Ribose-5-phosphate isomerase deficiency: new inborn error in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy

The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene-sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders.     

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541-547

Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.     

Genetic transformation of Alstroemeria using particle bombardment

Transgenic plants were obtained after particle bombardment of embryogenic callus derived from stem segments of two tetraploid Alstroemeria genotypes with plasmids containing different selection/reporter genes. Firstly, a plasmid containing a firefly luciferase reporter gene driven by the maize ubiquitin promoter (Ubi1), was bombarded into both friable embryogenic callus and proembryos. Transient and stable expression of luciferase was visually detected by a luminometer. This selection method is non-destructive and can be applied over the whole developmental process from callus to embryo and plantlet. Molecular proof of transformation was obtained both by PCR analysis and Southern hybridization. Secondly, a plasmid containing the bar gene together with an uidA gene coding for -glucuronidase both driven by the Ubi1 promoter was bombarded into proembryos. The transgenic callus was effectively selected from the callus clumps four months after bombardment on a medium containing 5 mg/l phosphinotricin (PPT). Selection by PPT was efficient and labour-saving. Stable expression of GUS was confirmed by the histochemical staining assay and molecular proof was obtained by PCR analysis.     

Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.     

Mobility and lifetime fecundity in new versus old populations of the Glanville fritillary butterfly

Life history theory often assumes a trade-off between dispersal and reproduction, and such a trade-off is commonly observed in wing-dimorphic insects. The results are less consistent for wing-monomorphic species, for which it is more difficult to assess dispersal capacity and rate. Three replicate experiments were carried out in consecutive years on the Glanville fritillary butterfly in a large outdoor population cage to study the relationship between lifetime egg production and mobility. The experimental material included females originating from newly-established and old populations, as previous studies have shown dispersal capacity to depend on population age. There was a consistent and significant interaction between mobility and population age, such that in newly-established populations mobile females had higher fecundity than less mobile females, while in old populations there was no such relationship. As selection favours individuals with the highest fecundity, selection pressure on mobility is likely to be different between the two population types, which may contribute to maintenance of variation in dispersal rate in the metapopulation as a whole. Several other female traits also affected lifetime fecundity, including lifespan, number of matings and date of eclosion, although these effects were not consistent across the years. These results highlight the importance of conducting experiments in more than one year before generalizing about patterns in life history variation.     

Tissue-specific changes in the hydroxylysine content and cross-links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock-out mice

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.     

Costs of dispersal

Dispersal costs can be classified into energetic, time, risk and opportunity costs and may be levied directly or deferred during departure, transfer and settlement. They may equally be incurred during life stages before the actual dispersal event through investments in special morphologies. Because costs will eventually determine the performance of dispersing individuals and the evolution of dispersal, we here provide an extensive review on the different cost types that occur during dispersal in a wide array of organisms, ranging from micro‐organisms to plants, invertebrates and vertebrates. In general, costs of transfer have been more widely documented in actively dispersing organisms, in contrast to a greater focus on costs during departure and settlement in plants and animals with a passive transfer phase. Costs related to the development of specific dispersal attributes appear to be much more prominent than previously accepted. Because costs induce trade‐offs, they give rise to covariation between dispersal and other life‐history traits at different scales of organismal organisation. The consequences of (i) the presence and magnitude of different costs during different phases of the dispersal process, and (ii) their internal organisation through covariation with other life‐history traits, are synthesised with respect to potential consequences for species conservation and the need for development of a new generation of spatial simulation models.     

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

Purpose

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Experimental design

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

Results

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20?μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9?%, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

Conclusions

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.     

Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.     

Increased Production of Folate by Metabolic Engineering of Lactococcus lactis

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.