na⁺– K⁺ ATPase), HgCl₃ (dose‐dependent inhibition of aquaporine channels), verapamil (inhibition of calcium and magnesium uptake) and amiloride (inhibition of sodium uptake) significantly decreased the per cent of embryos developing to the long‐pec stage in comparison to the same solutions without blockers. Ouabain and HgCl₃ also induced morphometric changes. For dechorionated embryos the survival rates in water and in the different test solutions were similar to untreated embryos.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Changes in DNA methylation during somatic embryogenesis in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 mM NH₄Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.Abbreviations 5-azaC 5-Azacytidine - CRED-RA Coupled restriction enzyme digestion and random amplification - 2,4-D 2,4-Dichlorophenoxyacetic acid - DNMRT Duncans new multiple range test - IAA Indole-3-acetic acid - 5-mC 5-Methylcytosine     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Lipid composition and turnover in heterotrophic cell suspension cultures of Saccharum officinarum

Utilizing radioactively labelled precursors we found that fully heterotrophic sugarcane cells without carotenoids and chlorophyll and absolutely dependent on sucrose for growth were capable of incorporating galactose into both mono- and di-galactosyldiacylglycerol (MGDG and DGDG), sulfate and galactose into sulfolipid (SL), and phosphate into phosphatidylglycerol (PG). All above-mentioned lipids showed turnover allowing the calculation of their half-lifes: τ_0.5 = 28 h for MGDG. There is an initial increment in the labelling of DGDG (at the expense of MGDG) and then a decrement with τ_0.5 = 27 h. The SL shows a τ_0.5 = 30 h and τ_0.5 = 28 h for PG. It is evident that during differentiation changes in lipid metabolism occur allowing the cell not only to increase, but to localize preferentially the biosynthetic machinery of the above-mentioned lipids in the chloroplasts and to make the process light-dependent.     

Treatment of Obese Adolescents: The Influence of Periodization Models and ACE Genotype

The aims of the present study were to compare the effects of two periodization models on metabolic syndrome risk factors in obese adolescents and verify whether the angiotensin‐converting enzyme (ACE) genotype is important in establishing these effects. A total of 32 postpuberty obese adolescents were submitted to aerobic training (AT) and resistance training (RT) for 14 weeks. The subjects were divided into linear periodization (LP, n = 16) or daily undulating periodization (DUP, n = 16). Body composition, visceral and subcutaneous fat, glycemia, insulinemia, homeostasis model assessment of insulin resistance (HOMA‐IR), lipid profiles, blood pressure, maximal oxygen consumption (VO_2max), resting metabolic rate (RMR), muscular endurance were analyzed at baseline and after intervention. Both groups demonstrated a significant reduction in body mass, BMI, body fat, visceral and subcutaneous fat, total and low‐density lipoprotein cholesterol, blood pressure and an increase in fat‐free mass, VO_2max, and muscular endurance. However, only DUP promoted a reduction in insulin concentrations and HOMA‐IR. It is important to emphasize that there was no statics difference between LP and DUP groups; however, it appears that there may be bigger changes in the DUP than LP group in some of the metabolic syndrome risk factors in obese adolescents with regard to the effect size (ES). Both periodization models presented a large effect on muscular endurance. Despite the limitation of sample size, our results suggested that the ACE genotype may influence the functional and metabolic characteristics of obese adolescents and may be considered in the future strategies for massive obesity control.     

Changes in the kinetics of plasma chemiluminescence as a measure of systemic oxidative stress in humans

Oxidative stress acts as a pathogenetic factor in many diseases; estimating its level is important for early diagnosis and therapy adjustment. The antioxidant status was evaluated for the blood plasma. A set of chemiluminescence (CL) kinetic-curve parameters (latent period τ_lat and analytical signal increment ΔI_CL) in a 2,2’-azo-bis(2-amidinopropane)dihydrochloride–luminol system were proposed for estimating the oxidative stress level. Uric acid and albumin were identified as major components that are responsible for the changes in the plasma CL kinetic curve. UV light caused oxidative modification of serum albumin in a dose-dependent manner, thus enhancing its antioxidant properties. Changes in plasma CL kinetics were proposed as a means to measure oxidative stress in the human body.     

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P₂‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P₂ generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P₂ in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P₂ exchange between late and recycling endosomes.     

Water chemistry in the microenvironment of rainbow trout Oncorhynchus mykiss embryos is affected by development,the egg capsule and crowding

The hypothesis tested was that embryonic metabolism affects the water chemistry in the boundary layer. In addition, embryo crowding would further compound the metabolic effect on the water chemistry in the boundary layer. As development progressed, the magnitude of the boundary layer gradients for O₂ and pH, but not for NH, increased. The presence of the egg capsule hindered the diffusion of O₂ into and H⁺ and NH out of the embryo. The magnitude of the O₂, pH and NH boundary layer gradient was significantly increased when embryos were surrounded by either sham embryos or live embryos. The majority of this crowding effect on embryo boundary layers was due to changes in water flow rather than due to metabolism directly. These results clearly show that the microenvironment adjacent to the developing rainbow trout Oncorhynchus mykiss embryo becomes more stagnant as development progresses in the presence of the egg capsule and is further intensified with embryo crowding.