Activated Partial Thromboplastin Time Monitoring of Unfractionated Heparin Therapy: Issues and Recommendations.

When administering unfractionated heparin (UFH), therapeutic levels of anticoagulation must be achieved rapidly and maintained consistently in the therapeutic range. The basic assays for monitoring UFH therapy are the activated partial thromboplastin time (APTT) and/or the chromogenic antifactor Xa or antithrombin assays. For many laboratories, the APTT is the preferred standard of practice; however, the APTT is a surrogate marker that only estimates the heparin concentration. Many factors, including patient variation, reagents of the APTT, UFH composition, and concentration can influence the APTT result. This article reviews various methods to determine the heparin therapeutic range and presents recommendations for the laboratory to establish an APTT heparin therapeutic range for all sizes of hospitals.

Biological activity of the Agrobacterium rhizogenes-derived trolC gene of Nicotiana tabacum and its functional relation to other plast genes.

Agrobacterium rhizogenes induces hairy roots through the activity of three essential T-DNA genes, rolA, rolB, and rolC, whereas the orf13 gene acts as an accessory root-inducing gene. rolB, rolC, and orf13 belong to the highly diverged plast gene family with remotely related representatives in the endomycorrhizal basidiomycete Laccaria bicolor. Nicotiana glauca and N. tabacum contain A. rhizogenes-derived T-DNAs with active plast genes. Here, we report on the properties of a rolC homolog in N. tabacum, trolC. Dexamethasone-inducible trolC and A4-rolC genes from A. rhizogenes A4 induce comparable, strong growth effects affecting all parts of the plants. Several have not been described earlier and were found to be very similar to the effects of the distantly related plast gene 6b. They include leaf chlorosis and starch accumulation, enations, increase of sucrose-dependent leaf disk expansion, growth of isolated roots on low-sucrose media, and stimulation of sucrose uptake by small root fragments. Collectively, our findings indicate that enhancement of sucrose uptake plays an important role in generating the complex 6b and rolC phenotypes and might be an ancestral property of the plast genes.

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth.

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.

The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco.

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.

The T-DNA oncogene A4-orf8 from Agrobacterium rhizogenes A4 induces abnormal growth in tobacco.

The related orf8 and iaaM T-DNA genes from Agrobacterium are each composed of two distinct parts. The 5' parts (called Norf8 or NiaaM) encode a 200-amino-acid (aa) sequence with homology to various T-DNA oncoproteins such as RolB, RolC, and 6b. The 3' parts (Corf8 or CiaaM) encode a 550-aa sequence with homology to IaaM proteins from Pseudomonas and Pantoea spp. Whereas iaaM genes encode flavin adenine dinucleotide (FAD)-dependent tryptophan 2-monooxygenases that catalyze the synthesis of indole-3-acetamide (IAM), A4-orf8 from Agrobacterium rhizogenes A4 does not. Plants expressing a 2x35S-A4-Norf8 construct accumulate soluble sugars and starch. We now have regenerated plants that express the full-size 2x35S-A4-orf8 and the truncated 2x35S-A4-Corf8 gene. 2x35S-A4-Corf8 plants accumulate starch and show reduced growth like 2x35S-A4-Norf8 plants but, in addition, display a novel set of characteristic growth modifications. These consist of leaf hypertrophy and hyperplasia (blisters); thick, dark-green leaves; thick stems; and swollen midveins. Mutations in the putative FAD-binding site of A4-Orf8 did not affect the blister syndrome. Plants expressing 2x35S-A4-Corf8 had a normal phenotype but contained less starch and soluble sugars than did wild-type plants. When 2x35S-A4-Corf8 plants were crossed to starch-accumulating 2x35S-A4-Norf8 plants with reduced growth, A4-Corf8 partially restored growth and reduced starch accumulation. A4-Corf8xA4-Norf8 crosses did not lead to the blister syndrome, suggesting that this requires physical linkage of the A4-NOrf8 and A4-COrf8 sequences.

New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter.

Agrobacterium 6b oncogenes induce tumours on Nicotiana glauca and enations and associated modifications in transgenic N. tabacum plants. 2x35S-AB-6b tobacco rootstocks produced a graft-transmissible factor that induced enations in wild-type scions; the nature of this enation factor remains to be identified. Here, we report on the properties of tobacco plants carrying a dexamethasone-inducible T-6b gene (dex-T-6b). Induction with dex led to complex growth modifications, many of which have not been reported previously. Modifications were only found in growing tissues; mature tissues remained unaffected. Growth could be either stimulated or inhibited. Dex induction of young plants led to morphogenetic gradients that included enations, tubular leaves and fragmented leaf primordia. Root elongation was increased or slowed down, while radial root growth was strongly enhanced. Additional cell divisions were found in the root pericycle and vasculature. Enation factor import from mature tissues did not have the same effects on growing tissues as local T-6b synthesis: normal scions grafted on induced dex-T-6b rootstocks formed enations, whereas local dex-T-6b induction at the shoot apex led to numerous dark-green spots on the abaxial side of the leaves. In leaf patch assays, the 23-kDa T-6b protein was found to move through leaves and to enter the vascular system. This and the fact that rootstocks of spontaneous tobacco enation mutants did not modify wild-type scions contrary to 6b plants indicate that the 6b protein might be the enation factor.

The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco.

Agrobacterium 6b oncogenes induce tumours and modify plant growth in various ways. Here we show that the AB-6b gene from strain AB4 placed under 2x35S promoter control (2x35S-AB-6b) induces a complex enation syndrome in transgenic Nicotiana tabacum plants, that also occurs in a few rare cases of genetic enations. In Arabidopsis thaliana, 2x35S-AB-6b induced radially symmetrical tubes on the abaxial side of the leaves, which must therefore be considered as the Arabidopsis equivalents of enations on other plant species. Tobacco and Arabidopsis 2x35S-AB-6b leaves contained small, supernumerary densely packed cells between the spongy mesophyll and the abaxial epidermis, close to vascular strands arising at an early stage of leaf development. On tobacco, the 2x35S-AB-6b enation syndrome could be transmitted across graft junctions to growing tissues of untransformed plants, both acropetally and basipetally. We propose that the AB-6b gene encodes the synthesis of one or more enation factor(s) that are transported by the phloem and modify the growth of developing tissues.

S phase and meristem-specific expression of the tobacco RNR1b gene is mediated by an E2F element located in the 5' leader sequence.

The RB/E2F pathway is involved in the control of the G(1)/S transition of the eukaryotic cell cycle where various S phase genes are activated by specific E2F factors. Ribonucleotide reductase (RNR) plays an essential role in the DNA synthesis pathway. Earlier studies showed that there are at least two RNR1 genes (RNR1a and RNR1b) and one RNR2 gene in tobacco. In synchronized tobacco BY2 cells, RNR1b gene expression is at its highest level in S phase. To investigate transcriptional regulation of the RNR1b gene, its promoter region was cloned and sequenced. Unlike its animal counterparts, the tobacco RNR1b promoter contains a consensus E2F-binding site. Surprisingly, this site is found in the leader sequence of the gene. We show here by gel shift analysis and antibody competition that one nuclear complex specifically binds this motif, and an E2F factor is part of this complex. Using reporter gene analysis, tobacco RNR1b promoter activity was detected during S phase in synchronized cells and in plant meristematic tissues. Mutation of the E2F element substantially reduced both activities. For the first time in plants, a single E2F motif found in the leader sequence plays an important role in the meristem and S phase-specific expression of the tobacco RNR1b gene.