Electrolyte monitoring during regional citrate anticoagulation in continuous renal replacement therapy.

Patients with acute kidney injury who need continuous renal replacement therapy with locoregional citrate anticoagulation are at risk of citrate accumulation with disruption of the calcium balance. We aimed to evaluate the safety of detecting citrate accumulation and adjusting electrolyte disbalances during continuous venovenous hemodialysis (CVVHD) in critically ill patients with acute kidney injury using a blood sample frequency every 6 h. A prospective single center study in critically ill intensive care unit patients who suffered from acute kidney injury with the need of renal replacement therapy. We evaluated the deviations in pH, bicarbonate and calcium during CVVHD treatment with local regional citrate anticoagulation. Values indicate median and interquartile range. Severe hypocalcemia (below 1.04 mmol/L) or hypercalcemia (above 1.31 mmol/L) occurred in 10.5% and 4.8% respectively. During treatment changes of systemic ionized calcium, post-filter ionized calcium, pH and bicarbonate were corrected with protocolized adjustments. No arrhythmias or citrate accumulation were seen. The values stabilized after 42 h and after that no statistically significant changes were observed. After 42 h of citrate CVVHD, systemic ionized calcium, pH and bicarbonate levels stabilized. A blood sample frequency every 6 h is probably safe to detect citrate accumulation and to adjust the settings of electrolytes to avoid serious electrolyte disturbances in ICU patients without severe metabolic acidosis or severe liver failure.

Expression of three ß-type carbonic anhydrases in tomato fruits.

Carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) activity were found in different Solanum lycopersicum fruit tissues, predominantly in the locular parenchyma and pericarp. The distribution of the CA and PEPC proteins in the tomato fruit tissues was examined by immunohistolocalization. CA and PEPC proteins were found in all fruit tissues examined as well as in the seeds. Three full length cDNA clones designated SlCA1, SlCA2 and SlCA3 coding for ß-carbonic anhydrases (CA; EC 4.2.1.1) were identified and characterized from tomato fruit. SlCA1 and SlCA3 encode two putative cytosolic isoforms whereas SlCA2 encodes a putative plastidial isoform. Quantitative real time RT-PCR analysis revealed that accumulation of SlCA1 mRNA transcripts was detected in all examined tomato fruit tissues or organs, whereas SlCA2 gene transcripts were found in abundance in leaves. Stems also had SlCA2 transcripts, with transcript levels being higher in flowers than in stems. The SlCA3 gene transcripts were found only in the flowers and the roots. The SlPEPC1 and SlPEPC2 gene transcript levels in different fruit tissues of the tomato were also examined. Τhe possible role of CA isoforms in relation to PEPC in tomato fruit is discussed.

Cloning and characterization of Lotus japonicus formate dehydrogenase: a possible correlation with hypoxia.

Formate dehydrogenases (FDHs, EC 1.2.1.2) comprise a group of enzymes found in both prokaryotes and eukaryotes that catalyse the oxidation of formate to CO(2). FDH1 from the model legume Lotus japonicus (LjFDH1) was cloned and expressed in E. coli BL21(DE3) as soluble active protein. The enzyme was purified using affinity chromatography on Cibacron blue 3GA-Sepharose. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of LjFDH1, based on the known structure of the Pseudomonas sp. 101 enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The temporal expression pattern of LjFDH1 gene was studied by real-time RT-PCR in various plant organs and during the development of nitrogen-fixing nodules. Furthermore, the spatial transcript accumulation during nodule development and in young seedpods was determined by in situ RNA-RNA hybridization. These results considered together indicate a possible role of formate oxidation by LjFDH1 in plant tissues characterized by relative hypoxia.

Isolation and characterisation of a non-polyadenylated mRNA species with an affinity for poly(adenylic acid) from Friend leukaemia cells.

Utilizing the technique of poly(A)-Sepharose affinity chromatography, it is possible to isolate a novel class of RNA molecules from polysomes of Friend leukaemia cells. These RNA species display messenger RNA-like behaviour. They are released from polysomes on treatment with EDTA and are able to direct polypeptide synthesis in a cell-free protein synthesising system. They appear to be distinct from the polyadenylated mRNAs, as judged by their lack of a 3'-terminal poly(A) tract, by their different size distribution, by their unusual base composition, by the presence of a possible 'uridylate rich' region towards their 3'-end, by their low sequence homology to polyadenylated mRNAs and by the difference in at least some of their translation products.

The metabolic behaviour of nuclear and cytoplasmic non-polyadenylated RNAs with an affinity for poly(adenylic acid) from Friend murine leukaemia cells.

Using poly(A)-Sepharose and poly(U)-Sepharose affinity chromatography, various classes of nuclear RNA can be distinguished in Friend leukaemia cells. One of these contains a poly(A) tract (poly(A)+-RNA) and another lacks a poly(A) tract but has an affinity for poly(A)-Sepharose (poly(A)-u+-RNA). The stability of these two particular nuclear RNA classes was examined by using a 'pulse-chase' technique involving D-glucosamine treatment. Nuclear poly(A)-u+-RNA was found to decay as a single component with a half-life of about 12 min. In contrast, nuclear poly(A)+-RNA appears to consist of at least two distinct metabolic components with half-lives of about 22 min and 120 min. Furthermore, poly(A)-u+-RNA is transported from the nuclei much more rapidly than the poly(A)+-RNA. The 'pulse-chase' approach also allowed a quantitative estimate to be made of the conversion of nuclear poly(A)+-RNA and poly(A)-u+-RNA to cytoplasmic poly(A)-RNA and poly(A)-u+-RNA.

Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings.

Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and ß-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

Carbon metabolism in developing soybean root nodules: the role of carbonic anhydrase.

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues.

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

Characterization of spermidine and spermine synthases in Lotus japonicus: induction and spatial organization of polyamine biosynthesis in nitrogen fixing nodules.

The biosynthesis of the polyamines spermidine (Spd) and spermine (Spm) from putrescine (Put) is catalysed by the consequent action of two aminopropyltransferases, spermidine synthase (SPDS EC: 2.5.1.16) and spermine synthase (SPMS EC: 2.5.1.22). Two cDNA clones coding for SPDS and SPMS homologues in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the encoded polypeptides was confirmed by their ability to complement spermidine and spermine deficiencies in yeast. The temporal and spatial expression pattern of the respective genes was correlated with the accumulation of total polyamines in symbiotic and non-symbiotic organs. Expression of both genes was maximal at early stages of nodule development, while at later stages the levels of both transcripts declined. Both genes were expressed in nodule inner cortical cells, vascular bundles, and central tissue. In contrast to gene expression, increasing amounts of Put, Spd, and Spm were found to accumulate during nodule development and after maturity. Interestingly, nodulated plants exhibited systemic changes in both LjSPDS and LjSPMS transcript levels and polyamine content in roots, stem and leaves, in comparison to uninoculated plants. These results give new insights into the neglected role of polyamines during nodule development and symbiotic nitrogen fixation (SNF).

The pattern of protein synthesis induced by heat-shock of the moderately halophilic bacterium Chromobacterium marismortui: protective effect of high salt concentration against the thermal shock.

The protein synthetic response to heat shock of the moderately halophilic bacterium Chromobacterium marismortui was examined. Upon exposure to elevated temperature there is an increased synthesis of a specific subset of proteins (heat shock proteins-hsps) in the molecular weight region of 15 to 90 kD, while normal protein synthesis is severely repressed. The synthesis of hsps reaches a maximum 5 min after heat shock at 42 degrees C. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. When cells grown in 2.5M NaCl were challenged with heat shock at 42 degrees C, the synthesis of some normal proteins was permitted. Furthermore, growth in high salt concentration resulted in an extension of the upper temperature limits at which C. marismortui could synthesize hsps. Adaptation of C. marismortui to decreasing salinity stimulated the synthesis of new proteins distinct from the hsps.

Heat shock proteins in the moderately halophilic bacterium Deleya halophila: protective effect of high salt concentration against thermal shock.

The moderately halophilic eubacterium Deleya halophila grown in medium containing 1 M or 2.5 M NaCl was heat-shocked at various temperatures and the electrophoretic patterns of pulse-labelled proteins were examined. Several polypeptides were induced (heat shock proteins, or hsp) at all temperatures. However, the level of induction of some hsp was dependent on the severity of the thermal shock as well as the salt concentration of the growth medium. Time course studies revealed that synthesis of some of the hsp was transient when cells were grown in 1 M NaCl, while growth at 2.5 M NaCl resulted in the synthesis of most of the hsp at almost maximal level for at least 60 min following temperature shift-up. When cells were returned to normal growth temperature (30 degrees C) after a heat shock treatment (47 degrees C for 5 min), normal protein synthesis resumed faster when cells were grown in 1 M than in 2.5 M NaCl. During the recovery period, several major hsp appeared to be synthesized at near maximal level at both salt concentrations.

Nodulin-24 gene of soybean codes for a peptide of the peribacteroid membrane and was generated by tandem duplication of a sequence resembling an insertion element.

A nodulin gene coding for a polypeptide with an apparent M(r) of 24,000 (nodulin-24) was isolated from soybean (Glycine max). DNA sequence analysis of this gene revealed that its coding capacity is for a polypeptide of only M(r) 15,100 and is interrupted by four introns. The three middle exons and their flanking segments appear to have been generated by duplications of a unit resembling an insertion sequence. This unit is bounded by a 12-base-pair inverted repeat and encompasses the 54-base-pair exon corresponding to each of three central hydrophobic domains of the protein, nodulin-24. The resulting repeated hydrophobic structure of this protein may be responsible for an apparent increase in M(r) from 15,100 to 24,000. In vitro translation and immunological studies suggest that nodulin-24 is a precursor and is processed cotranslationally into a M(r) 20,000 polypeptide. This polypeptide is a component of the membrane envelope enclosing the bacteroids (peribacteroid membrane) synthesized during symbiosis with Rhizobium. The low degree (<6%) of sequence divergence among the repeated units suggests that this gene has been generated recently during the evolution of symbiotic nitrogen fixation in soybean.

Phaseolus ENOD40 is involved in symbiotic and non-symbiotic organogenetic processes: expression during nodule and lateral root development.

ENOD40 is an early nodulin gene, recently isolated from legume species forming nodules either after Rhizobium infection or spontaneously. ENOD40 cDNAs from Phaseolus plants were isolated and nucleotide sequence determination revealed 85% and 88.5% homology with the reported soybean cDNA clones. The putative polypeptide deduced coincides with the soybean one but a stop codon, almost in the middle of the respective ORF, renders it much shorter. This polypeptide was overexpressed as a fusion protein in Escherichia coli. Although the spatial expression pattern of the gene in the root pericycle and nodule primordium at early stages of development as well as in the pericycle of the vascular bundles and uninfected cells in mature nodules is comparable to the gene's expression pattern in soybean, differences in developmental regulation are evident. We have shown that ENOD40 transcripts are also detected at very early stages of lateral root development, in the dividing pericycle cells of the root stele that give rise to the lateral root primordia. The presence of Rhizobium causes an enhancement of the gene's expression and also induction of the gene in the vascular tissues of developed lateral roots. Interestingly, a discrimination on the gene's expression level in adventious and acropetal incipient lateral root primordia, emerging in infected and uninfected roots, is observed. This indicates that the gene's product may be involved in the hormonal status of the plant and that ENOD40 may be used as a molecular marker in lateral root initiation.

Expression pattern of uricase II gene during root nodule development in Phaseolus vulgaris.

A Phaseolus vulgaris uricase II cDNA clone has been isolated and sequenced. Comparison on the nucleotide level between this clone and the soybean uricase II clone revealed 88.8% identity. The in situ hybridization technique was used to follow the expression pattern in developing root nodules of Phaseolus vulgaris. The uricase II transcripts were localized only in the uninfected cells of the central tissue and mainly in the periphery of the cell. Uricase II mRNA is first detected in nodules 12 days after infection. A maximum level of transcripts is reached in 21-day-old nodules, followed by a considerable reduction in 28-day-old nodules.

Identification and Characterization of a Bacteroid-Specific Dehydrogenase Complex in Rhizobium leguminosarum PRE.

In membranes of Rhizobium leguminosarum bacteroids isolated from nitrogen-fixing pea root nodules, two different protein complexes with NADH dehydrogenase activity were detected. One of these complexes, with a molecular mass of 110 kilodaltons, was also found in membranes of free-living rhizobia, but the other, with a molecular mass of 550 kilodaltons, appeared to be present only in bacteroids. The bacteroid-specific complex, referred to as DH1, probably consists of at least four different subunits. Using antibodies raised against the separate polypeptides, we found that a 35,000-molecular-weight polypeptide (35K polypeptide) in the DH1 complex is bacteroid specific, while the other proposed subunits were also detectable in cytoplasmic membranes of free-living bacteria. Dehydrogenase complex DH1 is also present in bacteroids of a R. leguminosarum nifA mutant, indicating that the synthesis of the dehydrogenase is not dependent on the gene product of this nif-regulatory gene. A possible involvement of the bacteroid-specific DH1 complex in electron transport to nitrogenase is discussed.

Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5'-flanking regions of nodulin and leghemoglobin genes.

The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium. Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library. Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp. The deduced protein sequence suggests that nodulin-23 may have a signal sequence. The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained. Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology. The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.

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.

Characterization and nucleotide sequence of a novel gene fixW upstream of the fixABC operon in Rhizobium leguminosarum.

On the Rhizobium leguminosarum PRE sym plasmid, fixABC and a novel gene fixW were identified upstream of the regulatory gene nifA. The molecular masses of FixABC, 29, 44 and 50 kDa respectively, were estimated by polyacrylamide gel electrophoresis (PAGE) and of FixW, 25 kDa, by PAGE and nucleotide sequencing. Hybridization studies using bacteroid mRNA as a probe showed that fixABC is one operon which can be transcribed independently of fixW. Nucleotide sequencing revealed that both fixW and fixA are preceded by a nif consensus promoter. The fixA promoter partly overlaps the 3'-terminal coding region of fixW, indicating that readthrough from fixW into fixA is possible. Two open reading frames, ORF71 and ORF79, precede fixW and form one operon with fixW. ORF71 contains sequences homologous to the fixA promoter and 5'-terminal coding region. One more duplication of fixA sequences was detected, also located within the sym plasmid nif/fix clusters. One duplication of fixW sequences was found. No fixW homologue could be found in other nitrogen fixing organisms except in a number of R. leguminosarum strains.

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum.

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta (35)S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.