The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.     

Origin of the glial processes responsible for the spontaneous postnatal phagocytosis of boutons on cat spinal motoneurons

Summary Previous studies have demonstrated that astrocyte processes are responsible for a spontaneously occurring phagocytosis of boutons on cat spinal motoneurons during the second postnatal week. In the present investigation, the astrocytes and the astrocyte processes in contact with the motoneurons were studied qualitatively and quantitatively during the early postnatal period. It could be concluded that the cells responsible for the phagocytosis of boutons are immature astrocytes. These cells were present not only during the period of phagocytosis but also prior to this period. The type of process responsible for the phagocytosis was present not only during the period of phagocytosis but also prior to and after that period although the relative contribution of such processes to the glia-covered membrane area of the motoneurons was reduced in the older animals. On the basis of these results, the possible specificity of the immature astrocyte as the element responsible for the phagocytosis of boutons during normal development is discussed.The author is indebted to Miss Maj Berghman, Mrs. Anna-Stina Höijer and Mrs. Lillebil Stuart for excellent technical assistance. This work was supported by grants from Karolinska Institutet and the Swedish Medical Research Council (proj. 2886).     

Comparison of PSA-specific CD8<Superscript>+</Superscript> CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins

The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.     

White matter lesions and soluble interleukin-1 receptor type II in CSF from demented and non-demented subjects

White matter lesions (WMLs) in the brain is a common, unspecific finding on magnetic resonance imaging appearing both in the healthy elderly as well as in a number of different diseases including dementia disorders. However, the pathophysiological and clinical significance of WMLs in dementia disorders is still unknown. In this study, we investigated the possibility of their origin being inflammatory by studying the correlation between WMLs and cerebrospinal fluid (CSF) levels of the proinflammatory cytokine soluble interleukin-1 receptor type II (sIL-1RII). The sIL-1RII is a member of the IL-1 family, and has been found to be elevated in CSF from Alzheimer's disease (AD) patients. In the present study, two groups of patients complaining of memory disturbances with little or extensive WMLs respectively, were examined, as well as healthy subjects. In accordance with other reports, WML scores (total, periventricular as well as deep lesions) were positively correlated with age but not mini mental state examination (MMSE) scores, and were significantly higher in patients with a dementia diagnosis as compared to non-demented subjects. There were no differences in sIL-1RII levels in CSF regardless of amount of total, periventricular or deep WMLs, nor were there any differences between demented and non-demented subjects. In conclusion, sIL-1RII levels in CSF are not correlated to magnetic resonance imaging WMLs in patients with dementia disorders or in healthy subjects.     

Poly-acid properties of biosynthetic hyaluronan studied by titration

In this study the poly-acid properties of biosynthetic hyaluronan produced by fermentation of Bacillus subtilis have been investigated. Potentiometric titration as well as ¹H NMR titration have been used to determine the dissociation constants of the carboxylic group on hyaluronic acid. The intrinsic pK_a and pK_{a, α=0.5} were determined in the presence of 0.1 M salt to be 2.99 and 3.37, respectively. The pK_{a, α=0.5} was found to be unaffected by variations in the ionic strength which is in good agreement with the fact that at α = 0.5, 50% of the carboxylic moieties on the hyaluronan are charged. On the other hand the intrinsic pK_a was found to be dependent on the ionic strength until the Debye-Hückel screening length approaches the length of repeating disaccharide unit of hyaluronic acid.Our findings are in good agreement with previously determined dissociation constants for other sources of hyaluronan. We have also shown that ¹H NMR spectroscopy is the preferred method for polyelectrolyte titration because of the ability to isolate the contribution of several ionisable groups on a polymer on molecular level.     

Discovery of cyclopentane- and cyclohexane-trans-1,3-diamines as potent melanin-concentrating hormone receptor 1 antagonists

We herein report the optimization of cyclopentane- and cyclohexane-1,3-diamine derivatives as novel and potent MCH-R1 antagonists. Structural modifications of the 2-amino-quinoline and thiophene moieties found in the initial lead compound served to improve its metabolic stability profile and MCH-R1 affinity, and revealed unprecedented SAR when compared to other 2-amino-quinoline-containing MCH-R1 antagonists.     

Plasma biomarkers of brain atrophy in Alzheimer's disease

Peripheral biomarkers of Alzheimer''s disease (AD) reflecting early neuropathological change are critical to the development of treatments for this condition. The most widely used indicator of AD pathology in life at present is neuroimaging evidence of brain atrophy. We therefore performed a proteomic analysis of plasma to derive biomarkers associated with brain atrophy in AD. Using gel based proteomics we previously identified seven plasma proteins that were significantly associated with hippocampal volume in a combined cohort of subjects with AD (N = 27) and MCI (N = 17). In the current report, we validated this finding in a large independent cohort of AD (N = 79), MCI (N = 88) and control (N = 95) subjects using alternative complementary methods—quantitative immunoassays for protein concentrations and estimation of pathology by whole brain volume. We confirmed that plasma concentrations of five proteins, together with age and sex, explained more than 35% of variance in whole brain volume in AD patients. These proteins are complement components C3 and C3a, complement factor-I, γ-fibrinogen and alpha-1-microglobulin. Our findings suggest that these plasma proteins are strong predictors of in vivo AD pathology. Moreover, these proteins are involved in complement activation and coagulation, providing further evidence for an intrinsic role of these pathways in AD pathogenesis.     

Effects of DHA-rich n-3 fatty acid supplementation on gene expression in blood mononuclear leukocytes: the OmegAD study

Background

Dietary fish oil, rich in n-3 fatty acids (n-3 FAs), e.g. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), regulate inflammatory reactions by various mechanisms, e.g. gene activation. However, the effects of long-term treatment with DHA and EPA in humans, using genome wide techniques, are poorly described. Hence, our aim was to determine the effects of 6 mo of dietary supplementation with an n-3 FA preparation rich in DHA on global gene expression in peripheral blood mononuclear cells.

Methods and Findings

In the present study, blood samples were obtained from a subgroup of 16 patients originating from the randomized double-blind, placebo-controlled OmegAD study, where 174 Alzheimer disease (AD) patients received daily either 1.7 g of DHA and 0.6 g EPA or placebo for 6 months. In blood samples obtained from 11 patients receiving n-3 FA and five placebo, expressions of approximately 8000 genes were assessed by gene array. Significant changes were confirmed by real-time PCR. At 6 months, the n-3 FAs group displayed significant rises of DHA and EPA plasma concentrations, as well as up- and down-regulation of nine and ten genes, respectively, was noticed. Many of these genes are involved in inflammation regulation and neurodegeneration, e.g. CD63, MAN2A1, CASP4, LOC399491, NAIP, and SORL1 and in ubiqutination processes, e.g. ANAPC5 and UBE2V1. Down-regulations of ANAPC5 and RHOB correlated to increases of plasma DHA and EPA levels.

Conclusions

We suggest that 6 months of dietary n-3 FA supplementation affected expression of genes that might influence inflammatory processes and could be of significance for AD.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00211159","term_id":"NCT00211159"}}NCT00211159