Phosphatidylcholine and phosphatidylethanolamine enhance the activity of the mammalian mitochondrial endonuclease in vitro

The purified endonuclease of bovine heart mitochondria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved sequence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial DNA replication (Low, R. L., Cummings, O. W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170). If the enzyme serves to provide an important nicking function at this site in vivo, then mitochondrial factors likely exist which further enhance the enzyme's recognition of this locus and prevent cleavage at other less favored sites. In this study, we report that specific membrane phospholipids appear to exert such effects in vitro. In standard endonuclease assays, low levels of phosphatidylcholine or phosphatidylethanolamine (0.5 mM) stimulate the purified enzyme activity 10-20-fold. However, at moderate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome. These findings suggest that an interaction of the endonuclease with major lipid components of the inner membrane could be an important determinant of the enzyme's specificity for mitochondrial DNA.     

Detection of endonuclease activity and several features of chromatin autolysis in brain cell nuclei]

The presence of Ca2+, Mg2+-dependent endonuclease activity in isolated brain cell nuclei was demonstrated and a comparison of some peculiarities of chromatin autolysis in rat brain and liver cell nuclei was carried out. Endogenous brain nuclease hydrolyzes chromatin into its structural subunits; its specific activity is 10,5 times as low as compared to the endogenous nuclease activity in rat liver nuclei. The dependency of the chromatin autolysis rate on pH and ionic composition of the incubation medium in isolated rate brain and liver nuclei appeared to be the same. The presence of Mn2+ changed the autolysis nature both in brain and in liver cell nuclei, the relative (as compared to Mg2+-dependent) Mn2+-dependent activity being higher in the brain cell nuclei. Possible differences of brain and liver chromatin structure (e. g. the presence of regions free of nucleosomic organization in brain chromatin) are assumed.     

Changes in endonuclease activity and in chromatin structure of rat hepatocytes during fetal and neonatal life

A developmental study of rat hepatic endonuclease has been performed. Nuclei, from different stages of hepatocyte maturation, were analyzed for endogenous endonuclease activity. The chromatin extracted from these nuclei does not show any fragmentation during the first 17 days of fetal development. On the 18th day of fetal life there is a massive increase in specific endonuclease activity. At birth this activity reaches a maximum level (3.5 units/mg DNA); thereafter it undergoes a gradual decrease. The size of the basic DNA repeats produced by the endonuclease action is 218.9 +/- 1.6 in 18-day-old fetuses and decreases to 204.9 +/- 2.5 in 19-day-old fetuses, a value which remains constant in the following fetal and postnatal life. This difference in monomer size is due to changes in the chromatin structure. Micrococcal nuclease digests show that the "nucleosome core" does not change during hepatocyte development. Therefore, the difference in size of the endonuclease DNA fragments must be due to the linker regions.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.     

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.     

Small coelomic epithelial cells of the starfish Asterias rubens L. that are able to proliferate in vivo and in vitro

Echinoderms, due to their outstanding potential for regeneration, are widely used as experimental models for research in regenerative biology. One of the main problems in this field concerns identification and characterization of cells responsible for the restoration of lost body parts and organs in adult animals. In this study, we analyze the probable candidates for this role in the starfish Asterias rubens L., namely, small coelomic epithelial cells with a high nuclear–cytoplasmic ratio that have the ability to proliferate. These cells are one of several cell types common to the coelomic epithelium (CE) and coelomic fluid (CF). They are analyzed with respect to morphology, proportion in the total cell pool, dynamics after injury and distribution between CE and CF. The results of whole-mount and scanning electron microscopy provide evidence that these small cells occupy a boundary position between CE and CF. Moreover, a novel subpopulation of CE cells is identified that is enriched (up to 50 %) with small epitheliocytes capable of migrating from CE into the CF. As shown in experiments with BrdU incorporation and anti-phospho-histone H3 antibody staining, small epitheliocytes cultured on laminin retain proliferative activity for at least 1 month and can form colony-like aggregates. Two types of small proliferating cells are distinguished by their behavior in culture: some cells remain attached to the substrate and form aggregates, while others detach from the substrate during culturing. The morphology of small epitheliocytes, their proliferative activity in vivo and in vitro and the ability to migrate suggest that they possess certain properties characteristic of stem cells.     

Variants able to cause growth hormone deficiency syndrome are present within the disease-nil WE strain of lymphocytic choriomeningitis virus.

Persistent infection of C3H/St mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong leads to disordered growth and hypoglycemia. Both host and viral determinants contribute to this growth hormone (GH) deficiency syndrome (GHDS). Development of the GHDS correlates with the virus's ability to replicate in the GH-producing cells and cause reduced levels of GH synthesis. LCMV strain WE infects few GH-producing cells and does not cause GHDS in C3H/St mice. We show here that clonal variants isolated from the GHDS-nil WE population are able to replicate at high levels in GH-producing cells and cause GHDS in C3H/St mice. These variants are stably maintained, but phenotypically silent, within the GHDS-nil WE population.     

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to beta4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.     

Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.     

Purified box C/D snoRNPs are able to reproduce site-specific 2'-O-methylation of target RNA in vitro

Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity.     

In vitro and in vivo dimerization of human endonuclease III stimulates its activity

Human endonuclease III (hNTH1), a DNA glycosylase with associated abasic lyase activity, repairs various mutagenic and toxic-oxidized DNA lesions, including thymine glycol. We demonstrate for the first time that the full-length hNTH1 positively cooperates in product formation as a function of enzyme concentration. The protein concentrations that caused cooperativity in turnover also exhibited dimerization, independent of DNA binding. Earlier we had found that the hNTH1 consists of two domains: a well conserved catalytic domain, and an inhibitory N-terminal tail. The N-terminal truncated proteins neither undergo dimerization, nor do they show cooperativity in turnover, indicating that the homodimerization of hNTH1 is specific and requires the N-terminal tail. Further kinetic analysis at transition states reveals that this homodimerization stimulates an 11-fold increase in the rate of release of the final product, an AP-site with a 3'-nick, and that it does not affect other intermediate reaction rates, including those of DNA N-glycosylase or AP lyase activities that are modulated by previously reported interacting proteins, YB-1, APE1, and XPG. Thus, the site of modulating action of the dimer on the hNTH1 reaction steps is unique. Moreover, the high intranuclear (2.3 microM) and cytosolic (0.65 microM) concentrations of hNTH1 determined here support the possibility of in vivo dimerization; indeed, in vivo protein cross-linking showed the presence of the dimer in the nucleus of HeLa cells. Therefore, it is likely that the dimerization of hNTH1 involving the N-terminal tail masks the inhibitory effect of this tail and plays a critical role in its catalytic turnover in the cell.     

A profile of research on the parasitic trypanosomatids and the diseases they cause

The parasitic trypanosomatids cause lethal and debilitating diseases, the leishmaniases, Chagas disease, and the African trypanosomiases, with major impacts on human and animal health. Sustained research has borne fruit by assisting efforts to reduce the burden of disease and by improving our understanding of fundamental molecular and cell biology. But where has the research primarily been conducted, and which research areas have received the most attention? These questions are addressed below using publication and citation data from the past few decades.     

Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo

Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including 'de novo' sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.     

Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins

Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We use our tagged chromosomal insertion site system to identify small sequences from borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identify YY1 (Ying-Yang1) binding sites as enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and trimethylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning.