Sustained oscillations in living cells

Glycolytic oscillations in yeast have been studied for many years simply by adding a glucose pulse to a suspension of cells and measuring the resulting transient oscillations of NADH. Here we show, using a suspension of yeast cells, that living cells can be kept in a well defined oscillating state indefinitely when starved cells, glucose and cyanide are pumped into a cuvette with outflow of surplus liquid. Our results show that the transitions between stationary and oscillatory behaviour are uniquely described mathematically by the Hopf bifurcation. This result characterizes the dynamical properties close to the transition point. Our perturbation experiments show that the cells remain strongly coupled very close to the transition. Therefore, the transition takes place in each of the cells and is not a desynchronization phenomenon. With these two observations, a study of the kinetic details of glycolysis, as it actually takes place in a living cell, is possible using experiments designed in the framework of nonlinear dynamics. Acetaldehyde is known to synchronize the oscillations. Our results show that glucose is another messenger substance, as long as the glucose transporter is not saturated.     

Three key residues form a critical contact network in a protein folding transition state

Determining how a protein folds is a central problem in structural biology. The rate of folding of many proteins is determined by the transition state, so that a knowledge of its structure is essential for understanding the protein folding reaction. Here we use mutation measurements--which determine the role of individual residues in stabilizing the transition state--as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures that make up the transition state. We apply this approach to the experimental data for the 98-residue protein acylphosphatase, and obtain a transition-state ensemble with the native-state topology and an average root-mean-square deviation of 6 A from the native structure. Although about 20 residues with small positional fluctuations form the structural core of this transition state, the native-like contact network of only three of these residues is sufficient to determine the overall fold of the protein. This result reveals how a nucleation mechanism involving a small number of key residues can lead to folding of a polypeptide chain to its unique native-state structure.     

Local cytoplasmic calcium gradients in living mitotic cells

Cytoplasmic free calcium has been proposed as a regulator of many microtubule-mediated processes, including mitosis. It has been difficult to test this hypothesis because methods for local measurement of free Ca2+ in the living cell have not been available. We have used the fluorescent calcium indicator dye Quin-2 (methoxyquinoline-1bis(o-aminophenoxy)ethane-N,N,N',N' -tetra acetic acid), which allows such observations to be made by digital processing of fluorescent images from the light microscope. Here we report the application of this technique to the study of local Ca2+ concentrations in mitotic endosperm cells of Haemanthus sp., and show that there is transient increase in free Ca2+ at the mitotic spindle poles during anaphase. This locally high Ca2+ may provide a mechanism for the regional control of microtubules and other cytoskeletal elements during anaphase.     

Direct observation of microtubule dynamics in living cells

The study of cell locomotion is fundamental to such diverse processes as embryonic development, wound healing and metastasis. Since microtubules play a role in establishing the leading lamellum and maintaining cell polarity, it is important to understand their dynamic behaviour. In vitro, subunits exchange with polymer by treadmilling and by dynamic instability. Disassembly events can be complete (catastrophic) or incomplete (tempered). In vivo, microtubules are in dynamic equilibrium with subunits with a half-time for turnover of 4-20 min. Microtubules grow by elongation of their ends and are replaced one by one with turnover being most rapid at the periphery. Although previous results are consistent with dynamic instability, we sought to directly test the mechanism of turnover. Direct observations of fluorescent microtubules in the fibroblast lamellum show that individual microtubules undergo rounds of assembly and disassembly from the same end. Reorganization of the microtubule network occurs by a tempered mode of dynamic instability.     

Subcellular localization of chitosan oligosaccharides in living cells

Chitosan oligosaccharides （COS） is derived from the chemical or enzymatic decomposition of chitosan. The exact mechanisms underlying many biological functions of COS have not been elucidated. Since subcellular localization is very important in determining biological activity in a number of molecules, we used fluorescein isothiocyanate-labeled chitosan oligosaccharides （FITC- COS） and investigated the localization of COS in living cells by laser scanning microscopy. We found that COS entered cells in a dose- and time-dependent manner, and we first showed that COS may enter cells by facilitated passive diffusion and was preferentially localized in the mitochondria. While in high concentration, it was also found in the cytoplasm and nucleus and was enriched in the nucleolus and karyotheca. The different subcellular localization of COS suggested that they played different effects. Determination of COS subcellular localization in cells is important to help us understand the potent mechanisms underlying its multiple functions.     

Star-shaped conjugated oligoelectrolyte for bioimaging in living cells

A new star-shaped oligoelectrolyte (TEFCOONa) with triphenylamine as the core, acetylene as linkage and anionic fluorenes as arms was obtained and used for direct imaging in living PANC-1 cells. Because of the hydrophobic conjugated groups of the oli-goelectrolyte, TEFCOONa can form nanospheres with an average diameter of~75 nm in 10 mmol/L PBS. These nanospheres possess a relatively high absolute quantum yield (16.5% in PBS), low cytotoxicity and can penetrate into the nucleus through the cytoplasm, which is essential for living cellular imaging. Collectively, these results validate our rational design of conjugated oligoelectrolyte and even hyper branched polymers-copolyelectrolyte as effective nanovectors for bioimaging and other clinical applications.     

Irreversible association of peptides with class II MHC molecules in living cells.

Functional, morphological and biochemical evidence indicates that class II major histocompatibility complex (MHC) molecules associate with processed peptides during biosynthesis. Peptide/MHC complexes in living cells have been reported to be less stable than similar complexes generated in vitro, which has led to the suggestion that there may be a peptide exchange mechanism operating in vivo. Although this could increase the capacity for binding incoming antigens, it would reduce the efficacy of processed antigenic peptides by exchanging these for self peptides. Here we measure the half-life of peptide/class II complexes in human antigen-presenting cells and find that it is very similar to the half-life of class II molecules themselves, indicating that peptides are bound irreversibly under physiological conditions. Thus class II MHC retains long-term 'memory' of past encounters with antigen to maximize the opportunity for T cell/antigen-presenting cell interaction.     

基因转录的活细胞成像研究技术

转录是基因表达过程中的一个关键调节步骤,也是人类发育和疾病的基础。最近,用活细胞成像方法研究基因转录引起了人们广泛的兴趣．综述了此领域当前的技术进展,并讨论了将来的发展趋势．     

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.     

Migratory neural crest-like cells form body pigmentation in a urochordate embryo

The neural crest, a source of many different cell types in vertebrate embryos, has not been identified in other chordates. Current opinion therefore holds that neural crest cells were a vertebrate innovation. Here we describe a migratory cell population resembling neural crest cells in the ascidian urochordate Ecteinascidia turbinata. Labelling of embryos and larvae with the vital lipophilic dye DiI enabled us to detect cells that emerge from the neural tube, migrate into the body wall and siphon primordia, and subsequently differentiate as pigment cells. These cells express HNK-1 antigen and Zic gene markers of vertebrate neural crest cells. The results suggest that migratory cells with some of the features of neural crest cells are present in the urochordates. Thus, we propose a hypothesis for neural crest evolution beginning with the release of migratory cells from the CNS to produce body pigmentation in the common ancestor of the urochordates and vertebrates. These cells may have gained additional functions or were joined by other cell types to generate the variety of derivatives typical of the vertebrate neural crest.     

New discoveries on the state of hemoglobin in living red blood cells

Professor Qin Wenbin from BaoTou Medical College first identified the hemoglobin(Hb)A2phenomenon 30 years ago.His first paper onthis phenomenon was published in Acta Biochimica et Biophysica Sinica,in Chinese,in1981.Subsequent research investigating its mecha-nism was published in Chinese in the Chinese Biochemical Journal and Progress Biochimica     

Patterns of elevated free calcium and calmodulin activation in living cells.

The temporal and spatial dynamics of intracellular signals and protein effectors are being defined as a result of imaging using fluorescent reagents within living cells. We have described a new class of fluorescent analogues termed optical biosensors, which sense chemical or molecular events through their effects on protein transducers. One example of this new class of indicators is MeroCaM, an environmentally sensitive fluorophore which when it is attached to calmodulin reflects the activation of calmodulin by calcium in vitro. We report here that the rise in free calcium and MeroCaM activation occur in the same period during serum stimulation of quiescent fibroblasts. MeroCaM activation also correlates with the spatial pattern of increased free calcium and the contraction of transverse fibres during wound healing. Finally, migrating fibroblasts in the later stages of wound-healing exhibit an increasing gradient of free calcium and MeroCaM activation from the front to the rear.