Inwardly rectifying K(+) current and differentiation of human placental cytotrophoblast cells in culture.

Ion transport is important for driving nutrient transport across the syncytiotrophoblast and yet is poorly understood. We have examined K(+)currents under basal conditions in cultured cytotrophoblast cells, at various stages of differentiation, using the whole cell patch clamp technique. Cytotrophoblast cells were isolated from human term placenta and maintained in culture for up to 3 days. Cells were studied at four stages of progressive morphological differentiation: (i) mononuclear cells, (ii) mononuclear cells in aggregates, (iii) small multinucleate cells and (iv) large multinucleate syncytiotrophoblast-like cells. In the conditions of whole cell recording the only K(+) selective current identified in all cell types was a strong inwardly rectifying current which was sensitive to Ba(2+) and Cs(+). This current was unaffected by intracellular ATP whereas intracellular GTPgammas caused either run down of the current or activated a linear current. The characteristics of the current described are consistent with those of the inwardly rectifying K(+) channel Kir2.1. The inwardly rectifying K(+) current was observed in three out of 19 (16 per cent ) mononuclear cells, seven out of 21 (33 per cent ) mononuclear aggregates, eight out of 21 (38 per cent ) small multinucleate cells and 16 out of 19 (84 per cent ) large multinucleate cells. This inwardly rectifying K(+) current is likely to have an important role in determining net K(+) diffusion across the syncytiotrophoblast cell membrane, perhaps increasing in importance as the cells terminally differentiate.

Transgenic tobacco plants expressing the maize Cat2 gene have altered catalase levels that affect plant-pathogen interactions and resistance to oxidative stress.

Transgenic tobacco genotypes expressing the maize Cat2 gene were developed with altered catalase (CAT) levels that resulted in a moderate increase of CAT activity in two transgenic lines. Bacterial infection, with a pathogen that does not share homology with the transgene, caused local and systemic down-regulation of the steady state mRNA levels of the 35S-driven transgene in a manner resembling post-transcriptional gene silencing (PTGS). Phenotypic symptoms of hypersensitive response (HR) and systemic acquired resistance (SAR) were similar in control SR1 and the transgenic genotypes. Induction of hin1, used as a molecular marker of plant responses to invading bacteria, displayed a similar pattern between control and transgenic lines, but some variation in the levels of expression was observed. The major difference was recorded in the ability of the plants to restrict bacterial growth during HR. All transgenic lines were more sensitive than control SR1, with two lines exhibiting a significantly reduced capacity to inhibit bacterial growth. This is consistent with the putative enhanced capacity of transgenic lines containing the maize Cat2 gene to more effectively remove H2O2, which may act as a direct antimicrobial agent. Steady state mRNA levels of PR-1 and PR-5 varied among the genotypes, possibly indicating differences in strength of the SAR signal. Transgenic line 2, which was the most sensitive during HR, was most effective in restricting bacterial growth during SAR. This indicates that a reverse correlation might exist between the severity of infection during HR and the ability to inhibit bacterial growth during SAR. Growth under high light conditions affected plant-pathogen interactions in control SR1, as well as in transgenic line 8. Early induction and higher expression of PR-1 and PR-5 was detected in both SR1 and line 8 in high light-grown plants as compared with their low light-grown counterparts. Our data indicate that growth under high light conditions can predispose plants to better resist pathogen attack, and may amplify local and systemic defense signals. Finally, one transgenic line, which exhibited 1.3-fold higher average CAT activity in comparison with the untransformed SR1 control, suffered significantly less methyl viologen (MV) damage than untransformed control plants at moderate and high MV concentrations.

Differential response of antioxidant genes in maize leaves exposed to ozone.

Antioxidant enzymes function to eliminate reactive oxygen species (ROS) produced as a consequence of normal metabolic functions as well as environmental stress. In these studies, the responses of catalase (Cat), superoxide dismutase (Sod) and glutathione S-transferase (Gst), as well as D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) genes were analyzed in 9- and 15-day postimbibition maize seedlings exposed to various ozone (O3) concentrations and time periods. After a single (acute) 6 h exposure, or 3, 6 and 10 consecutive days (chronic) exposure to O3, Cat1, Cat3, Gst1, Sod3, Sod4 and Sod4A transcript levels generally increased, while Cat2, RbcS and Sod1 levels decreased. Such changes in mRNA levels do not necessarily reflect parallel changes in the protein products of these genes. Changes in transcript levels seemed to be correlated with the spatial location of the isozymes encoded by the genes. The results are discussed with respect to gene regulation and expression, and the localization and function of these antioxidant enzymes during ozone-mediated oxidative stress.

Sites of mRNA expression of the cystic fibrosis (CF) and multidrug resistance (MDR1) genes in the human placenta of early pregnancy: No evidence for complementary expression.

The aims of this study were to establish the sites of mRNA expression of both the cystic fibrosis (CF) and multidrug resistance (MDR1) genes in human placental sections from early pregnancy (first, early and mid-second trimesters). Riboprobes specific for each of these two genes were generated and used for in situ hybridization experiments. The results show parallel mRNA expression for the CF and MDR1 genes, with the signal detected in the syncytiotrophoblast and cytotrophoblast cells of the placental villi. Other cell types within the villous core were negative. Similar results were obtained at all stages of pregnancy studied.

Update of REACH-1 and MERIT-HF clinical trials in heart failure. Cardio.net Editorial Team.

BACKGROUND: This article reviews the design and results of REACH-1 (Research on Endothelin Antagonism in Chronic Heart Failure) and MERIT (Metoprolol controlled and Extended release, Randomised Intervention Trial in congestive Heart Failure), two recently reported clinical trials that investigated, respectively, the role of a non-selective endothelin antagonist (bosentan) and of a beta-selective blocker for the treatment of heart failure. RESULTS: The REACH-1 trial demonstrated that initiation of bosentan therapy is associated with an increased risk of worsening heart failure. However, long-term therapy with bosentan may have improved symptoms and favourably altered the progression of heart failure. The MERIT-HF clinical trial indicated that beta-blockade using metoprolol confers a significant beneficial effect on total mortality in patients with stable chronic heart failure.

Detection of a high-frequency silent polymorphism (C-->T) in the kir2.1 (KCNJ2) inwardly rectifying potassium channel gene by polymerase chain reaction and single strand conformation polymorphism.

The aim of this work was to determine the frequency of a base substitution (C-->T) identified in the Kir2.1 gene (approved gene symbol: KCNJ2; OMIM number: 600681). Polymerase chain reaction (PCR) of the area of the Kir2.1 gene containing this substitution was performed on 52 genomic DNA samples. Using single strand conformation polymorphism (SSCP) analysis, the genotype and allele frequencies were subsequently determined and the polymorphism identified in this study was verified by cycle sequencing. The data demonstrate that the C-->T nucleotide change identified corresponds to a silent polymorphism with a relatively high frequency. The deduced genotype frequencies of homozygotes and heterozygotes were: C/C: 73%; T/T: 2% and C/T: 25%. The deduced allele frequencies were C: 85.6% and T: 14.4%.

Modulation of antioxidant responses by arsenic in maize.

The effects of arsenic on the expression of the antioxidant genes encoding superoxide dismutase, catalase, and glutathione S-transferase, as well as the activity of SOD and CAT enzymes, were examined at different developmental stages and in different tissues. Both CAT and SOD activities increased in response to low concentrations (0.01-0.1 mM) of arsenic in developing maize embryos. In germinating embryos both CAT and SOD activities increased in response to a wide range of arsenic concentrations (0.01-10 mM). Cat1 transcript increased in response to arsenic in developing and germinating embryos and in young leaves. Conversely, Cat2 increased at low concentrations of arsenic only in germinating embryos. Cat3 transcript levels increased in response to low concentrations of arsenic only in developing embryos. Sod3 transcript increased at low concentrations of arsenic in developing, germinating embryos and in leaves. The cytosolic Sod4 and Sod4A increased in response to arsenic in germinating embryos, while only Sod4 transcript increased in response to arsenic in leaves. Expression of Gst1 was similar to that of Cat1 in all tissues examined. These results indicate that arsenic triggers tissue and developmental stage specific defense responses of antioxidant and detoxification related genes in maize.

Expression of the Kir2.1 (inwardly rectifying potassium channel) gene in the human placenta and in cultured cytotrophoblast cells at different stages of differentiation.

The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.

Expression of the cystic fibrosis (CF) and multidrug resistance (MDR1) genes during development and differentiation in the human placenta.

The aims of this study were to establish whether both the cystic fibrosis (CF) and multidrug resistance (MDR1) genes are expressed in the human placenta during development and differentiation. To study their pattern of expression during development, RNA was extracted from first, second and third trimester human placentas. To investigate differentiation, RNA was extracted from cytotrophoblast cells isolated from human term placentas and maintained in culture for 18, 66, 90 and 114 h and from the undifferentiated choriocarcinoma cell line JAr. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene specific, intron spanning primers, a cDNA product of 1 kb, as expected for CF expression, was detectable following 35 cycles of PCR from all RNA samples except those from JAr; in the latter a product was only detected in one sample out of four separate passages and this was only just detectable after 40 cycles of PCR. RT-PCR using MDR1 specific primers resulted in a product from all samples at 0.34 kb as expected if this gene is expressed. These results demonstrate that both the CF and MDR1 genes are expressed in the human placenta at all stages of development and differentiation, although the expression of the CF, but not the MDR1, gene appears to be much weaker in the undifferentiated JAr cells in comparison with cytotrophoblast cells.

The human placenta expresses transcription enhancer factor-1 but there is no correlation with the expression of placental lactogen.

A transcriptional enhancer which has a consensus binding sequence for transcription enhancer factor-1 (TEF-1) has been found 3' of the hPL(3) gene. We examined whether TEF-1 is expressed by the human placenta and whether such expression is co-ordinated with that of human placental lactogen (hPL). Probing Northern blots of total RNA from first trimester and term placenta, the choriocarcinoma-derived cell line JAr and primary cultured cytotrophoblast cells with a cDNA for TEF-1 revealed transcripts of 12-13 kb and 3-4 kb. The level of TEF-1 expression was the same in first trimester as compared with term placenta and in undifferentiated JAr as compared with differentiated cytotrophoblast cells. hPL expression was tenfold higher in term compared with first trimester placenta and, whilst detectable in cytotrophoblast cells, was undetectable in JAr cells. These data show that TEF-1 is expressed by the placenta but is not co-ordinated with hPL expression.

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii.

vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3.

Expression of type VI collagen mRNAs in human endometrium during the menstrual cycle and first trimester of pregnancy.

The pattern of type VI collagen deposition in human endometrium throughout the menstrual cycle and in decidua of the first trimester of pregnancy was studied. Immunohistochemical analysis demonstrated a dense microfibrillar network of collagen VI in the stroma of preimplantation endometrium which was reduced during the peri-implantation period and no longer detected in first trimester decidua. However, type VI collagen was consistently present within blood vessel walls in both endometrium and decidua. Using in situ hybridization, mRNAs encoding alpha 1(VI), alpha 2(VI) and alpha 3(VI) chains within endometrial stromal and vascular cells were identified. All three mRNA species are abundant in the villous mesenchyme of the first trimester placenta. The detection of collagen VI mRNA species in the endometrium throughout the menstrual cycle suggests that the apparent decrease in abundance of extracellular immunoreactive fibrils may be a consequence of translational control, matrix redistribution or turnover. In contrast, in the first trimester of pregnancy, collagen VI protein was mainly absent from the decidual stroma and amounts of mRNA were very low, indicating a significant reduction in production. Loss of stromal type VI collagen contributes to the remodelling of the maternal extracellular matrix of pregnancy.

The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene.

Two-dimensional gel electrophoresis of pea root and root hair proteins revealed the existence of at least 10 proteins present at elevated levels in root hairs. One of these, named RH2, was isolated and a partial amino acid sequence was determined from two tryptic peptides. Using this sequence information oligonucleotides were designed to isolate by PCR an RH2 cDNA clone. In situ hybridization studies with this cDNA clone showed that rh2 is not only expressed in root hairs, but also in root epidermal cells lacking these tubular outgrowths. During post-embryonic development the gene is switched on after the transition of protoderm into epidermis and since rh2 is already expressed in a globular pea embryo in the protoderm at the side attached to the suspensor, we conclude that the expression of rh2 is developmentally regulated. At the amino acid level RH2 is 95% homologous to the pea PR protein I49a. These gene encoding I49a is induced in pea pods upon inoculation with the pathogen Fusarium solani [12]. We postulate that rh2 contributes to a constitutive defence barrier in the root epidermis. A similar role has been proposed for chalcone synthase (CHS) and chitinase, pathogenesis-related protein that are also constitutively present in certain epidermal tissues.

Characterization of the soybean early nodulin cDNA clone GmENOD55.

Two cDNA clones of the soybean early nodulin GmENOD55 were characterized. These clones may represent two members of the soybean early nodulin gene family GmENOD55. GmENOD55 has an N-terminal signal peptide and it contains an internal domain consisting of proline and serine residues. Analyses of nodules lacking infection threads and intracellular bacteria suggest that the GmENOD55 gene is first expressed after release of Bradyrhizobium japonicum in plant cells. This conclusion is supported by in situ hybridization studies showing that the expression is restricted to the infected cell type.