酶消化结合组织块培养法用于颞下颌关节盘组织工程细胞扩增的初步研究

目的采用酶消化结合组织块培养法对山羊颞下颌关节（temporomandibular joint，TMJ）盘细胞进行体外培养和扩增，探索TMJ关节盘细胞体外培养及扩增的新方法。方法在无菌条件下，切取一月龄山羊TMJ关节盘，剪至1．0mm^3的碎块，用0．25％胰酶、0．01％I型胶原酶消化关节盘组织块，将消化好的组织块置入6孔板中培养。在倒置显微镜下连续观察细胞的形态变化及贴壁率，甲苯胺蓝染色、I型胶原免疫组化染色进行细胞鉴定，测定其生长曲线。结果原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞，7天贴壁细胞逐渐增多，第10天时细胞彼此相连，铺满平底，细胞以梭形为主，部分多角形。传代后12小时贴壁率达90％，大部分为多角形，4～5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒，胶原免疫纽化染色胞浆内可见棕黄色颗粒。结论酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力，可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。     

Relationship between action potential, contraction-relaxation pattern, and intracellular Ca2+ transient in cardiomyocytes of dogs with chronic heart failure

Abnormalities of contractile function have been identified in cardiomyocytes isolated from failed human hearts and from hearts of animals with experimentally induced heart failure (HF). The mechanism(s) responsible for these functional abnormalities are not fully understood. In the present study, we examined the relationship between action potential duration, pattern of contraction and relaxation, and associated intracellular Ca²⁺ transients in single cardiomyocytes isolated from the left ventricle (LV) of dogs (n = 7) with HF produced by multiple sequential intracoronary microembolizations. Comparisons were made with LV cardiomyocytes isolated from normal dogs. Action potentials were measured in isolated LV cardiomyocytes by perforated patch clamp, Ca²⁺ transients by fluo 3 probe fluorescence, and cardiomyocyte contraction and relaxation by edge movement detector. HF cardiomyocytes exhibited an abnormal pattern of contraction and relaxation characterized by an attenuated initial twitch (spike) followed by a sustained contracture ('dome') of 1 to 8 s in duration and subsequent delayed relaxation. This pattern was more prominent at low stimulation rates (58% at 0.2 Hz, n = 211, 21% at 0.5 Hz, n = 185). Measurements of Ca²⁺ transients in HF cardiomyocytes at 0.2 Hz manifested a similar spike and dome configuration. The dome phase of both the contraction/relaxation pattern and Ca²⁺ transients seen in HF cardiomyocytes coincided with a sustained plateau of the action potential. Shortening of the action potential duration by administration of saxitoxin (100 nM) or lidocaine (30 μM) reduced the duration of the dome phase of both the contraction/relaxation profile as well as that of the Ca²⁺ transient profile. An increase of stimulation rate up to 1 Hz caused shortening of the action potential and disappearance of the spike-dome profile in the majority of HF cardiomyocytes. In HF cardiomyocytes, the action potential and Ca²⁺ transient duration were not significantly different from those measured in normal cells. However, the contraction-relaxation cycle was significantly longer in HF cells (314 ± 67 ms, n = 21, vs. 221 ± 38 ms, n = 46, mean ± SD), indicating impaired excitation-contraction uncou pling in HF cardiomyocytes. The results show that, in cardiomyocytes isolated from dogs with HF, contractile abnormalities and abnormalities of intracellular Ca²⁺ transients at low stimulation rates are characterized by a spike-dome configuration. This abnormal pattern appears to result from prolongation of the action potential. Received 22 January 1998; received after revision 16 March 1998; accepted 27 March 1998     

Down-regulation of sodium current in chronic heart failure: effect of long-term therapy with carvedilol

Evidence has accumulated recently about the importance of alterations in Na⁺ channel function and slow myocardial conduction for arrhythmias in the infarcted and failing heart. The present study tested a hypothesis that Na⁺ current (I_Na/C) density decreases in chronic heart failure (HF) and that Na⁺ channel (NaCh) functional density can be restored by long-term therapy with carvedilol, a mixed α- and β-adrenergic blocker. Studies were performed using a canine model of chronic HF produced in dogs by sequential intracoronary embolizations with microspheres. HF developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 28 ± 1 %). Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium, and I_Na was measured by whole-cell patch-clamp. The maximum I_NA/C was decreased in failing (n = 19) compared to normal (n = 12) hearts (33.1 ± 1.6 vs 48.5 ± 5.1 pA/pF, mean ± SE, p < 0.001). The steady-state inactivation and activation of I_Na remained unchanged in failing compared to normal hearts. Long-term treatment with carvedilol (1 mg/kg, twice daily for 3 months) normalized I_Na/C in dogs with HF. I_Na/C in HF dogs (n = 6) treated with carvedilol was higher compared to that of non-treated HF dogs (n = 6) (49.4 ± 0.9 vs 29 ± 4.8 pA/pF, p < 0.007). In vitro culture of VCs of failing hearts for 24 h did not restore I_Na/C. However, I_Na/C was partially restored when VCs were incubated for 24 h with BAPTA-AM, an intracellular Ca²⁺ buffer. Thus, we conclude that experimental chronic HF in dogs results in down-regulation of the functional density of NaCh that can be restored by long-term therapy with carvedilol. The mechanism of NaCh down-regulation in HF may be linked to poor Ca²⁺ handling in this stage of disease. Received 4 June 2002; received after revision 1 July 2002; accepted 17 July 2002 RID="*" ID="*"Corresponding author.     

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. Received 14 November 1997; received after revision 16 March 1998; accepted 16 March 1998     

The structure of human interferon-β: implications for activity

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-β (HuIFN-β), in particular in relation to its activity. The recently determined crystal structure of HuIFN-β provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-β, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-β. Received 12 June 1998; received after revision 16 July 1998; accepted 16 July 1998     

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K _m 366 ± 118 nM, V _max 2.1 ± 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids. Received 31 March 1998; accepted 8 May 1998     

Visual pigment: G-protein-coupled receptor for light signals

The visual pigment present in photoreceptor cells is a prototypical G-protein-coupled receptor (GPCR) that receives a light signal from the outer environment using a light-absorbing chromophore, 11-cis-retinal. Through cis-trans isomerization of the chromophore, light energy is transduced into chemical free energy, which is in turn utilized for conformational changes in the protein to activate the retinal G-protein. In combination with site-directed mutagenesis, various spectroscopic and biochemical studies identified functional residues responsible for chromophore binding, color regulation, intramolecular signal transduction and G-protein coupling. Extensive studies reveal that these residues are localized into specific domains of visual pigments, suggesting a highly manipulated molecular architecture in visual pigments. In addition to the recent findings on dysfunctional mutations in patients with retinitis pigmentosa or congenital night blindness, the mechanism of intramolecular signal transduction in visual pigments and their evolutionary relationship are discussed. Received 20 July 1998; received after revision 9 September 1998; accepted 23 September 1998     

Pycnogenol enhances immune and haemopoietic functions in senescence-accelerated mice

Pycnogenol (procyanidin extracted from Pinus maritima) has been shown to be a potent free radical scavenger and an antioxidant phytochemical. The effects of pycnogenol on immune and haemopoietic dysfunction in senescence-accelerated mice (SAM), as a murine model of accelerated ageing, were determined. SAMP8, a strain of senile-prone mice, exhibit learning and memory deficits, immunodeficiency and dysfunction of the haemopoietic system. Oral feeding with pycnogenol for 2 months significantly improved their T- and B-cell function. Pycnogenol also augmented the proliferative capacity of haemopoietic progenitors of bone marrow in SAMP8. These data suggest that pycnogenol may be useful for either retardation or restoration of parameters associated with ageing. Received 3 July 1998; accepted 28 July 1998     

Hyperthermia-induced apoptosis and the inhibition of DNA laddering by zinc supplementation and withdrawal of calcium and magnesium in suspension culture of tobacco cells

In the present paper we report examination of stereotypic hallmarks of apoptosis in heat-treated tobacco cells. Hyperthermia (44 °C, 4 h) caused apoptosis in 53.6% of cells when assayed 24 h after heat treatment. The induction of apoptosis by heat treatment was confirmed by flow cytometric assay. Cytological observations revealed condensation of the cytoplasm and nucleus, as well as nuclear collapse. DNA ladders were observed in DNA extracted from heat-treated cells, whereas DNA from control cells remained undegraded. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay revealed that 51.8% of the heat-treated cells (44 °C, 4 h) show positive reaction after a 24-h recovery. When cells were cultured in a medium supplemented with 0.4–5.0 mM ZnSO₄, internucleosomal DNA fragmentation induced by heat shock was completely negated. Strikingly, when cells were cultured in Ca²⁺ and/or Mg²⁺ free medium for 44 h followed by heat treatment, DNA laddering was not observed. The results suggest hyperthermia-induced apoptosis and a correlation between the regula tion of endonucleases and heat shock signal in apoptotic tobacco cells. Received 17 September 1998; received after revision 4 January 1999; accepted 4 January 1999     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

Significance and molecular targets of protein kinase A during cAMP-mediated protection of cold stored liver grafts

The use of marginal donor livers is followed by a higher frequency of primary dys- or nonfunction after transplantation. The present study was designed to test the hypothesis that stimulation of the cAMP second-messenger signal pathway might protect the liver from ischemic injury, laying emphasis on the role of protein kinase A-mediated signal transduction.?Rat livers were harvested after 45 min of cardiac arrest and preserved in HTK solution for 24 h. Hepatic integrity was assessed thereafter using a blood-free reperfusion model.?Supplementation of the preservation solution with dibutyryl-cAMP (db-cAMP) promoted phosphorylation of BAD at Ser 112 and concomitantly mitigated mitochondrial release of cytochrome c into the cytosol. Apoptotic cell transformation was evident in reperfused livers by positive TUNEL-staining of sinusoidal lining cells and the detection of cleaved poly(ADP-ribose) polymerase (PARP) in tissue homogenates by western analysis. Treatment with db-cAMP was effective in minimizing both TUNEL staining and PARP cleavage and significantly reduced postischemic enzyme leakage of alanine aminotransferase to one half, while hepatic bile production was enhanced by approximately 60% when compared to untreated livers. This functional improvement was accompanied by a net amelioration of portal vascular conductivity. Inhibition of A kinase-anchoring protein with HT31 completely reversed any of the observed effects obtained by db-cAMP.?We conclude that enhancement of cellular cAMP signal maintains hepatic integrity during and after ischemic preservation which may be attributed to protein kinase A dependent phosphorylation of BAD in line with subsequent inhibition of mitochondria-initiated apoptosis of sinusoidal lining cells. Received 12 July 2001; received after revision 14 August 2001; accepted 14 August 2001     

Ventral neural tube cells differentiate into hepatocytes in the chick embryo

A population of ventral neural tube cells has recently been shown to migrate out of the hind brain neural tube via the vagus nerve and contribute to the developing gastrointestinal tract. Since liver is also innervated by the vagus nerve, we sought to determine if these cells also migrate into the liver. Ventral neural tube cells in the caudal hindbrain of chick embryos were tagged with a replication-deficient retroviral vector containing the LacZ gene on embryonic day 2. Embryos were processed for detection of labeled cells on embryonic day 5 and 11. Labeled cells were seen in the liver on both days and identified as hepatocytes. Previously, it was believed that all hepatocytes develop from the gut endoderm. Results of the present study show an additional source for the formation of liver cells. Received 25 August 1998; received after revision 5 November 1998; accepted 5 November 1998     

Phylogeny and rapid Northern and Southern Hemisphere speciation of goldfinches during the Miocene and Pliocene Epochs

Mitochondrial cytochrome b (cyt b) from 25 out of 31 extant goldfinches, siskins, greenfinches and redpolls (genus Carduelis) has been sequenced from living samples taken around the world, specimens have also been photographed. Phylogenetic analysis consistently gave the same groups of birds, and this grouping was generally related to geographical proximity. It has been supposed that Pleistocene glaciations played a crucial role in the origin of extant diversity and distribution of Northern Hemisphere vertebrates. Molecular comparison of most extant songbird species belonging to the genus Carduelis does not support this assertion. The fossil record of chicken and pheasant divergence time has been used to calibrate the molecular clock; cyt b DNA dendrograms suggest that speciation in Carduelinae birds occurred during the Miocene and Pliocene Epochs (9 – 2 million years ago) in both the Northern and Southern Hemispheres. Only about 4% average amount of nucleotide substitution per lineage is found between the most distant Carduelis species; this suggests a remarkably rapid radiation when compared with the radiation of other passerine songbird genera. In addition, a continuum of small songbird speciation may be found during the Miocene Epoch in parallel with speciation of other orders (i.e. Galliformes, chicken/pheasant). Pleistocene glaciations may have been important in subspeciation (i.e. Eastern European grey-headed goldfinches/Western European black-headed goldfinches) and also in ice-induced vicariance (isolation) (i.e. siskin in Western Europe vs. siskin in Far East Asia) around the world. European isolated Serinus citrinella (citril finch) is not a canary, but a true goldfinch. South American siskins have quickly radiated in the last 4 million years coinciding with the emergence of the Isthmus of Panama; probably, a North American siskin related to C. notata invaded a suitable and varied biotope (the South American island) for Carduelis birds. North American goldfinches may be renamed as siskins, because they have a distant genetic relationship with European goldfinches. Genus Acanthis could be dropped, and thus redpolls should be separated from twite and linnet, the latter (Europeans) probably being related to American goldfinches. Also, reproductive barriers are observed between closely related species and not between other more distant ones. Finally, a tentative classification for genus Carduelis species is suggested. Received 6 March 1998; received after revision 3 July 1998; accepted 7 July 1998     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same α β dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992), Science 255: 54–63]. The implications of this model and the relevant experiments will be reviewed here. Received 27 April 1998; received after revision 17 July 1998; accepted 10 August 1998     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

Structural view of cadherin-mediated cell-cell adhesion

Following the multiplication of biochemical, biophysical and structural studies describing cadherin molecules and their interactions, several ideas have emerged to explain the mechanisms of cadherin-mediated cell adhesion. Although different models were proposed for cadherin interactions, a consensus has come forth considering lateral dimerization of cadherins as being a central component of the cell-cell adhesion process. This review summarizes the recent development in structural studies of cadherins. Received 14 September 1998; received after revision 14 November 1998; accepted 16 November 1998     

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of α-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes. Received 30 December 1998; accepted 12 January 1999