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PubMed1999

Characterization of a brain-specific nuclear LIM domain protein (FHL1B) which is an alternatively spliced variant of FHL1.

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Alternative Splicing↗
PubMed2004

Exploring the quantitative relationship between the level of MDR1 transcript, protein and function using digoxin as a marker of MDR1-dependent drug efflux activity.

A limited number of gene expression studies have investigated the quantitative relationships between the amount of transcript, level of protein or activity/function, with disparate conclusions regarding these relationships. Collectively these studies indicate that the relevance of quantitative transcript analysis as a predictor of phenotype has to be evaluated on a gene-by-gene or even a case-by-case basis. The purpose of this study was to define a suitable marker for MDR1-dependent drug efflux, and to quantitatively investigate the relationships between the amount of transcript, protein and drug efflux in the frequently used Caco-2 cell model. The substrate specificity of digoxin, a commonly used marker for MDR1, was investigated using transgenic MDCK II or LLC-PK1 cell lines expressing the efflux proteins MDR1, BCRP and MRP2, since these proteins are localised to the apical part of the enterocyte plasma membrane and exhibit comparatively high transcript levels in the human small intestine. Relationships between levels of transcript, protein and function were investigated quantitatively using real-time RT-PCR, ECL western blot analysis and basolateral-to-apical and apical-to-basolateral efflux ratios. Our results indicate that digoxin is a specific marker for MDR1-dependent drug efflux in the Caco-2 cell drug absorption model and that MDR1 transcript abundance is at least as valid as MDR1 protein abundance as a predictor of MDR1 efflux activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Affinity capture of specific DNA-binding proteins for mass spectrometric identification.

We describe a general approach for affinity microcapture of site-specific, nucleic acid-binding proteins. The major difficulties to developing this method into a widely applicable protocol derived from the need for a massive enrichment and the inadvertent, extensive binding of nonspecific proteins to the bait. On the basis of a detailed analysis, we propose (i) a one-step fractionation of crude extracts on P11 phosphocellulose, followed by (ii) a discrete series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format, with cobalt magnets, concatamerized and biotinylated ligands, selective salt conditions, and improved competitor DNAs. We also present rules for determining the precise number and order of selections. The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2 x 10(9) cultured leukemia cells. Captured proteins were 10-20,000-fold enriched from the nuclear extract, in a form and amounts that permitted facile MALDI-TOF and TOF/TOF MS-based protein identification. This is 1-2 orders of magnitude better than many previous efforts and in a fraction of the time (approximately 1 factor/week). The method can be applied to any protein that binds DNA, including those with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regulation, and DNA repair with the analytical power of mass spectrometry-based proteomics.

Cell Line, Tumor↗
PubMed1997

Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.

Amino Acid Sequence↗
PubMed1998

The molecular basis of C4 photosynthesis in sorghum: isolation, characterization and RFLP mapping of mesophyll- and bundle-sheath-specific cDNAs obtained by differential screening.

C4 photosynthesis depends upon the strict compartmentalization of the CO2-assimilatory enzymes of the C4 and Calvin cycle in two different cell types, mesophyll and bundle-sheath cells. A differential accumulation is also observed for enzymes of other metabolic pathways, and mesophyll and bundle-sheath chloroplasts of NADP-malic enzyme type C4 plants differ even in their photosynthetic electron transport chains. A large number of studies indicate that this division of labour between mesophyll and bundle-sheath cells is the result of differential gene expression. To investigate the extent of this differential gene expression and thus gain insight into the genetic basis of C4 photosynthesis, genes that are differentially expressed in the mesophyll and bundle-sheath cells were catalogued in the NADP-malic enzyme type C4 grass Sorghum bicolor. A total of 58 cDNAs were isolated by differential screening. Using a tenfold difference in transcript abundance between mesophyll and bundle-sheath cells as a criterion, 25 cDNAs were confirmed to encode mesophyll-specific gene sequences and 8 were found to encode bundle-sheath-specific sequences. Eight mesophyll-specific cDNAs showed no significant similarities within GenBank and may therefore represent candidates for the elucidation of hitherto unknown functions in the differentiation of mesophyll and bundle-sheath cells. The chromosomal location of 50 isolated cDNAs was determined by RFLP mapping using an interspecific sorghum cross.

Base Sequence↗
PubMed1998

The expression of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed-specific and related to dormancy.

We have isolated a gene, AtPer1, from the dicotyledon Arabidopsis thaliana, which shows similarity to the 1-cysteine (1-Cys) peroxiredoxin family of antioxidants. In higher plants, members of this group of antioxidants have previously only been isolated from monocotyledons. It has been suggested that seed peroxiredoxins protect tissues from reactive oxygen species during desiccation and early imbibition and/or are involved in the maintenance of/protection during dormancy. AtPer1 expression is restricted to seeds. Despite differences in seed development between monocots and dicots, AtPer1 shows an expression pattern during seed development and germination similar to the dormancy-related transcript Per1 in barley. In situ hybridization identifies AtPer1 as the first aleurone-expressed transcript characterized in developing Arabidopsis seeds. The transcript is also expressed in the embryo. AtPer1 expression in seeds is unaltered in an ABA-deficient mutant (aba-1) during seed development, while expression in seeds of an ABA-insensitive mutant (abi3-1) is reduced. The transcript is not induced in vegetative tissue in response to stress by ABA or drought. AtPer1 transcript levels are correlated to germination frequencies of wildtype seeds, but AtPer1 transcript abundance is not sufficient for expression of dormancy in non-dormant mutants. Hypotheses on peroxiredoxin function are discussed in view of the results presented here.

Amino Acid Sequence↗
PubMed2001

Identical promoter elements are involved in regulation of the OPR1 gene by senescence and jasmonic acid in Arabidopsis.

Like other developmental processes, the terminal phase of leaf development, generally referred to as leaf senescence. regulates a subset of genes whose transcript abundances are increased during senescence. Jasmonic acid (JA), a plant growth regulator, also regulates the expression of subsets of genes in many aspects of plant growth and development, including leaf senescence. However, the underlying molecular mechanisms by which senescence and JA modulate gene expression are poorly understood. During an effort to isolate senescence-associated genes, we identified an Arabidopsis enhancer trap line in which the reporter gene GUS is up-regulated by both senescence and JA. The T-DNA tagged gene was subsequently cloned using thermal asymmetric interlaced PCR (TAIL-PCR). This gene encodes a 12-oxo-phytodienoic acid-10,11-reductase (OPR1). Consistent with the GUS expression data, RNA gel blot analysis showed that OPR1 was indeed up-regulated by both senescence and JA. Promoter deletion analysis and linker-scanning mutagenesis assays were employed to unveil the molecular bases of OPR1 regulation by senescence and JA. Two regulatory cis elements, namely JASE1 (5'-CGTCAATGAA-3') and JASE2 (5'-CATACGTCGTCAA-3'), in the promoter region of the gene, were identified. While JASE2 contains a mixed A/C box-like motif, JASE1 represents a new motif without any signature sequence so far reported. Both elements were required for the up-regulation of OPR1 by leaf senescence and JA. suggesting that leaf senescence and JA may share a common molecular mechanism for modulating OPR1.

Arabidopsis↗
PubMed2002

Lysosomal enzymes, cathepsins in brain tumour invasion.

The expression patterns of different classes of peptidases in central nervous system (CNS) tumours have been most extensively studied in astrocytomas and meningiomas. Although the two types of tumours are very different in most respects, both may invade locally into normal brain. This process of invasion includes increased synthesis and secretion of lysosomal proteolytic enzymes - cathepsins. Aspartic endopeptidase cathepsin (Cat) D levels were found to be elevated in high-grade astrocytoma and partial inhibition of glioblastoma cell invasion by anti-Cat D antibody suggests that the enzyme activity is involved in the invasion process. Several studies on cysteine endopeptidase (CP) Cat B in gliomas agreed that transcript abundance, protein level and activity of Cat B increased in high-grade astrocytoma cultures compared with low-grade astrocytoma cultures and normal brain. Moreover, in glioma biopsies Cat B levels correlated with evidence of clinical invasion and it has been demonstrated that Cat B both in tumour cells and in endothelial cells can serve as a new biological marker for prognosis in glioblastoma patients. A high level of Cat B protein was also a diagnostic marker for invasive types of meningioma, distinguishing between histomorphologically benign, but invasive meningiomas and noninvasive, so-called clear-benign meningiomas. Cat L was also significantly increased in high-grade astrocytoma compared with low-grade astrocytoma and normal brain. Specific Cat L antibodies and antisense Cat L RNA transfection significantly lowered glioblastoma cell invasion. In meningioma, Cat L was a less-significant marker of invasion than Cat B. In contrast to cathepsins, the activities of endogenous cysteine peptidase inhibitors (CPIs), including stefins, cystatins and kininogens, were significantly higher in benign and atypical meningioma cell extracts than in malignant meningioma, and low-grade compared to high-grade astrocytoma. However, very low levels of stefins A and B were found in meningioma and glioblastoma tissues. Further studies on the expression levels and balance between cysteine endopeptidases (CPs) and CPIs would improve the clinical application of cathepsins in prognosis, which would lead to more-informed therapeutic strategies.

Animals↗
PubMed2003

Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes.

We previously isolated a partial soybean cDNA clone whose transcript abundance is increased upon infection by the sedentary, endoparasitic soybean cyst nematode Heterodera glycines. We now isolated the corresponding full-length cDNA and determined that the predicted gene product was similar to the group of cofactor-dependent phosphoglycerate mutase/bisphosphoglycerate mutase enzymes (PGM/bPGM; EC 5.4.2.1/5.4.2.4). We designated the corresponding soybean gene GmPGM. PGM and bPGM are key catalysts of glycolysis that have been well characterized in animals but not plants. Using the GmPGM cDNA sequence, we identified a homologous Arabidopsis thaliana gene, which we designated AtPGM. Histochemical GUS analyses of transgenic Arabidopsis plants containing the AtPGM promoter ::GUS construct revealed that the AtPGM promoter directs GUS expression in uninfected plants only to the shoot and root apical meristems. In infected plants, GUS staining also is evident in the nematode feeding structures induced by the cyst nematode Heterodera schachtii and by the root-knot nematode Meloidogyne incognita. Furthermore, we discovered that the AtPGM promoter was down-regulated by abscisic acid and hydroxyurea, whereas it was induced by sucrose, oryzalin, and auxin, thereby revealing expression characteristics typical of genes with roles in meristematic cells. Assessment of the auxin-inducible AUX1 gene promoter (a gene coding for a polar auxin transport protein) similarly revealed feeding cell and meristem expression, suggesting that auxin may be responsible for the observed tissue specificity of the AtPGM promoter. These results provide first insight into the possible roles of PGM/bPGM in plant physiology and in plant-pathogen interactions.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of a NaCl-inducible vacuolar Na+/H+ antiporter in Beta vulgaris.

We have cloned, by RT-PCR and the use of degenerate oligonucleotide primers, a Na+/H+ antiporter from Beta vulgaris that is homologous to NHX1 of Arabidopsis thaliana and is a member of the family of recently cloned plant NHX-genes. This antiporter, BvNHX1, partially complements the salt-sensitive phenotype of a Deltaena1-4Deltanhx1 yeast strain. Antibodies were raised against a central portion of the BvNHX1 open reading frame that was predicted from the cloned cDNA. This antiporter was found to be highly enriched in tonoplast membranes isolated from plant tissues. BvNHX1 transcript abundance increased after salt treatments, in both suspension-cell cultures and whole plants. BvNHX1 protein abundance in the tonoplast-enriched membranes was also elevated after salt treatments. The vacuolar Na+/H+ antiporter activity increased up to 3-fold when the cell were exposed to 100 mM NaCl. The increase in protein abundance in response to the salt treatment, together with the salt-induced vacuolar Na+/H+ antiporter activity in B. vulgaris suggests that BvNHX1 plays an important role in salinity tolerance.

Journal Article↗
PubMed2003

Differential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development.

In this study, we report the cloning of the three-member LePS2 gene family of acid phosphatases via subtractive screening of a cDNA library of Pi-starved cultivated tomato cells (Lycopersicon esculentum Mill. cv. Lukullus). As members of the plant Pi-starvation response, LePS2 genes were tightly regulated in cultivated cells and tomato seedlings by Pi availability. The LePS2 enzymes which are most likely expressed in the cytoplasma could be involved in processes that are accompanied by degradation of phosphorylated organic substrates. Independently from exogenous phosphate supply LePS2 expression was detected in tomato endosperm during germination. LePS2 genes were differentially induced after infection with the bacterial pathogen Pseudomonas syringae and in the early stages of flower development. Using RT-PCR it was found that the gene LePS2B was the most abundant transcript in phosphate-depleted cells, but a reduced expression was determined in floral buds and it was not found during pathogen interaction. In this respect, it is interesting that the promoter sequences of the LePS2 genes are also divergent. LePS2 gene products may have functions in developmental processes which are restricted to distinct plant tissues or cell types.

Journal Article↗
PubMed2005

A mutation accumulation assay reveals a broad capacity for rapid evolution of gene expression.

Mutation is the ultimate source of biological diversity because it generates the variation that fuels evolution. Gene expression is the first step by which an organism translates genetic information into developmental change. Here we estimate the rate at which mutation produces new variation in gene expression by measuring transcript abundances across the genome during the onset of metamorphosis in 12 initially identical Drosophila melanogaster lines that independently accumulated mutations for 200 generations. We find statistically significant mutational variation for 39% of the genome and a wide range of variability across corresponding genes. As genes are upregulated in development their variability decreases, and as they are downregulated it increases, indicating that developmental context affects the evolution of gene expression. A strong correlation between mutational variance and environmental variance shows that there is the potential for widespread canalization. By comparing the evolutionary rates that we report here with differences between species, we conclude that gene expression does not evolve according to strictly neutral models. Although spontaneous mutations have the potential to generate abundant variation in gene expression, natural variation is relatively constrained.

Animals↗
PubMed2004

Gene expression and adiposity are modified by soy protein in male Zucker diabetic fatty rats.

It has earlier been demonstrated that soy protein diets ameliorate the diabetic phenotype in obese Zucker rats. In this study, we further investigated physiological changes related to adiposity in male Zucker diabetic fatty rats consuming soy-based diets and compared these diets with the insulin-sensitizing drug, rosiglitazone. Transcript abundance of known genes was assessed in the livers to identify potential molecular connections between soy diets and adiposity. Male Zucker diabetic fatty rats were assigned to casein (C) protein, low-isoflavone soy (LIS) protein, high-isoflavone soy (HIS) protein, or C + rosiglitazone (CR) diets. Compared with the C diet, the LIS diet decreased plasma lipids and increased body weight, but did not change liver weight or carcass adiposity. HIS decreased plasma lipids, liver weight, and body weight. CR decreased plasma lipids and increased carcass adiposity and body weight with no effect on liver weight. In LIS livers, 15 genes involved in signaling and lipid metabolism were up-regulated 2-fold or higher. In HIS livers, seven genes had a 2-fold or higher change in abundance. However, in CR livers, none of the genes was significantly changed compared with the C diet. There appears to be a distinct change in gene expression associated with soy diets as compared with C-based diets and rosiglitazone treatment.

Adipose Tissue↗
PubMed2025

Genome-wide identification of potassium transporters and channels in Malus domestica genome.

Potassium (K+) is an essential nutrient for plants. It contributes to most physiological and biochemical pathways for plant metabolism, growth, and development. It is the most available plant nutrient, comprising 10–15% of plant weight. Plants have a sophisticated system of K+ transporters and channels for distribution in plant body. Apple is one of the most consumed fruits in the world. Its fruit quality and yield are positively affected by K+. However, limited information is available about K+ transport systems in Apple. In this study, 47 candidate genes (26 K+ transporters and 21 K+ channels) have been identified in Apple (Malus domestica) genome. The phylogenetic comparisons with other plants (Glycine max, Arabidopsis thaliana, and Oryza sativa) indicated that the K+ transport system is much conserved among different plants. The analysis of Gene structure showed the presence of specific introns and exon patterns for these gene families. Transcriptomic data analysis and RT-qPCR demonstrated significant variations in the transcript abundance of these genes in response to abiotic stresses. The current project represents the first report about the K+ transport system in Apple. Therefore, it may act as a starting point for further functional characterizations.

Malus↗
PubMed2025

Long non-coding RNA NEAT1 promotes colorectal cancer progression via interacting with SIRT1.

Nuclear-enriched abundant transcript 1 (NEAT1), a long noncoding RNA, is found to be significantly dysregulated in different types of cancer, including colorectal cancer (CRC). Nevertheless, there is still much to learn about the precise functions and processes of NEAT1 in the progression of CRC. Using The Cancer Genome Atlas (TCGA) database and 50 CRC specimens from the First Affiliated Hospital of Dali University, we assessed the expression of NEAT1 to determine its clinical impact. Through gene set enrichment analysis (GSEA), Cancer Single-cell State Atlas (CancerSEA), and immune infiltration studies, we elucidated key functions of NEAT1. We utilized Cell Counting Kit-8 (CCK8), wound healing, and Transwell assays to investigate the role of NEAT1 in the progression of CRC. Through the use of GSEA and immunohistochemistry, additional investigations were conducted to unveil the downstream targets of NEAT1 and gain insights into their regulatory dynamics. Our in vitro studies confirmed the regulatory role of NEAT1 in CRC. Findings indicate that increased NEAT1 expression correlates with adverse outcomes in colorectal tissues. In the CRC model, reduced levels of NEAT1 lead to reduced cell proliferation, invasion, and migration. Additionally, NEAT1 influenced immune cell infiltration in CRC and functioned as an oncogene by upregulating Sirtuin 1 (SIRT1) expression. This study demonstrates that NEAT1 promotes CRC progression and metastasis through a SIRT1-mediated mechanism, suggesting its potential as a prognostic biomarker and therapeutic target for CRC.

RNA, Long Noncoding↗
PubMed2003

Tachykinins and tachykinin receptors in human uterus.

(1) Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell-surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. (2) The neurokinin B (NKB) precursor PPT-B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT-A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT-C, the gene encoding the novel tachykinin peptide hemokinin-1 (HK-1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. (3) Tachykinin NK(2) receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK(1) receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK(3) receptor was present only in tissues from nonpregnant women. (4) The mRNA expression of the tachykinin-degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. (5) Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA>>SP>/=NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. (6) The tachykinin NK(2) receptor-selective agonist [Lys(5)MeLeu(9)Nle(10)]NKA(4-l0) was approximately equipotent with NKA, but the tachykinin NK(1) and NK(3) receptor-selective agonists [Sar(9)Met(O(2))(11)]SP and [MePhe(7)]NKB were ineffective in the myometrium from nonpregnant women. (7) The uterotonic effects of [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) were antagonized by the tachykinin NK(2) receptor-selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys(5)MeLeu(9)Nle(10)]NKA(4-10). (8) These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK(2) receptors in the regulation of myometrial contraction.

Adult↗
PubMed2006

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Animals↗
PubMed2006

ABC transporter expression profiling after ischemic reperfusion injury in mouse kidney.

Renal ATP binding cassette (ABC) transporters have an important role in the elimination of metabolic waste products and compounds foreign to the body. The kidney has the ability to tightly control the expression of these efflux transporters to maintain homeostasis, and as a major mechanism of adaptation to environmental stress. In the present study, we investigated the expression of 45 ABC transporter genes in the mouse kidney under basal conditions, after induction of ischemia and after regeneration. Two days after clamping, mice showed a 76% decrease in renal creatinine clearance, which improved clearly within 7 days. This was confirmed by histological examinations. Seven days after ischemia, real-time quantitative Polymerase chain reaction data showed that transcript abundance of abcb1, abcb11, and abcc4 was increased, and that of abca3, abcc2, and abcg2 decreased. Expression of all transporters returned to baseline after 14 days, except for abcb11, which was reduced. Abcb11 is the major liver canalicular bile salt export pump. Here we show for the first time expression in the kidney and localization of the transporter to the apical membrane of proximal tubules. The presence of another novel renal transporter, abca3, was confirmed by Western blotting. Immunohistochemistry showed that abca3 is localized to the peritubular capillaries and apical membrane of proximal tubules. In conclusion, after inducing ischemic reperfusion injury in the kidney, ABC transporters appear to be differentially regulated, which might be associated with the renal regeneration process. Furthermore, we showed for the first time expression and subcellular localization of abcb11 and abca3 in mouse kidney.

ATP Binding Cassette Transporter, Subfamily B↗