Choline-Containing Compounds in Human Astrocytomas Studied by 1H NMR Spectroscopy In Vivo and In Vitro

Abstract: We have studied 14 patients with different grades of astrocytomas using ¹H NMR spectroscopy in vivo. Typically, astrocytomas exhibited a low N -acetyl-aspartate peak, a prominent signal from choline group-containing compounds, and lactate in the ¹H NMR spectra in vivo. The uncorrected choline/creatine + phosphocreatine peak area ratios were higher in tumors than in normal brain tissue. Absolute concentration of choline-containing compounds (1.74 ± 0.09 mmol/L) in the normal brain tissue was not different in any grade of astrocytoma, but total creatine concentration in healthy brain (7.49 ± 0.30 mmol/L) was higher than that in grade IV astrocytomas (4.84 ± 0.89 mmol/L). Relaxation constants of choline-containing compounds did not differ in tumors from those determined in normal brain. Perchloric acid extracts of biopsy samples from 35 astrocytomas and 13 samples of normal temporal white matter were analyzed with ¹H NMR. Total concentration of choline-containing compounds did not differ between controls and any grade of astrocytoma when the quantification was done in vitro. It is interesting that phosphorylcholine concentration was about twofold greater in grade IV astrocytomas than in controls or other grades of astrocytomas. We conclude that high phosphorylcholine in grade IV astrocytomas may be an indicator of degree of malignancy. The proportional changes within the group of choline-containing compounds observed in vitro were not reflected in the NMR properties of choline signal in vivo.     

Phylogenetic analysis of cuckoo wasps (Hymenoptera: Chrysididae) reveals a partially artificial classification at the genus level and a species‐rich clade of bee parasitoids

Cuckoo wasps (Hymenoptera: Chrysididae) are a species‐rich family of obligate brood parasites (i.e. parasitoids and kleptoparasites) whose hosts range from sawflies, wasps and bees, to walking sticks and moths. Their brood parasitic lifestyle has led to the evolution of fascinating adaptations, including chemical mimicry of host odours by some species. Long‐term nomenclatural stability of the higher taxonomic units (e.g. genera, tribes, and subfamilies) in this family and a thorough understanding of the family's evolutionary history critically depend on a robust phylogeny of cuckoo wasps. Here we present the results from phylogenetically analysing ten nuclear‐encoded genes and one mitochondrial gene, all protein‐coding, in a total of 186 different species of cuckoo wasps representing most major cuckoo wasp lineages. The compiled data matrix comprised 4946 coding nucleotide sites and was phylogenetically analysed using classical maximum‐likelihood and Bayesian inference methods. The results of our phylogenetic analyses are mostly consistent with earlier ideas on the phylogenetic relationships of the cuckoo wasps' subfamilies and tribes, but cast doubts on the hitherto hypothesized phylogenetic position of the subfamily Amiseginae. However, the molecular data are not fully conclusive in this respect due to low branch support values at deep nodes. In contrast, our phylogenetic estimates clearly indicate that the current systematics of cuckoo wasps at the genus level is artificial. Several of the currently recognized genera are para‐ or polyphyletic (e.g. Cephaloparnops, Chrysis, Chrysura, Euchroeus, Hedychridium, Praestochrysis, Pseudochrysis, Spintharina, and Spinolia). At the same time, our data support the validity of the genus Colpopyga, previously synonymized with Hedychridium. We discuss possible solutions for how to resolve the current shortcomings in the systematics of cuckoo wasp genera and decided to grant Prospinolia the status of a valid genus (Prospinolia stat.n. ) and transferring Spinolia theresae [du Buysson 1900] from Spinolia to Prospinolia (Prospinolia theresae stat.restit. ). We discuss the implications of our phylogenetic inferences for understanding the evolution of host associations in this group. The results of our study not only shed new light on the evolutionary history of cuckoo wasps, but also set the basis for future phylogenomic investigations on this captivating group of wasps by guiding taxonomic sampling efforts and the design of probes for target DNA enrichment approaches.     

Cell-mediated immunity in human acute myeloblastic leukemia

Summary Lymphocyte stimulation tests with autologous myeloblasts were performed in 31 patients with nonlymphatic acute leukemia. Twenty-five patients were receiving chemotherapy combined with immunotherapy; six received chemotherapy only. Thirteen nonleukemic patients with various disorders and six healthy control patients were also studied.It was found that 4/15 patients in 9/38 tests had autologous lymphocytes stimulated by autologous circulating myeloblasts. Bone marrow cells also effected stimulation, significantly more often if the marrow was taken in relapse than when it was taken in remission.However, so-called immunotherapy with allogeneic leukemic myeloblasts and BCG could not be shown to increase these frequencies. Nor did it significantly increase the degree of stimulation measured as DNA synthesis in lymphocytes.Moreover, nonleukemic bone marrow cells from patients with other disorders also stimulated autologous lymphocytes in 1/13 patients. No recognition of autologous myeloblasts was observed when the responding lymphocytes were taken in incomplete remission or during the month preceding relapse.with the technical assistance of T. Lehtinen and A. M. SjögrenSupported by the Swedish Cancer Research Foundation Grant no. 699-B76-04XA     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.     

Structure of the human argininosuccinate synthetase gene and an improved system for molecular diagnostics in patients with classical and mild citrullinemia

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia, an autosomal recessive inherited defect of the urea cycle. Most patients described so far have presented with the classical form of the disease. There are also patients with a mild form of citrullinemia in whom the exact molecular basis and clinical relevance are uncertain. Mutations in the human ASS gene have not yet been described in mildly affected or asymptomatic patients with citrullinemia. The genomic sequence of the human ASS gene is not precisely known making mutation analysis difficult. Here, the entire genomic DNA sequence and mutations in the ASS gene of patients with the classical and mild form of the disease are described. The mutations c.1168G-->A (G390R) and IVS13+5 G-->A and the novel mutation c.323G-->T (R108L) have been found to be associated with classical citrullinemia, whereas the novel mutations c.535T-->G (W179R), and c.1085G-->T (G362V) have been detected on alleles of the mildly affected patients. Thus, mutations found in the human ASS gene of asymptomatic children with biochemical abnormalities and in some cases enzymatically proven citrullinemia have allowed us to classify these cases as ASS-deficient patients. The elucidation of the structure of the human ASS gene has made possible the use of intronic primers for molecular analysis of patients with mild disease and the classical form, and provides another option for prenatal diagnostics in affected families with the severe type.     

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.     

Precipitation of porcine insulin with carbon dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO₂) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO₂ as an acid‐precipitating agent. The solubility of porcine insulin in NaHCO₃ solutions in pressurized CO₂ was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self‐association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO₂, although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909–919. © 2009 Wiley Periodicals, Inc.     

Polyphosphate accumulation among denitrifying bacteria in activated sludge

Bacterial polyphosphate accumulation and denitrification are important processes in biological removal of nutrients from wastewater. It has been suggested that phosphorus accumulators are able to denitrify. However, the bacteria known as the most important phosphorus accumulators, belonging to the genus Acinetobacter are generally not known to denitrify. To clarify how commonly both physiological traits are present in the same organism, we screened 165 isolates from activated sludge and wastewater for their ability to denitrify, and the ability of the denitrifying isolates to accumulate polyphosphate. Of the 165 isolates, 149 were from acetate mineral medium (87 of these identified as Acinetobacter by the API 20 NE identification system) and 16 were from nutrient broth and nitrate medium. Only 15 of 165 isolates tested showed true respiratory denitrification activity. In the presence of acetylene they converted more than 80% of 5mM NO3- to N2O in 6 days. None of the Acinetobacter isolates were among the 15 respiratory denitrifiers. The denitrifying isolates were identified as species of Pseudomonas, Agrobacterium, Pasteurella, Sphingomonas or could not be identified by the API 20 NE identification system. According to the BIOLOG identification system the denitrifiers were species of Pseudomonas, Hydrogenophaga, Citrobacter, Xanthomonas or they could not be identified. The ability of confirmed denitrifiers to accumulate phosphate was measured in experiments where cells pregrown under phosphorus limitation were exposed to phosphate (8 mg P/L) under aerobic conditions. The rates of excess phosphate uptake varied from 0.3 to more than 23 mg P/g dry matter/h. Rates for four isolates were higher than those reported for Acinetobacter strains. These results show that polyphosphate accumulation and denitrification in activated sludge can be carried out by the same organisms.     

Olfactory and gustatory sensitivity in adults with attention-deficit/hyperactivity disorder

Recently, research on olfactory functions in attention-deficit/hyperactivity disorder (ADHD) has become prominent, whereas gustation has never been investigated. Increased odor sensitivity was found in medication-na?ve children with ADHD, but not in adult ADHD, which might be due to a dopaminergic dysregulation presumed to underlie this disorder. Taste sensitivity, in particular bitter sensitivity as a hereditary trait, also might be altered in ADHD. To examine olfactory and gustatory functions in adult ADHD patients, we assessed odor sensitivity by Sniffin' Sticks, taste sensitivity by taste strips, and bitter sensitivity by the one-solution test in women with ADHD (n = 12), Bulimia Nervosa (n = 12), and healthy control women (n = 12). Bulimia Nervosa as second patient group was included to control for effects of impulsivity. Preliminary results indicate that ADHD patients were significantly more often classified as tasters, i.e. perceived the bitter taste as more intense, compared to both bulimic patients and healthy controls. No group differences were found with regard to general odor and taste sensitivity. It is proposed that the higher frequency of tasters in ADHD patients might underlie a genetic variation of the bitter receptor-dependent signaling pathway associated with ADHD.