Endocytosis is involved in DNA uptake in yeast

The structurally related mammalian alpha and beta isoforms of phosphatidylinositol (PtdIns) transfer protein (PITP) bind reversibly a single phospholipid molecule, preferably PtdIns or phosphatidylcholine (PtdCho), and transport that lipid between membrane surfaces. PITPbeta, but not PITPalpha, is reported extensively in the scientific literature to exhibit the additional capacity to bind and transport sphingomyelin (CerPCho). We undertook a detailed investigation of the lipid binding and transfer specificity of the soluble mammalian PITP isoforms. We employed a variety of donor and acceptor membrane lipid compositions to determine the sensitivity of recombinant rat PITPalpha and PITPbeta isoforms toward PtdIns, PtdCho, CerPCho, and phosphatidate (PtdOH). Results indicated often striking differences in protein-phospholipid and protein-membrane interactions. We demonstrated unequivocally that both isoforms were capable of binding and transferring CerPCho; we confirmed that the beta isoform was the more active. The order of transfer specific activity was similar for both isoforms: PtdIns>PtdCho>CerPCho>PtdOH. Independently, we verified the binding of CerPCho to both isoforms by showing an increase in holoprotein isoelectric point following the exchange of protein-bound phosphatidylglycerol for membrane-associated CerPCho. We conclude that PITPalpha and PITPbeta are able to bind and transport glycero- and sphingophospholipids.     

Genome-wide screening of yeast metal homeostasis genes involved in mitochondrial functions

Metal ions are essential for mitochondria to execute their roles. Yeast mutants that are sensitive to metals (either excess or deficiency) on non-fermentable media but not on fermentable media may carry mutations in genes that participate in metal homeostasis involving mitochondrial functions. A collection of approximately 4,800 haploid yeast deletion mutants was screened for metal ion homeostasis genes linked to mitochondrial respiration. In addition to several well-characterized metal homeostasis genes, 45 new mutants, impaired in various molecular functions, were identified on non-fermentable media that were sensitive to adscititious metals or metal deficiency. While 35 of these mutants displayed metal-sensitivity only on non-fermentable media, the remaining 10 also exhibited metal sensitivity on fermentable media, suggesting metal-sensitivity of the latter is not due to mitochondrial dysfunction. Inductively coupled plasma optical emission spectrometry (ICP-OES) was conducted for 12 mutants that were sensitive to metal excess to analyze their metal contents. Among these 12 mutants 7 were sensitive to metal excess on non-fermentable but not on fermentable media. All the seven respiration-dependent mutants displayed abnormal levels of metal ions inside mitochondria, indicative of disrupted mitochondrial metal homeostasis. This study therefore effectively identified multiple new genes involved in metal homeostasis pathways possibly pertinent to mitochondrial functions, and should be helpful for future studies to further understand their molecular roles.     

Synthetic peptides as carriers for cellular import of drugs

Summary We describe the solid phase synthesis of an amphipathic peptide C-terminated by a cysteamide group which allows further addition after removal from the resin and cleavage of the side-chain protecting groups. The peptide is shown to be rapidly internalized by cells with a nuclear localization of the peptide. When the peptide is linked to an oligonucleotide, the conjugate is also internalized with a final localization that is mainly cytoplasmic.     

Synthetic peptides as carriers for cellular import of drugs

We describe the solid phase synthesis of anamphipathic peptide C-terminated by a cysteamide groupwhich allows further addition after removal from theresin and cleavage of the side-chain protectinggroups. The peptide is shown to be rapidlyinternalized by cells with a nuclear localization ofthe peptide. When the peptide is linked to anoligonucleotide, the conjugate is also internalizedwith a final localization that is mainly cytoplasmic.     

Proteins involved in binding and cellular uptake of nucleic acids

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.     

Rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth. The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions.     

A microfluidic system for investigation of extravascular transport and cellular uptake of drugs in tumors

Three-dimensional (3D) tumor models have been established in various microfluidic systems for drug delivery and resistance studies in vitro. However, one of the main drawbacks of these models is non-uniform distribution of cells, leaving regions with very low cell density within the 3D structures. As a result, molecular diffusion in the cell compartments is faster than that observed in solid tumors. To solve this problem, we developed a new technique for preparation of 3D tumor models in vitro. It was based on a microfluidic device containing three parallel channels separated by narrowly spaced posts. Tumor cells were loaded into the central channel at high density. To test the system, B16.F10 melanoma cells were perfusion-cultured overnight and the resulting 3D structure was characterized in terms of viability, density, and morphology of cells as well as transport properties of small fluorescent molecules. Immediately upon loading of tumor cells, the cell density was comparable to those observed in B16.F10 tumor tissues in vivo; and the viability of tumor cells was maintained through the overnight culture. The tumor model displayed low extracellular space and high resistance to diffusion of small molecules. For membrane-permeant molecules (e.g., Hoechst 33342), the rate of interstitial penetration was extremely slow, compared to membrane-impermeant molecules (e.g., sodium fluorescein). This versatile tumor model could be applied to in vitro studies of transport and cellular uptake of drugs and genes.     

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.     

Genome-wide screen reveals novel mechanisms for regulating cobalt uptake and detoxification in fission yeast

Cobalt is an essential micronutrient but is toxic when present in excess. To study cobalt homeostasis we performed a genome-wide screen for deletion strains that show sensitivity or resistance to CoCl(2). Among 54 cobalt-sensitive strains, 18 are supersensitive strains, which are involved in histidine biosynthetic process, ubiquitination, mitochondria function, membrane trafficking, transporter and a variety of other known functions or still unknown functions. Furthermore, we identified 56 cobalt-resistant deletion strains, which are mainly involved in mitochondria function, signal transduction, ubiquitination, and gene expression and chromatin remodeling. Notably, deletion of the zhf1 (+) gene, encoding a zinc ion transporter, confers supersensitivity to cobalt and overexpression of the zhf1 (+) gene confers marked tolerance to cobalt, indicating that Zhf1 play key roles in cobalt detoxification. Interestingly, all the histidine-auxotrophic mutants displayed cobalt sensitivity and deletion of cationic amino acid transporter Cat1, which was shown to be involved in histidine uptake, suppressed the CoCl(2)-sensitive growth defect of the his2 mutants, suggesting that CoCl(2) may be transported into the cell together with histidine via histidine transporters including Cat1. In addition, we obtained results suggesting that the E2 ubiquitin conjugating enzyme Rhp6 and Sty1 stress MAP kinase pathway are involved in the regulation of cobalt homeostasis. Altogether, our genome-wide study demonstrates for the first time the mechanisms of cobalt homeostasis, particularly its uptake and detoxification in fission yeast.     

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.     

Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin

Clinical application of platinum-based anticancer drugs is largely limited by severe general toxicity and drug resistance. Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of these drugs. This study describes an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin and oxaliplatin) by encapsulating each of them in the cavity of apoferritin (AFt). The encapsulation was achieved through manipulating the pH-dependent unfolding-refolding process of AFt at pH 2.0 and 7.4, respectively, in saturated drug solution. UV-vis spectrometry, circular dichroism spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry were used to characterize the AFt-drug complexes. The loading capacity of AFt varies with respective drugs and the structural integrity of the protein shell remains intact after encapsulation. In vitro assays on the rat pheochromocytoma cell line (PC12) show that AFt-cisplatin inhibits the cells in a slow but sustaining mode and the cellular uptake of platinum is enhanced by AFt. AFt-carboplatin and AFt-oxaliplatin complexes only exhibit a marginal cytotoxicity towards this cell line under similar concentrations.     

Secondary conformational conversion is involved in glycosaminoglycans-mediated cellular uptake of the cationic cell-penetrating peptide PACAP

Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell-penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine-rich CPP pituitary adenylate-cyclase-activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil-to-α-helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs-deficient cells, PACAP α-helix was essential for GAGs-dependent uptake.     

Metal cation uptake by yeast: a review

This review addresses metal uptake specifically by yeast. Metal uptake may be passive, active or both, depending on the viability of the biomass, and is influenced by a number of environmental and experimental factors. Uptake is typically accompanied by a degree of ion exchange and, under certain conditions, may be enhanced by the addition of an energy source, Intracellularly accumulated metal is most readily associated with the cell wall and vacuole but may also be bound by other cellular organelles and biomolecules. The intrinsic biochemical, structural and genetic properties of the yeast cell along with environmental conditions are crucial for its survival when exposed to toxic metals. Conditions of pH, temperature and the presence of additional ions, amongst others, have varying effects on the metal uptake process. We conclude that yeasts have contributed significantly to our understanding of the metal uptake process and suggest directions for future work.     

Genome-wide patterns of histone modifications in yeast

Post-translational histone modifications and histone variants generate complexity in chromatin to enable the many functions of the chromosome. Recent studies have mapped histone modifications across the Saccharomyces cerevisiae genome. These experiments describe how combinations of modified and unmodified states relate to each other and particularly to chromosomal landmarks that include heterochromatin, subtelomeric chromatin, centromeres, origins of replication, promoters and coding regions. Such patterns might be important for the regulation of heterochromatin-mediated silencing, chromosome segregation, DNA replication and gene expression.     

Cell-cycle-dependent regulation of CNT1, a concentrative nucleoside transporter involved in the uptake of cell-cycle-dependent nucleoside-derived anticancer drugs

Most nucleoside-derived anticancer drugs are taken up by the high-affinity Na-dependent nucleoside transporter CNT1. Since such drugs are to some extent cell-cycle-dependent in their cytotoxic action, we examined the relationship between CNT1 expression and cell-cycle progression in the rat hepatoma cell line FAO. Cell cultures were synchronized either at late G1 or early S stages by combining mimosin treatment with either previous synchronization or not by serum starvation. Cell-cycle progression was then assessed by measuring [methyl-3H]thymidine incorporation into DNA and monitoring cyclin E and A protein levels. In these conditions, CNT1 protein amounts increase at the G1-S transition. When cells were synchronized using hydroxyurea (HU), which directly interacts with nucleotide metabolism by inhibiting ribonucleotide reductase, CNT1 protein amounts increased in synchronized cells and remained high during cell-cycle progression. These data indicate that CNT1 adapts to cell-cycle progression and responds to nucleos(t)ide metabolism status, a feature that might contribute to the cytotoxic action of cell-cycle-dependent anticancer drugs.     

Human beta-secretase activity in yeast detected by a novel cellular growth selection system

Sequential processing of the transmembrane amyloid precursor protein (APP) by the beta-secretase BACE and by the gamma-secretase causes secretion of Abeta peptides. Extracellular aggregation of these peptides in the brain is a major hallmark of Alzheimer's disease. For therapeutic purposes and the development of specific inhibitors, it is important to characterize these secretases. We have established a cellular growth selection system for functional expression of human BACE in the yeast Saccharomyces cerevisiae. A fragment of APP bearing the beta-site, the transmembrane domain and the cytosolic tail was fused to the C-terminus of the yeast enzyme invertase, which is normally secreted to allow cell growth in the presence of sucrose as the sole carbon source. The resulting invertase-APP fusion protein was expressed as a type-I transmembrane protein in intracellular compartments of yeast cells lacking endogenous invertase. In these cells, co-expression of human BACE restored cell growth on selective plates upon cleavage of the invertase-APP fusion protein. The cellular growth selection system presented here can be generally applied to screen for secretases that specifically cleave membrane-bound substrates. Furthermore, this system provides the basis for a high-throughput screen for identifying secretase inhibitors that are active in eukaryotic cells.