Common co-lipids, in synergy, impart high gene transfer properties to transfection-incompetent cationic lipids

Efficacious cationic transfection lipids usually need either DOPE or cholesterol as co-lipid to deliver DNA inside the cell cytoplasm in non-viral gene delivery. If both of these co-lipids fail in imparting gene transfer properties, the cationic lipids are usually considered to be transfection inefficient. Herein, using both the reporter gene assay in CHO, COS-1 and HepG2 cells and the whole cell histochemical X-gal staining assay in representative CHO cells, we demonstrate that common co-lipids DOPE, Cholesterol and DOPC, when act in synergy, are capable of imparting improved gene transfer properties to a novel series of cationic lipids (1-5). Contrastingly, lipids 1-5 became essentially transfection-incompetent when used in combination with each of the pure co-lipid components alone.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

Mitochondrial functional interactions between frataxin and Isu1p, the iron-sulfur cluster scaffold protein, in Saccharomyces cerevisiae

Here we investigated a biological association of constitutively active Src with TrkA in SK-N-MC human neuroblastoma cells. Activation of TrkA and extracellular signal-regulated kinase (ERK) by nerve growth factor (NGF) was inhibited by pretreatment with PP2, an inhibitor of Src family kinases. Moreover, NGF-induced phosphorylation of TrkA and ERK was also attenuated by the transfection with a dominant-negative src construct. On the other hand, the transfection with a constitutively active src construct enhanced these phosphorylations. In addition, we showed that active Src phosphorylates TrkA directly in vitro, and that Src associates with TrkA through Grb2 after NGF stimulation. These results suggest that constitutively active Src that associates with TrkA through Grb2 after NGF stimulation participates in TrkA phosphorylation and in turn enhances the mitogen-activated protein kinase signaling in SK-N-MC cells.     

Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.     

Gsh-2, a murine homeobox gene expressed in the developing brain

A novel murine dispersed homeobox gene, designated Gsh-2, is described. Analysis of cDNA sequence, including the full open reading frame, reveals an encoded homeodomain that is surprisingly similar to those of the Antennapedia-type clustered Hox genes. In addition, the encoded protein includes polyhistidine and polyalanine tracts, as observed for several other genes of developmental significance. In situ hybridizations showed Gsh-2 expression in the developing central nervous system, including the ganglionic eminences of the forebrain, the diencephalon, which gives rise to the thalamus and hypothalamus, and in the hindbrain. Furthermore, a random oligonucleotide selection and PCR amplification procedure was used to define a target DNA binding sequence, CNAATTAG, as a first step towards the identification of downstream target genes.     

Flavonoids inhibit high glucose-induced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids—hesperidin, naringin, and resveratrol—on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.     

Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations.

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.     

Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity.     

OsLTP47 may function in a lipid transfer relay essential for pollen wall development in rice

In plants, lipid transfer proteins(LTPs) transport pollen wall constituents from the tapetum to the exine, a process essential for pollen wall development. However, the functional cooperation of different LTPs in pollen wall development is not well understood. In this study, we have identified and characterized a grassspecific LTP gene, Os LTP47, an important regulator of pollen wall formation in rice(Oryza sativa). Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays conf...     

Zinc deficiency in rats decreases thrombin-stimulated platelet aggregation by lowering protein kinase C activity secondary to impaired calcium uptake

Aggregation of rat platelets, when stimulated by adenosine diphosphate (ADP) or fluoride, is impaired by zinc deficiency, and the defect is associated with a decreased uptake of external Ca²⁺. Zinc deficiency also impairs the aggregatory response of platelets to phorbol myristate acetate (PMA), an activator of protein kinase C, but low zinc status decreases the PMA response only when calcium is added to the external medium. The purpose of this study was to determine the role of protein kinase C in rat platelet function and its relationship to the zinc deficiency pathology observed in platelets stimulated by thrombin (THR). The percent of maximal aggregation and the concentration of cytosolic-free Ca²⁺ were measured in washed platelets stimulated by THR and PMA. For the protein kinase C experiments platelets were obtained from rats fed a grain-based diet, and for the thrombin experiments they were from rats fed purified diets. In the latter experiments, immature male rats were fed for 2 weeks a low zinc diet (<1 mg/kg) ad libitum or a zinc adequate (100 mg/kg) diet either ad libitum or pair-fed. Zinc deficiency impaired the aggregation of platelets stimulated by 0.045 U/mL of THR by approximately 40%, and the external calcium uptake (0.03 U/mL of THR) was decreased by approximately 30%. Staurosporine, a protein kinase C inhibitor, decreased thrombin-induced aggregation in a concentration-dependent manner, but it had no effect on the external calcium uptake. While PMA had a synergistic effect with thrombin in the stimulation of platelet aggregation, it actually decreased the cytosolic-free calcium response to thrombin. It is concluded that zinc deficiency impairs thrombin-stimulated platelet aggregation and calcium uptake and that protein kinase C activity is essential for rat platelet aggregation. Protein kinase C does not stimulate calcium uptake and must act downstream of the calcium uptake defect. A model of rat platelet activation is presented depicting impaired Ca²⁺ uptake as the primary defect in zinc deficiency.     

Saccharomyces cerevisiae CSM1 gene encoding a protein influencing chromosome segregation in meiosis I interacts with elements of the DNA replication complex

We present the characteristics of the Csm1 (Spo86) protein of Saccharomyces cerevisiae that are important for meiotic division. The level of Csm1p does not change throughout the cell cycle, but this protein is absent in mature spores. Deletion of CSM1 causes incorrect spore formation and meiotic chromosome missegregation together with increased sensitivity of vegetative cells to benomyl and manganese. In a two-hybrid analysis with Csm1p as bait, we detected interactions with three members of the Mcm2-7 family of proteins involved in the initiation of DNA replication, and with Clf1p also implicated in replication. The Csm1p-Mcm3, Mcm5 and Mcm7p interactions were confirmed by co-immunoprecipitation. Three other interacting proteins, Mgs1p, Ulp2, and Plp2, participate in chromosome assembling and segregation, whereas the function of two others has not been established. Genetic experiments showed that the two-hybrid isolates MGS1, CLF1, MCM3, 5, 7 (CDC47), and YDL089w, when overexpressed, partially suppress the csm1Delta/csm1Delta sporulation defect. We propose that, besides its other functions, Csm1p may be involved in premeiotic DNA replication.     

Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.     

A Xenopus c-kit-related receptor tyrosine kinase expressed in migrating stem cells of the lateral line system

The mammalian c-kit receptor tyrosine kinase gene is required during embryogenesis for the survival and/or proliferation of three migrating stem cell populations: primordial germ cells, haematopoietic stem cells and neural crest-derived melanoblasts. We have cloned a Xenopus gene, XKrkl, whose closest relative is c-kit. Differences in the expression pattern suggest that XKrkl is not the Xenopus homologue of c-kit; however, it is expressed in a migrating stem cell population, the precursor cells for the mechanosensory lateral line system. XKrkl is the first reported marker for lateral line stem cells.     

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.     

Ectopic lens induction in fish in response to the murine homeobox gene Six3

Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.     

Tectal responses to potassium loads and subsequent visual stimuli in the toad, Bufo bufo

Calling male toads were tested behaviourally for their prey catching responses to wormlike stimuli and assigned to groups of non-hungry and hungry depending on their prey catching motivation before being prepared for visual unit, massed unit and slow potential shift (SPS) recording from the optic tectum. Control recordings to visual stimuli were made before recording the effects of application of isotonic solutions containing concentrations of 0-41 mM K(+). Application of solution was followed by presentation of the visual stimulus while the solution still bathed the tectum. The best tectal responses were made to large square visual stimuli in the non-hungry toads, perhaps because recordings were made in the breeding season. Responses of the tectum to solution addition were significant in the concentration range of 7-17 mM K(+). Hungry toads showed an earlier, smaller response than non-hungry (sexually motivated) animals. When the visual stimulus was presented, there were unit and massed unit responses at all bathing solution concentrations, which were larger in non-hungry animals. These experiments revealed that toads motivated to feed respond earlier than non-hungry toads to application of artificial CSF to the tectum, though non-hungry toads responded best to the subsequent visual stimulus.