On the binding of actinomycin and of daunomycin to DNA: a calorimetric and spectroscopic investigation

The “strong” binding of two antibiotics, actinomycin D and daunomycin, to native DNA (calf-thymus) in dilute aqueous solution has been studied by means of calorimetric and spectroscopic measurements. In essence our results show: (1) Daunomycin interaction with DNA is an exothermic process, all features of which depend in a discontinuous way on the fraction of DNA binding sites engaged by the drug. Fluorescence data indicate that such a discontinuous trend should be independent of the GC content of DNA. (2) Actinomycin binding to DNA is, on the contrary, characterized by a positive enthalpy. For such binding, no discontinuity appears discernible with increasing the molar ratio of drug to DNA (phosphorous) on the basis of calorimetric and fluorescence data. (3) Both antibiotics can be bound simultaneously to DNA: our results would suggest that their binding sites on the biopolymer are independent.Discussion is focussed on the possible information derivable from our data on whether or not intercalation may indeed be the main process through which each antibiotic considered “strongly” interacts with DNA.     

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.     

Guilt by association: the nuclear envelope proteome and disease

The discovery that many inherited diseases are linked to interacting nuclear envelope proteins has raised the possibility that human genetic studies could be assisted by a fusion with proteomics. Two principles could be applied. In the first, the proteome of an organelle associated with a genetically variable disease is determined. The chromosomal locations of the genes encoding the organellar proteins are then determined. If a related disease is linked to a large chromosomal region that includes a gene identified in the organelle, then that gene has an increased likelihood of causing the disease. Directly sequencing this allele from patient samples might speed identification compared with further genetic linkage studies as has been demonstrated for multiple diseases associated with the nuclear envelope. The second principle is that if an organelle has been implicated in the pathology of a particular disorder, then comparison of the organelle proteome from control and patient cells might highlight differences that could indicate the causative protein. The distinct, tissue-specific pathologies associated with nuclear envelope diseases suggest that many tissues will have a set of disorders linked to this organelle, and there are numerous as yet unmapped or partially mapped syndromes that could benefit from such an approach.     

Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

The K edges of the heme iron in the x-ray absorption spectra of native and carboxymethylated cytochrome c

The K-absorption edges of the heme iron have been determined from the x-ray absorption spectra of native (Nat) and carboxymethylated (Cm) cytochrome c. The data collected at pH 6.5 and 10 and in both the oxidation states of the hemes indicate that the latter parameter is the one responsible for the observed chemical shifts of the absorption edges in both the cytochromes. The implications regarding a proposed structural model for Cm cytochrome c are discussed     

Control of the yeast cell cycle by protein synthesis

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.     

Three-dimensional reconstruction and quantitative analysis of rat lung type II cells: a computer-based study

The three-dimensional structure of alveolar epithelial type II cells was imaged using a computer-based system designed for reconstruction and quantitative analysis of serially sectioned specimens. Six type II cells were reconstructed from serial ultrathin sections of lungs from two Sprague Dawley male rats and the results were compared to standard morphometric estimates of type II cell composition from five other Sprague Dawley male rats. A minor portion of the type II cell surface was in contact with the alveolar airspace while most of the cell surface was embedded in the alveolar septal interstitium. The type II cells contained multiple Golgi regions located close to the nucleus. Mitochondria formed a few branching filamentous networks extending throughout the cell. The reconstructed cells appeared to represent a homogeneous population having fractional volumes of intracellular organelles very similar to those found by morphometric techniques. The spatial distribution of secretory organelle volume suggests that the organization of this cell type reflects an ordered progression of secretory particle maturation which is consistent with earlier hypotheses of lamellar body assembly.     

Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury.     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

Organelle acidification and disease

A subset of cellular compartments maintain acidic interior environments that are critical for the specific functions of each organelle and for cell growth and survival in general. The pH of each organelle reflects the balance between proton pumping, counterion conductance, and proton leak. Alterations in steady-state organelle pH due to defects in either proton pumping activity or counterion conductance have been suggested to contribute to the pathology of several diseases; however, definitive evidence remains elusive. This review describes recent evidence for the misregulation of organelle pH in the progression of cancer, Dent's disease, and cystic fibrosis .     

Modelling the mechanical properties of single suspension-cultured tomato cells

BACKGROUND AND AIMS: The relationship between composition and structure of plant primary cell walls, and cell mechanical properties is not fully understood, partly because intrinsic properties of walls such as Young's modulus cannot be obtained readily. The aim of this work is to show that Young's modulus of walls of single suspension-cultured tomato cells can be determined by modelling force-deformation data. METHODS: The model simulates the compression of a cell between two flat surfaces, with the cell treated as a liquid-filled sphere with thin compressible walls. The cell wall and membrane were taken to be permeable, but the compression was so fast that water loss could be neglected in the simulations. Force-deformation data were obtained by compressing the cells in micromanipulation experiments. RESULTS:Good fits were obtained between the model and low-strain experimental data, using the modulus and initial inflation of the cell as adjustable parameters. The mean Young's modulus for 2-week-old cells was found to be 2.3 +/- 0.2 GPa at pH 5. This corresponds to an instantaneous bulk modulus of elasticity of approx. 7 MPa, similar to a value found by the pressure probe method. However, Young's modulus is a better parameter, as it should depend only on the composition and structure of the cell wall, not on bulk cell behaviour. This new method has been used to show that Young's modulus of cultured tomato cell walls is at its lowest at pH 4.5, the pH optimum for expansin activity. CONCLUSIONS:The linear elastic model is very suitable for estimating wall Young's modulus from micromanipulation experiments on single tomato cells. This is a powerful method for determining cell wall material properties.     

Localization of Mycoplasma pneumoniae cytadherence-associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure

The terminal organelle of the cell wall-less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane-bound extension of the mycoplasma cell possesses a characteristic electron-dense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence-accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody-based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild-type M. pneumoniae and the hmw2- mutant I-2. The fusion protein was produced in both backgrounds at wild-type levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I-2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electron-dense core of the terminal organelle.     

Reassessment of morphological characteristics in freshwater eels (genus Anguilla, Anguillidae) shows congruence with molecular phylogeny estimates

The morphology-based phylogeny of freshwater eels, proposed by V. Ege in 1939, has been accepted as the basis of eel classification since that time. However, this has been called into question by recent molecular studies. Most of the morphological characteristics recognized by Ege are morphometric. Since methods for the application of morphometric data to phylogeny construction have not been fully established, it is unclear whether the observed discrepancies between morphological and molecular data arise from intrinsic differences or from flawed analyses. Here, we have used two methods to assemble evolutionary trees from distance matrices constructed according to Ege's data, the neighbor-joining (NJ) method and the minimum network (MinNet) method; the latter is based on an evolutionary algorithm. After reanalysing Ege's morphological data, we found that both methods gave results consistent with those based on molecular data, although not with Ege's original classification. Therefore, we speculate that some morphological features Ege used to subdivide the eel groups may not be synapomorphic as he proposed, but symplesiomorphic or convergent . The method developed here may prove useful for constructing phylogeny for taxon groups where only continuous morphometric characteristics are recognized, such as the freshwater eels.     

Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide

The results of an investigation on the interaction of proflavine and of ethidium bromide with DNA (calf thymus) in dilute aqueous solution are reported. The binding of the two dyes by DNA has been studied by means of microcalorimetric and of equilibrium dialysis measurements. Data on the thermodynamics of dimerization of both proflavine and ethidium bromide in aqueous solution obtained on the basis of spectroscopic and/or calorimetric experiments are also reported.The enthalpy data show that dye-dimerization and dye “strong” interaction with DNA are energetically favourable and quite similar while only in the latter case the phenomenon is also entropy driven. This is taken as further evidence in support of the concept that “strong” interaction-of both proflavine and ethidium bromide with DNA means dye molecules intercalation into the native, double helical structure of the biopolymer.     

黑麦雄性细胞发育过程中细胞器DNA动态的荧光研究

DAPI(4’,6-diamidino-2-phenylindole)是一种DNA特异结合的荧光染料,可以用于在荧光显微镜下观察和检测各种DNA,尤其是细胞内含量甚微的DNA,包括质体DNA和线粒体DNA,其灵敏性和可靠性是被公认的,并得到了越来越多的Southern杂交实验的证明,而且实验操作简便易行。近几年,DAPI荧光技术已在细胞质遗传的研究领域获得了成功的应用。     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

The Drosophila fusome,organelle biogenesis and germ cell differentiation: If you build it…

From stem cells to oocyte, Drosophila germ cells undergo a short, defined lineage. Molecular genetic analyses of a collection of female sterile mutations have indicated that a germ cell-specific organelle called the fusome has a central role at several steps in this lineage. The fusome grows from a prominent spherical organelle to an elongated and branched structure that connects all mitotic sisters in a germ cell syncytium. The organelle is assembled from proteins normally found in the membrane skeleton and, additionally, contains an extensive membranous reticulum, the probable product of differentiation-dependent vesicle trafficking. This review briefly summarizes a current view of the processes that drive germ cell differentiation, particularly the various roles that the fusome might play in regulating the developmental events. Future efforts will consider to what extent an organelle assembly-dependent model for differentiation is heuristic and whether the Drosophila fusome represents a homolog of a similar organelle in vertebrate lymphocytes.