The role of alanine and aspartate aminotransferases in C4 photosynthesis.

Alanine and aspartate are essential transfer metabolites for C4 species of the NAD-malic enzyme and phosphoenolpyruvate carboxykinase subtype. To some degree both amino acids are also part of the metabolite shuttle in NADP-malic enzyme plants. In comparison with C3 species, the majority of C4 species are therefore characterised by enhanced expression and activity of alanine and aspartate aminotransferases (AT) in the photosynthetically active tissue. Both enzymes exist in multiple copies and have been found in different subcellular compartments. We tested whether different C4 species show preferential recruitment of enzymes from specific lineages and subcellular compartments. Phylogenetic analysis of alanine and aspartate ATs from a variety of monocot and eudicot C4 species and their C3 relatives was combined with subcellular prediction tools and analysis of the subsequent transcript amounts in mature leaves. Recruitment of aspartate AT from a specific subcellular compartment was strongly connected to the biochemical subtype. Deviation from the main model was however observed in Gynandropsis gynandra. The configuration of alanine AT generally differed in monocot and eudicot species. C4 monocots recruited an alanine AT from a specific cytosolic branch, but eudicots use alanine AT copies from a mitochondrial branch. Generally, plants display high plasticity in the setup of the C4 pathway. Beside the common models for the different C4 subtypes, individual solutions were found for plant groups or lineages.

Short-term growth hormone treatment in children with Hurler syndrome after hematopoietic cell transplantation.

Children with Hurler syndrome experience progressive growth failure after hematopoietic cell transplantation (HCT). The goal of this study was to review the safety and efficacy of growth hormone (GH) in eight children with Hurler syndrome who were treated at our institution with GH for short stature or GH deficiency between 2005 and 2008. The age at initiation of treatment with GH was 9.6+/-2.3 years and time since HCT was 7.5+/-1.5 years. Mean GH dose was 0.32 mg/kg/week. Baseline growth velocity was 3.5+/-1.5 cm/year (-2.6+/-1.9 s.d.), and it increased to 5.2+/-3.0 cm/year (-0.1+/-3.6 s.d.) after 1 year of treatment. Of the six patients with radiographic data, there was one progression of scoliosis, one progression of kyphosis and one progression of genu valgum. No patient discontinued treatment due to progression of skeletal disease. One patient discontinued GH due to slipped capital femoral epiphysis. Preliminary data suggest that 1-year GH treatment may modestly improve growth velocity in children with Hurler syndrome.

Postoperative deep wound infections in adults after spinal fusion: management with vacuum-assisted wound closure.

OBJECTIVE: Vacuum-assisted wound closure (VAC) exposes the wound bed to negative pressure, resulting in removal of edema fluid, improvement of blood supply, and stimulation of cellular proliferation of reparative granulation tissue. It has been used to treat open wounds in the extremities, open sternal wounds, pressure ulcers, and abdominal wall wounds. This study retrospectively reviewed instrumented spine fusions complicated by surgical wound infection and managed by a protocol including the use of VAC in order to evaluate the efficacy of applying vacuum therapy on patients with deep spine infections and exposed instrumentation. METHODS: Twenty consecutive patients with deep wound infections after undergoing spinal fusion procedures were studied. There were 12 men and 8 women with an average age of 55 years (31-81 years). Eight patients had undergone concomitant anterior and posterior arthrodesis, nine patients had a posterior spinal fusion, and three patients had a transforaminal lumbar interbody fusion. Seven patients had a decompression with exposed dura. Sixteen patients presented with a draining wound within the first 6 weeks postoperatively (average 24 days). There were four patients who presented with back pain and temperature after 1 year postoperatively (average 3 years). All patients were taken to the operating room for irrigation and debridement followed by placement of the VAC with subsequent delayed closure of the wound. RESULTS: There was an average of 1.8 (1-8) irrigation and debridement procedures prior to placement of the VAC. Once the VAC was initiated, there was an average of 2.2 (2-3) procedures until and including closure of the wound. The wound was closed an average of 7 days (5-14 days) after the placement of the initial VAC in the wound. All patients tolerated the VAC without adverse effects. All patients were kept on a 6-week course of intravenous antibiotic therapy. The average follow-up was 10 months (6-24 months). There were no cases of uncontrolled sepsis once the VAC was initiated. All patients achieved a clean closed wound without removal of instrumentation at a minimum follow-up of 6 months. CONCLUSION: VAC therapy is an effective adjunct in closing complex deep spinal wounds with exposed instrumentation.

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids.

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.

Coronal plane imbalance in adolescent idiopathic scoliosis with left lumbar curves exceeding 40 degrees: the role of the lumbosacral hemicurve.

STUDY DESIGN: Retrospective radiographic analysis of the potential role the lumbosacral hemicurve has on adolescent idiopathic scoliosis coronal trunk imbalance. OBJECTIVE: To determine if the lumbosacral hemicurve predisposes adolescent idiopathic scoliosis to coronal decompensation preoperatively and postoperatively. SUMMARY OF BACKGROUND DATA: Although coronal decompensation remains a clinical problem in adolescent idiopathic scoliosis, the literature regarding the role of potential intrinsic structural properties of the lumbosacral hemicurve is sparse. METHODS: Fifty patients with adolescent idiopathic scoliosis were used to measure several potential parameters predisposing to coronal decompensation including lumbosacral hemicurve magnitude and flexibility, sacral and iliac obliquity. RESULTS: Overall, 84% (42/50) demonstrated preoperative decompensation. A more rigid lumbosacral hemicurve was significantly related to preoperative coronal decompensation in the combined and the King I groups. Preoperatively, significant correlation with decompensation was observed for sacral and iliac obliquity in the King I group and for iliac obliquity in the combined group. Postoperatively, coronal decompensation remained significantly correlated to sacral obliquity in the combined group and King I groups. Iliac obliquity was significantly related to postoperative decompensation in the combined group. CONCLUSIONS: The lumbosacral hemicurve represents an important structure predisposing to left coronal plane imbalance in adolescent idiopathic scoliosis that includes a large left lumbar curve as a component of the curve pattern. "At-risk" signs for persistent postoperative coronal decompensation include iliac and sacral obliquity noted on the preoperative standing full-length radiographs.

The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite.

Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis.

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.

The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria.

Two plant test systems are presented in the search for new inhibitors of the non-mevalonate isoprenoid pathway. A derivative of clomazone appears to be an inhibitor of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate (DOXP/MEP) pathway of isoprenoid formation.

Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana.

Various plant isoprenoids are synthesized via the non-mevalonate pathway of isopentenyl diphosphate formation. In this pathway, 1-deoxy-D-xylulose 5-phosphate (DOXP), the first intermediate, is transformed to 2-C-methyl-D-erythritol 4-phosphate (MEP) by an enzyme which was recently cloned from Escherichia coli. In order to find a plant homologue of this 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) we cloned a cDNA fragment from Arabidopsis thaliana which has high homology to the E. coli DXR. By expression of this fragment in E. coli we could demonstrate that it encodes a protein which transforms DOXP to MEP. The antibiotic fosmidomycin specifically inhibits this DXR enzyme activity.

Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714.

Isopentenyl diphosphate, the universal isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1-13C]glucose and analysis of the resulting labelling patterns found in the isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for isoprenoid biosynthesis. All isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus, which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast isoprenoids (phytol in Cy. caldarium and O. danica and beta-carotene in O. danica) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium Synechocystis PCC 6714 using [1-13C]- and [6-13C]-glucose. The two isoprenoids examined, phytol and beta-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route.

Incorporation of 1-deoxy-D-xylulose into isoprene and phytol by higher plants and algae.

In further substantiating the novel mevalonate-independent pathway for isoprenoid biosynthesis, which generates isopentenyl diphosphate (IPP) via 1-deoxy-D-xylulose-5-phosphate, labeling experiments with 1-[2H(1)]deoxy-D-xylulose were performed with various higher plants and algae: efficient incorporation was observed into isoprene emitted by Populus, Chelidonium, and Salix, into the phytol moiety of chlorophylls in a red alga (Cyanidium), in two green algae (Scenedesmus, Chlamydomonas), and a higher plant (Lemna). By contrast, 13C-mevalonate applied was incorporated into isoprene and phytol to a much lower extent or not at all. This demonstrates that this '1-deoxy-D-xylulose-5-phosphate pathway' for biosynthesis of plastidic isoprenoids is widely distributed in photosynthetic organisms.

Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway.

Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids. By labeling experiments using [1-(13)C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (beta-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.

Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus.

Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliquus grown heterotrophically on 13C-labelled glucose and acetate. Several isoprenoid compounds were isolated and investigated by 13C-NMR spectroscopy. According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl side-chains of chlorophylls and plastoquinone-9, and the carotenoids beta-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria. Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.