Effects of emodin on human erythroleukemia cell line HEL / 中国实验血液学杂志

This study was aimed to investigate the inhibitory effect of emodin on the proliferation of HEL cells, the inducing apoptosis effect of HEL cells and their mechanisms. The proliferation inhibition was detected by MTT method; the change of morphology was observed by AO/EB stains; the cell cycle and apoptosis was analyzed by flow cytometry; the expressions of Akt, P-Akt, P-GSK3β and HSP70 proteins were determined by Western blot. The results indicated that emodin displayed significant anti-proliferative effect on HEL cells in a dose dependent manner(r = 0.99), with IC(50) value of 4.19 µmol/L; AO/EB stains showed that the morphology of HEL cells obviously changed after emodin treatment for 24 hours, and at 24 and 48 hours the apoptosis rates of HEL cells treated by emodin were (27.35 ± 1.68)% and (58.49 ± 1.55)% respectively. Compared with blank control group, the cell ratio in G(0)/G(1) phase increased while that in S phase decreased (p < 0.01); the expression of Akt protein was not changed (p > 0.05), and that of P-Akt, P-GSK3β and HSP70 proteins were down-regulated (p < 0.05). It is concluded that emodin efficiently inhibits the HEL cell proliferation and induces apoptosis of HEL cells, which may be related to the down-regulation of P-Akt, P-GSK3β and HSP70 proteins expression.

Voices of the dead: complex nonlinear vocal signals from the larynx of an ultrasonic frog.

Most anurans are highly vocal but their vocalizations are stereotyped and simple with limited repertoire sizes compared with other vocal vertebrates, presumably because of the limited mechanisms for fine vocal motor control. We recently reported that the call of the concaveeared torrent frog (Amolops tormotus Fei) is an exception in its seemingly endless variety, musical warbling quality, extension of call frequency into the ultrasonic range and the prominence of subharmonics, chaos and other nonlinear features. We now show that the major spectral features of its calls, responsible for this frog's vocal diversity, can be generated by forcing pressurized air through the larynx of euthanized males. Laryngeal specializations for ultrasound appear to include very thin portions of the medial vocal ligaments and reverse sexual size dimorphism of the larynx--being smaller in males than in females. The intricate morphology of the vocal cords, which changes along their length, suggests that nonlinear phenomena probably arise from complex nonlinear oscillatory regimes of separate elastically coupled masses. Amolops is thus the first amphibian for which the intrinsic nonlinear dynamics of its larynx--a relatively simple and expedient mechanism--can account for the species' call complexity, without invoking sophisticated neuromuscular control.

Six specific lysine residues are crucial in maintaining the structure and function of soluble manganese stabilizing protein.

When manganese stabilizing protein (MSP) was treated with 0.5 mM N-succinimidyl propionate (NSP), the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered, in spite of changes of MSP surface Lys residues. Furthermore, far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondary and tertiary structure. Mapping of the sites of NSP modification by Staphylococcus V(8) protease digestion of the modified protein, as well as analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry, indicated that seven Lys residues were modified. The results suggested that these residues are not absolutely essential to the structure and function of MSP. However, when the NSP concentration was increased to 4 mM, the modified MSP was unable to bind photosystem II and completely lost its reactivating capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clear conformational change in MSP after 4 mM NSP treatment, suggesting that some Lys residues are involved in maintaining the structure and function of MSP. Analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that another six Lys residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66 (or Lys76), were modified by 4 mM NSP. Therefore, these six Lys residues are crucial in maintaining the structure and function of soluble MSP.

Ultrasonic communication in frogs.

Among vertebrates, only microchiropteran bats, cetaceans and some rodents are known to produce and detect ultrasounds (frequencies greater than 20 kHz) for the purpose of communication and/or echolocation, suggesting that this capacity might be restricted to mammals. Amphibians, reptiles and most birds generally have limited hearing capacity, with the ability to detect and produce sounds below approximately 12 kHz. Here we report evidence of ultrasonic communication in an amphibian, the concave-eared torrent frog (Amolops tormotus) from Huangshan Hot Springs, China. Males of A. tormotus produce diverse bird-like melodic calls with pronounced frequency modulations that often contain spectral energy in the ultrasonic range. To determine whether A. tormotus communicates using ultrasound to avoid masking by the wideband background noise of local fast-flowing streams, or whether the ultrasound is simply a by-product of the sound-production mechanism, we conducted acoustic playback experiments in the frogs' natural habitat. We found that the audible as well as the ultrasonic components of an A. tormotus call can evoke male vocal responses. Electrophysiological recordings from the auditory midbrain confirmed the ultrasonic hearing capacity of these frogs and that of a sympatric species facing similar environmental constraints. This extraordinary upward extension into the ultrasonic range of both the harmonic content of the advertisement calls and the frog's hearing sensitivity is likely to have co-evolved in response to the intense, predominantly low-frequency ambient noise from local streams. Because amphibians are a distinct evolutionary lineage from microchiropterans and cetaceans (which have evolved ultrasonic hearing to minimize congestion in the frequency bands used for sound communication and to increase hunting efficacy in darkness), ultrasonic perception in these animals represents a new example of independent evolution.

Folding studies of two hydrostatic pressure sensitive proteins.

High hydrostatic pressure combined with various spectroscopies is a powerful technique to study protein folding. An ideal model system for protein folding studies should have the following characteristics. (1) The protein should be sensitive to pressure, so that the protein can be unfolded under mild pressure. (2) The folding process of the protein should be easily modulated by several chemical or physical factors. (3) The folding process should be easily monitored by some spectroscopic parameters. Here, we summarized the pressure induced folding studies of two proteins isolated from spinach photosystem II, namely the 23-kDa and the 33-kDa protein. They have all the characteristics mention above and might be an ideal model protein system for pressure studies.

[Progresses in the study on light harvesting pigment protein complexes and reaction centers from purple bacteria].

This review describes the recent progress in understanding of light harvesting complexes and reaction centers from purple bacteria. Emphasis is paid on the structure of two light harvesting complexes, inner or outer, and the mechanism of the transfer of excited energy among relative pigments (Fig.1). At the same time, it is detailedly stated about the understanding of the structure of the reaction center and the transform mechanism from light energy to chemical energy, usable for life system (Fig.2).

Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

Acetonitrile-induced unfolding of the photosystem II manganese-stabilizing protein studied by electrospray mass spectrometry.

In this paper an acetonitrile-induced unfolding of the manganese-stabilizing protein (MSP) of photosystem II was discovered. More distinct unfolding states of MSP were identified than previously by using mainly electrospray ionization mass spectrometry (ESI-MS), together with fluorescence spectra and far-UV circular dichroism (CD) at pH 2.0, 6.2 or 11.6, and with acetonitrile concentrations from 0 to 50%. At pH 6.2 with acetonitrile concentration changing from 0 to 10%, relatively broad charge-state distributions and poor intensity were observed in ESI-MS, indicating the presence of coexisting conformers. It was concluded that the structure of the MSP protein is unlikely to be a tightly folded form. When the concentration of acetonitrile was 20-40%, simulating the state in the biological membrane, changes in the state of unfolding of MSP were observed to a certain extent using ESI-MS, fluorescence and CD spectroscopy. The charge-state distribution in ESI-MS was found to move toward high states (from 13+ to 27+ to 15+ to 31+) with increasing acetonitrile concentration. At pH 2.0, the MSP structure is rearranged into an unfolded state, and at pH 11.6 the MSP structure is induced to assume another unordered state by deprotonation of appropriate residues. An interesting observation was that a second peak envelope emerged with 20-50% acetonitrile in the medium at pH 11.6.

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II.

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

[Different effects of illumination with xenon and sulfur lamp on growth and development of cotton plants].

Experiments were carried out with cotton (Gossgpium hirsutum cv. Xuzhou 142) plants to study the effects of illumination with xenon and sulfur lamp on development of cotton plants. The results showed that, compared with xenon lamp, illumination with sulfur lamp inhibited excessive elongation of hypocotyl via promotion of longitudinal elongation of epidermis and cortex cells, increased the numbers of branches, buds and bolls significantly. It suggested that illumination with sulfur lamp rendered cotton photomorphogenesis more favorable to yield formation.

[Differences in plant hormone level of cucumber seedlings grown under sulfur or xenon lamp].

The primary mechanism of growth difference of cucumber (Cucumis sativus L.) seedlings cultured under sulfur lamp and xenon lamp in a phytotron was investigated. Compared with cucumber seedlings grown under xenon lamp, those under sulfur lamp were shorter, and the cell number in the middle hypocotyls epidermis and cortex of them were more (Fig. 1, Table 1). Endogenous hormone analysis indicates that the content of IAA and GA(3) of seedlings under sulfur lamp were 17% and 24% lower, while ABA content was 31% higher than that under xenon lamp (Fig. 2). Based on these results, it is suggested that the growth difference between cucumber seedlings grown under sulfur lamp and under xenon lamp might be related to the control of endogenous hormones.

[Thermodynamic and kinetic analysis of unfolding of P23k protein isolated from spinach photosystem II].

The unfolding of 23kD (P23k) protein isolated from spinach photosystem II particle was studied by high pressure and fluorescence spectroscopy. The thermal equilibrium study indicated that the protein could be totally unfolded by 180 or 160 MPa at 20 degrees C and 3 degrees C, respectively. The standard free energy and standard volume change of the protein for unfolding at 20 degrees C is 23.45 kJ/mol and -150.3 ml/mol, respectively. Kinetics study indicated that at 20 degrees C the activation volume for unfolding, delta V(u)(++), was negative (-66.2 ml/mol), meanwhile the activation volume for folding, deltaV(f)(++), was positive (84.1 ml/mol). The rate constants for folding and unfolding (K(0f), K(0u)) were 1.87 s(-1) and 1.3x10(-4) s(-1), respectively, these results provide some clues to explain why the protein is so sensitive to pressure.

Vocal acrobatics in a Chinese frog, Amolops tormotus.

Although amphibians are highly vocal, they generally emit only a limited number of acoustic communication signals. We report here the extraordinarily rich vocal repertoire of Amolops tormotus, a ranid species in China. These frogs produce countless vocalizations, some of which share features of birdsong or primate calls, e.g., ultrasonic frequency components, multiple upward and downward FM sweeps, and sudden onset and offset of selective harmonic components within a call note. Frame-by-frame video analysis of the frog's calling behavior suggests the presence of two pairs of vocal sacs that may contribute to the remarkable call-note complexity in this species. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00114-002-0335-x.

[Fourier transform infrared spectroscopy study on the protein secondary structure of photosystem II reaction center].

A successful study on the secondary structure of the isolated photosystem II (PSII) particles with the Fourier transform infrared spectroscopy is reported in this paper. The beta condensation effect is obviously characterized by infrared absorption spectra. The infrared spectra of both living protein and beta condensed protein samples are measured at room temperature. The amide I band in infrared spectrum is used to perform the quantitative analysis of the sample properties. The recorded spectra show the irreversible effect for the PSII particles after the 400 K heating. A rather strong change of the infrared spectra is observed due to the beta condensation of PSII protein. All the spectra are well fitted by 3-Lorentz-peak. The FTIR spectroscopy shows its effectiveness in studying the heating effect on the PSII particles.

The Ultrafast Energy Transfer Process in Purple Bacteria Photosynthetic Reaction Center.

The ultrafast energy transfer process, which takes place in femtosecond time range, in bacterial photosynthetic reaction center RS601 was investigated using femtosecond pump-probe technique with selective excitation. Upon 755 nmexcitation, the excited state of bacteriopheophytin H decayed to bacteriochlorophyll B with a time constant of about 130 fs, while the excited state of B transported the energy to its energy acceptor, the dimeric bacteriochlorophyll P, in about 240 fs with the 800 nm excitation. The internal conversion process between the upper and lower exciton levels of special pair P might exist upon the excitation of 850 nm pulses. In addition, from the results obtained in our experiments, the charge separation and electron transfer from P to the acceptor H was also observed via the real intermediate B within a few picoseconds.

The Photochemical Change in Photosynthetic Reaction Center of Purple Bacterium with Pheophytin Substitution.

The reaction centers are isolated from chromatophores of Rhodobacter sphaeroides 601 by detergent LDAO, and purified by chromatography on a DEAE-52 cellulose column. In the presence of acetone and an access of free pheophytins (Phes), bacteriopheophytins (Bphes) in reaction centers are replaced by pheophytins at sites H(A) and H(B) when incubated under high temperature. The substituting amounts are about 50% and 71% Bphes in reaction centers with incubation of fifteen and sixty minutes respectively. In the absorption spectra of reaction centers containing Phes (Phe RC), the Q(X) 537 nm and Q(Y) 758 nm bands of Bphe disappeared, three distinct bands assigned to the Q(X 509/542 nm and QY) 674 nm bands of phe appeared. Compared to reaction centers in control, the photochemical activities of Phe RCs, with incubating time of fifteen and sixty minutes, drop to 78 and 71% of that in control respectively.

The Influence of NBS Modification of 241Trp on the Reconstitution of 33 kD Protein with PS and on the Recovery of Oxygen-evolving Activity.

The extrinsic 33 kD protein of photosystemII(PSII) plays an important role in the stabilizing of manganese cluster and maintaining high oxygen-evolving activity of PSII. In this research, (241)Trp, the only tryptophan in the 33 kD protein, was modified by N-Bromosuccinimide. The pH-dependence of modification suggests that this tryptophan is buried in the hydrophobic interior of the protein. The protein's capability of reconstitution to the PSII ecreased after modification, and no oxygen-evolving activity of PS was recovered after the reconstitution. Results suggest that (241)Trp of the 33 kD protein is essential for the binding of the protein to the PS and the normal oxygen-evolving activity.

The Characteristics of DCPIPH(2) right curved arrow MV Electron Transport in Rb. sphaeroides 601.

The absorption spectrum and fluorescence emission spectrum of RS601 were found to keep the typical characteristics of those of the purple nonsulfide bacteria Rb. sphaeroides. Under illumination, methyl viologen was reduced by RS601 chromatophores in the presence of DCPIPH(2) as the electron donor, setting up a standard noncyclic electron transport. o-phenanthroline with I(50) of 1.0 mM inhibited the DCPIPH(2) right curved arrow MV electron transport. Antimycin A did not inhibit the DCPIPH(2) right curved arrow MV electron transport and had no I(50). The results suggested that the exact site where methyl viologen accepted electron should locate between the secondary electron acceptor, Q(B), and cyt b, but not at the Q(A) binding site as indicated before. The difference of electron transport between reduced sides of reaction centers of Rb. sphaeroides and Rs. rubrum was discussed.

Comparison of Different Effects of Chloroacetates on Electron Transports in PS II and in the Reaction Center of Rb. sphaeroides 601.

Chloroacetates displayed different effects on electron transports in the photosynthetic reaction center of purple bacteria (Rb.sphaeroides 601) and in the photosystem II (PS II) of higher plants. Decays of chlorophyII a fluorescence measured after actinic flashes show that chloroacetates inhibit the electron transport from Q(A)(-) to Q(B) (Q(B)(-)) and the equilibrium between Q(A)(-)Q(B) (Q(B)(-)) and Q(A)Q(B)(-) (Q(B)(2-)), acting on electron transport as well as proton transduction. The study on PSII electron transport indicates another inhibition site of chloroacetate at the oxidation side of PSII. Chloroacetates up to 500 mM have no inhibition on the DCPIPH(2) right curved arrow MV electron transport in the RS601's chromatophores. Dissociation constants of OP and trichloroacetates from RS601 reaction center were assessed to be 1.1x10(-3) M and from Dixon curve respectively. The differences on aspects of structures and functions between RS601 reaction center and PS II were discussed.

Studies on the Stimulating Nature of Uncouplers on the Electron Transport in BBY Particles.

BBY particles, which have kept the physicochemical property of PSII, were shown by transmission electron microscopy to possess no intact thylakoid membranes. The results of measuring 9-AA fluorescence quenching and millisecond Chl alpha delayed light emission proved that BBY particles were also unable to establish proton gradient across the membranes (deltapH) in light. Moreover, uncouplers gramicidin D and NH(4)Cl increased PSII electron transport in BBY particles only at low pH. This stimulation was more obvious around pH 6.0 than at other pH. The consistent stimulating value and pH-dependence indicated that the stimulating mechanisms of the two uncouplers are similar. From above, we infer that the uncouplers can bypass the proton transfer of localized pathway in BBY particles, stimulating the corresponding electron transport.