Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

用快速琼脂糖层析纯化磷酸甘油酸激酶及磷酸甘油醛脱氢酶

研究了用快速琼脂糖离子交换层析(DEAE—Fast Flow sepharose)结台PEG 4000／Reppal PES双水相体系从黄豆中分离纯化磷酸甘油酸激酶(PGK)及磷酸甘油醛脱氢酶(GAPDH)。控制床层高度(10～20cm),径向放大具有压降低的优点．设计多点进料取代传统的中心管进料,解决了径向流场不均匀的问题。GAPr)H的总收率及纯化倍数分别为58％和144,PGK的总收率及纯化倍数分别为41％和44。工艺成本为2．92美元／ku GPADH,具有一定的实用价值。     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .     

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Eclosion hormone-stimulated cGMP levels in the central nervous system of Manduca sexta: inhibition by lipid metabolism blockers,increase in inositol(1,4,5)trisphosphate and further evidence against the involvement of nitric oxide

Previous studies have shown that the neuropeptide, eclosion hormone, stimulates a nitric oxide-independent increase in the levels of cGMP in the nervous system of Manduca sexta. By contrast, recent results in Bombyx mori suggest that eclosion hormone increases cGMP via the production of nitric oxide. In view of these conflicting results we have carried out additional studies to test whether nitric oxide is involved in this process in Manduca. Evidence presented here supports our earlier observations that in Manduca the eclosion hormone-stimulated increase in cGMP is nitric oxide-and carbon monoxide-independent. In addition, we show that a wide variety of inhibitors of lipid metabolism block the eclosion hormone-stimulated cGMP increase. This supports the hypothesis that the activation of the guanylate cyclase is mediated by a lipid messenger. We also show that eclosion hormone stimulates an increase in the levels of inositol(1,4,5)trisphosphate. The time-course of this increase is consistent with the hypothesis that eclosion hormone stimulation of a phospholipase C is an early event in the cascade that results in an increase in cGMP. Receptor-mediated lipid hydrolysis is often mediated by G protein-coupled receptors. Experiments using pertussis toxin show that the eclosion hormone-stimulated increase in cGMP is not mediated by a pertussis toxin-sensitive G protein.Abbreviations AACOCF ₃ arachidonyl trifluoromethyl ketone - 4-BPB 4-bromophenacyl bromide - cGMP guanosine 3,5 cyclic monophosphate - D609 tricyclodecan-9-yl-xanthogenate - DEDA 7,7 dimethyleicosadienoic acid - DAG diacylglycerol - EH eclosion hormone - ET-18-OCH ₃ 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine - ETYA 5,8,11,14-eicosatetraynoic acid - InsP ₃ inositol(1,4,5)trisphosphate - LO lipoxygenase - Lyso-PA lysophosphatidic acid - HPLC highpressure liquid chromatography - NDGA nordihydroguaiaretic acid - NOS nitric oxide synthase - OEPC oleoxyethyl phosphorylylcholine - ONO-RS-082 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid - oxo-M oxotremorine-M - PAF platelet-activating factor - PKC protein kinase C - PLA ₂ phospholipase A₂ - PLC phospholipase C - PLD phospholipase D - PPH phosphatidate phosphohydrolase - PtdIns(4,5)P ₂ phosphatidylinositol bisphosphate - PTX pertussis toxin - TEA triethylamine - TFA trifluoroacetic acid - U-73122 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione     

Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages

Intracellular Ca²⁺ (Ca_i) signaling following the binding of surface receptors activates a Ca²⁺ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH_o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca²⁺-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP₃), (ii) intracellular dialysis with high concentrations of the Ca²⁺ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca²⁺-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca²⁺ selective with a selectivity sequence of Ca²⁺ > Sr²⁺ Mg²⁺ = Mn²⁺ = Ni²⁺. The Ca²⁺ influx current was modulated by changes in pH_o; modulation was not produced as a consequence of changes in internal pH (pH_i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH_o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH_o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca_i transient associated with receptor activation by regulating the influx of Ca²⁺ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823.     

Immobilization of Haloferax mediterranei aldolase by cross-linking in a proteinic matrix: stability and halophilic characteristics

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) of Haloferax mediterranei was immobilized by treating the cell extract in the presence of 10% BSA, with the cross-linking reagent, 0.5% glutaraldehyde for 15min, with the retention of 60% of its original activity. The immobilized preparation exhibited a shift in the temperature optimum from 55°C to 65°C. The enzyme showed enhanced stability towards inactivation by radiation and storage (0–5°C) on immobilization. Immobilization also made the enzyme less halophilic, reducing its denaturation on prolonged storage in a non-salt medium, as well as exhibiting optimal activity at a lower KCl concentration (0.5m) as compared to the soluble enzyme (1–2m).     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.