BBA - Molecular Basis of Disease (v.1586, #1)
Human α-fetoprotein as a Zn2+-binding protein. Tight cation binding is not accompanied by global changes in protein structure and stability by Serge E. Permyakov; Keith A. Oberg; Alexandra M. Cherskaya; Mikhail M. Shavlovsky; Eugene A. Permyakov; Vladimir N. Uversky (1-10).
The binding of zinc to human α-fetoprotein (AFP) isolated from human umbilical cord serum was studied by fluorimetric Zn2+-titration. We found that the total number of strong binding sites for zinc on this protein was 5: AFP has one very strong (dissociation constant, K d<10−8 M) and at least four lower affinity zinc binding sites (K d<10−5 M). Fourier transform infrared (FTIR) analysis revealed that aspartate and histidine residues could be involved in the strong coordination of zinc. Intriguingly, binding of zinc to the protein does not induce structural changes that can be detected by circular dichroism, FTIR, intrinsic fluorescence or (1,1′)-bi-(4-anilino)naphthalene-5,5′-disulfonic acid (bis-ANS) binding. Finally, scanning microcalorimetry measurements showed that stability of the protein is also unaffected by zinc binding in spite of the strength of the coordination. Such strong interactions without major structural consequences are highly unusual, and AFP may therefore be the first characterized representative of a new class of ligand-binding proteins.
Keywords: α-Fetoprotein; Carcinoembryonic protein; Protein structure; Structural transition; Conformational stability; Zn2+ binding;
The inhibitory effects of immunosuppressive factors, dexamethasone and interleukin-4, on NF-κB-mediated protease production by oral cancer by Mahiro Beppu; Tetsuro Ikebe; Kanemitsu Shirasuna (11-22).
Matrix metalloproteinase-9 (MMP-9) produced by tumor cells is known to be implicated in the invasion of squamous cell carcinoma (SCC). In the process of searching for agents to inhibit MMP-9 in cancer, immunosuppressive factors, dexamethasone (DEX) and interleukin-4 (IL-4) were found to inhibit protein production as well as gene expression of MMP-9 in tumor necrosis factor α (TNFα)-stimulated SCC cells. DEX and IL-4 could also suppress the expression of urokinase type plasminogen activator (uPA) to prevent the conversion from the proenzyme form of MMP-9 to its active form. Regarding their mechanisms to inhibit the expression of MMP-9 and uPA, DEX and IL-4 had no effect on the cell surface levels of TNFα receptors, but inhibited the activation of NF-κB and NF-κB-dependent gene expression. DEX, but not IL-4, could strongly augment the TNFα-induced expression of IκBα in SCC cells. These results suggest that DEX and IL-4 suppress not only immunological reactions, but also tumor invasion by targeting NF-κB.
Metabolic consequence of long-term exposure of pancreatic β cells to free fatty acid with special reference to glucose insensitivity by Katsumi Iizuka; Hiromu Nakajima; Mitsuyoshi Namba; Jun-ichiro Miyagawa; Junichi Miyazaki; Toshiaki Hanafusa; Yuji Matsuzawa (23-31).
Long-term exposure of the pancreatic β cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic β cells with special reference to insulin secretion. Pancreatic β-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of β-oxidation. Intracellular content of triglyceride and FFA were elevated, β-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to β cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.
Keywords: Palmitate; Insulin; Lipotoxicity; Malate pyruvate shuttle; Pyruvate carboxylase;
Characterization of a missense mutation at histidine-44 in a pyruvate dehydrogenase-deficient patient by Scott J Jacobia; Lioubov G Korotchkina; Mulchand S Patel (32-42).
Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the α subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient’s missense mutation αH44R in E1α by creating and purifying three recombinant human E1s (αH44R, αH44Q, and αH44A). Substitutions at histidine-15 resulted in decreased V max values (6% αH44R; 30% αH44Q; 90% αH44A) while increasing K m values for thiamine pyrophosphate (TPP) compared to wild-type (αH44R, 3-fold; αH44Q, 7-fold; αH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of αH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the αH44R E1 patient.
Keywords: Pyruvate dehydrogenase; Site directed mutagenesis; Inborn error; Pyruvate dehydrogenase complex deficiency;
A novel splicing mutation causes an undescribed type of analbuminemia by Monica Campagnoli; Antonio Rossi; Lars Palmqvist; Anders Flisberg; Aimon Niklasson; Lorenzo Minchotti; Monica Galliano (43-49).
Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous subjects. In this report we describe a new molecular defect that caused the analbuminemic trait in a newborn of Iraqi origin. When the parents’ DNA was analyzed, both subjects were found to be heterozygous for the same mutation found in the infant. All the 14 exon and flanking intron sequences of the albumin gene were amplified via PCR and screened for mutations by SSCP and heteroduplex analysis. A mutation in the DNA region encoding exon 1 and its flanking intron was revealed by the presence of a heteroduplex. The fragment, which was directly DNA sequenced, contains a previously unreported single nucleotide change, consisting in a G to A substitution at nucleotide 118 in the structural gene of the human protein. This mutation, involving the first base of intron 1, destroys the GT dinucleotide consensus sequence found at the 5′ end of most intervening sequences and causes the defective pre-mRNA splicing responsible for the analbuminemic trait.
Keywords: Analbuminemia; Human serum albumin; Splicing mutation;
Presence of BRCA1 and BRCA2 proteins in human milk fat globules after delivery by Cécile Vissac; Didier Lémery; Ludovic Le Corre; Pierre Fustier; Pierre Déchelotte; Jean-Claude Maurizis; Yves-Jean Bignon; Dominique J. Bernard-Gallon (50-56).
We evaluated BRCA1 and BRCA2 oncosuppressor protein expression in 26 milk samples in women just after delivery. The quantification of BRCA1 and BRCA2 proteins was performed in isolated milk fat globules using an affinity chromatography strategy. The amounts of BRCA1 and BRCA2 proteins were found to be similar. We explained the presence of BRCA1 and BRCA2 proteins in human milk fat globules by the fact that they are formed by exocytosis of lipids from epithelial cells of the mammary gland and are enveloped by plasma membrane from the apical part of the milk-secreting cells. This raises the possibility that BRCA1 and BRCA2 proteins are a protective response to proliferation and play a possible role in newborn nutrition.
Keywords: Oncosuppressor; BRCA1; BRCA2; Quantification of protein; Human milk fat globule; Delivery; Affinity perfusion chromatography;
Effects of regional hypoxia and acidosis on Rb+ uptake and energetics in isolated pig hearts: 87Rb MRI and 31P MR spectroscopic study by V.V Kupriyanov; B Xiang; J Sun; O Jilkina; R Deslauriers (57-70).
The study compared the effects of regional hypoxia and acidosis on Rb+ uptake and energetics in isolated pig hearts perfused by the Langendorff method. The left anterior descending artery (LAD) was cannulated and the LAD bed was perfused with the same specific flow as the whole heart. Following equilibration with normal Krebs–Henseleit buffer (KHB, pO2 568 mm Hg, pH 7.42) the perfusate was switched to one that contained Rb+ (Rb-KHB). Simultaneously, perfusion through the LAD was carried out with hypoxic (pO2=31 mm Hg), an acidemic (pH 7.12) or normal (pO2=550 mm Hg) Rb-KHB for 120 min. 87Rb images of the entire heart or localized 31P spectra from the left ventricular anterior wall were acquired. Hypoxia decreased the maximal 87Rb image intensity and Rb+ flux in the anterior wall to 79±9% and 85±7%, respectively, of that in the posterior wall. Extracellular acidosis did not affect 87Rb image intensity and reduced Rb+ flux (83±10%). During hypoxia phosphocreatine and ATP decreased to 36±10 and 50±15% of baseline, respectively and intracellular pH (pHi) decreased to 6.90±0.05. Extracellular acidosis did not affect the phosphocreatine or ATP levels but reduced pHi (7.06±0.18 vs. 7.26±0.06 in control). We suggest that intracellular acidosis plays a role in the inhibition of Rb+ uptake during hypoxia.
Keywords: Hypoxia; Acidosis; Rb+ uptake; Energy metabolism; 87Rb magnetic resonance imaging; 31P nuclear magnetic resonance;
Renal function and cortical (Na++K+)-ATPase activity, abundance and distribution after ischaemia–reperfusion in rats by Gabriela Coux; Laura Trumper; M.Mónica Elı́as (71-80).
The effects of ischaemic injury and reperfusion on renal function, cortical ATP content, alkaline phosphatase activity and (Na++K+)-ATPase activity and abundance in cortical homogenates and isolated basolateral and apical membranes were examined. Rats were submitted to 5 or 40 min of right renal artery occlusion and 60 min of reperfusion. Renal function of the ischaemic–reperfused kidney was studied by conventional clearance techniques. Our results show that 1 h of reperfusion after a short period of renal ischaemia (5 min) allows the complete restoration of the biochemical features of cortical cells and functional properties of the injured kidney. A longer period of ischaemia, such as 40 min, followed by 1 h of reperfusion showed functional and biochemical alterations. ATP recovered from 26% after 40 min of ischaemia to 50% of control values after 1 h reperfusion. However, renal function was strongly impaired. Brush border integrity was compromised, as suggested by AP excretion and actin appearance in urine. Although total cortical (Na++K+)-ATPase activity was not different from controls, its distribution in isolated apical and basolateral membranes was abnormal. Remarkably, we detected an increase in α-subunit protein abundance that may suggest that (Na++K+)-ATPase synthesis is promoted by ischaemia–reperfusion. This increase may play an important role in the pathophysiology of ischaemic acute renal failure.
Keywords: Rat kidney; Renal function; (Na++K+)-ATPase; Ischemia–reperfusion; Acute renal failure; Basolateral membrane; Apical membrane;
Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by d-2-hydroxyglutaric acid in vitro by Cleide G da Silva; César A.J Ribeiro; Guilhian Leipnitz; Carlos S Dutra-Filho; Ângela T.S Wyse; Clóvis M.D Wannmacher; João J.F Sarkis; Cornelis Jakobs; Moacir Wajner (81-91).
l-2-Hydroxyglutaric (LGA) and d-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as l-2-hydroxyglutaric aciduria and d-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO2 production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO2 production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 22.214.171.124) in a dose-dependent manner (35–95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by d-2-hydroxyglutaric aciduria.
Keywords: l-2-Hydroxyglutaric acid; d-2-Hydroxyglutaric acid; Brain metabolism; Cytochrome c oxidase;
Changes in gene expression in rat thymocytes identified by cDNA array support the occurrence of oxidative stress in early magnesium deficiency 1 Presented in part at the 9th International Magnesium Symposium, 10–15 September 2000, Vichy, France. 1 by Isabelle Petrault; Wioletta Zimowska; Jacques Mathieu; Dominique Bayle; Edmond Rock; Alain Favier; Yves Rayssiguier; Andrzej Mazur (92-98).
Magnesium deficiency in experimental animals leads to inflammation, exacerbated immune stress response and a decrease of specific immune response. It also results in a significant increase in free radical species and subsequent tissue injury. An accelerated thymus involution was observed in Mg-deficient rats in relation to enhanced apoptosis and enhanced susceptibility to oxidative stress. To examine the stress-inducing effects of low Mg status on thymocytes, cDNA arrays were used to evaluate changes in gene expression in weaning rats submitted to Mg deficiency of short duration (2 days). Several genes exhibited changes in their expression caused by Mg deficiency before any perceptible modification in cell integrity and functions. The up-regulated genes included cytochrome c oxidase, glutathione transferase, CuZn superoxide dismutase, genes associated with the stress response (HSP70 and HSP84) and a gene involved in DNA synthesis and repair (GADD45). The down-regulated genes included Na/P cotransporter 1. These findings are consistent with altered cell growth, modifications of ion fluxes and oxidative stress described during Mg deficiency. The observation of induction of genes involved in protection and repair in cells from Mg-deficient animals provides additional evidence of the role of oxidative stress in the pathobiology of this deficiency.
Keywords: Magnesium; Thymocyte; cDNA array; Gene expression; Oxidative stress;
X Chromosomal short tandem repeat polymorphisms near the phosphoglycerate kinase gene in men with chronic prostatitis by Donald E. Riley; John N. Krieger (99-107).
Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11–13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11–13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.
Keywords: Prostatitis; Short tandem repeat; X Chromosome; Prostate disease;
Systemic lupus erythematosus patients under immunosuppressive treatment express high levels of the immunoglobulin lambda variable IGLV8S1 gene with silent somatic mutations 1 1 This project was approved by the Ethics Commission, University Hospital, Faculty of Medicine of Ribeirão Preto (USP) (no. 4953/98). by Marcia Cristina Tamia-Ferreira; Glauce L. Trevisan; Ivan Fiore de Carvalho; Geraldo A.S. Passos (108-112).
Systemic lupus erythematosus (SLE) patients express high titers of somatically mutated serum autoantibodies against nuclear structures including double-stranded DNA. These somatic mutations accumulate codons for basic amino acids in the immunoglobulin variable regions of both, heavy and light chains, facilitating binding to nucleic acids. The variable (V) immunoglobulin lambda 8 (IGLV8S1) gene contributes to autoreactive B-cell repertoire of auto-immune patients. Accumulation of immune complexes of these anti-DNA autoantibodies causes severe systemic inflammation in SLE. The current treatment of lupus disease is based on immunosuppressive drugs, but the precise role for this therapy remains to be defined. To evaluate the in vivo effect of combined immunosuppressive treatment on B-lymphocytes repertoire of SLE patients, we have developed an approach using the IGLV8S1 gene as a marker. The transcription of this gene in treated SLE patients was increased. However, we observed a trend, in these patients, to conserve complementarity determining regions (CDRs) and framework regions (FRs) of Vλ8 polypeptide light chain deduced sequence, from its germline counterpart. Sequencing IGLV8S1 cDNA of untreated SLE patients, taken as a control for treatment effect, displayed a decreased frequency of silent somatic mutations (consequently high frequency of replacement mutations) in the Vλ8 polypeptide chain deduced sequence. These data suggest that the immunosuppressive drug treatment modulates the positive selection of somatically mutated Vλ8 light chain.
Keywords: Systemic lupus erythematosus; Immunosuppression; Immunoglobulin lambda variable gene; IGLV8 gene expression; cDNA; Somatic mutation; Antibody repertoire;
P24, a glycogen synthase kinase 3 (GSK 3) inhibitor by Concepción Pérez Martı́n; Jesús Vázquez; Jesús Avila; Francisco J. Moreno (113-122).
A heat resistant glycogen synthase kinase 3 (GSK 3) binding protein, p24, that inhibits its kinase activity at a low magnesium concentration (in a way similar to that of lithium) was found in microtubules from adult rat brains. This protein associates with GSK 3 in microtubules and corresponds to one previously described in the literature as p25, although it has a relative molecular weight of 23 472. p24 is a poor substrate for GSK 3 but it could be phosphorylated by other protein kinases such as cAMP dependent protein kinase and cdk 5. Since p24 could form complexes with GSK 3, it may not only regulate GSK 3 activity but also it might act as an anchoring protein for the kinase.
Keywords: p24; Glycogen synthase kinase 3 inhibitor; GSK 3 binding protein; Protein kinase; Alzheimer’s disease;
Sialyltransferase activity of human plasma and aortic intima is enhanced in atherosclerosis by Elena V Gracheva; Nelya N Samovilova; Natalia K Golovanova; Olga P Il’inskaya; Edward M Tararak; Peter P Malyshev; Valery V Kukharchuk; Nina V Prokazova (123-128).
Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients’ plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K m) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V max) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients’ plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients’ plasma.
Keywords: Sialyltransferase activity; Human blood plasma; Human aortic intima; Membrane fraction; Atherosclerosis;