Increased plasma levels of soluble CD40, together with the decrease of TGF beta 1, as possible differential markers of Alzheimer disease.

Alzheimer's disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinson's disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.

A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.

Cysteine proteinases are responsible for characteristic transketolase alterations in Alzheimer fibroblasts.

Cultured fibroblasts from patients affected by Alzheimer's disease (AD) exhibited peculiar alterations of the enzyme transketolase (TK). Abnormalities (dubbed alkaline bands, ab) consisted of enzyme forms having unusually high pl and were proposed as a marker of the disease in living patients. The mechanisms of TK-ab expression were investigated with the use of cysteine proteinase inhibitors and purified preparations of either rat liver or human cysteine proteinases. The cysteine proteinase inhibitors N-acetyl-leu-leu-norleucinal (ALLN), L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane (E-64), and egg white cystatin added to AD cells just prior to extraction abolished TK abnormalities. Moreover, 1 day incubation of AD cultures with either ALLN (10 micrograms/ml), NH4Cl (10 mM), or KCl (30 mM) prevented TK-ab generation, due, presumably, to an impairment of lysosomal functions. Isolated rat liver cysteine proteinases were able to degrade TK in normal extracts and reproduce the characteristic TK-ab of AD fibroblasts. Moreover, pure human cathepsin H was also shown to partially induce an Alzheimer-like TK pattern and cleave normal TK to a 35 kDa fragment as spontaneously occurring in AD fibroblasts. The explanation of mechanisms of TK-ab formation provided evidence for an underlying imbalance of proteolysis in AD fibroblasts due to a relative increase/derangement of the cysteine proteinases cathepsins which might be also involved in the reported abnormal processing of multiple cellular components.

Megakaryocyte-like increase in ploidy of Friend's erythroleukemia cells induced to endoreplication by colcemid.

Previous work from this laboratory has shown that Friend's murine erythroleukemia cells (MELCs) express some bio-chemical traits of the megakaryocytic lineage. The supposed mixed erythroid/megakaryocytic nature of these cells has been investigated further by challenging MELCs with the antimicrotubule agent colcemid. This compound, at the concentration of 40 nM, was found to induce a striking arrest of cell growth without significant effects on viability. At the same time, the bulk of treated MELCs underwent a large increase in size to contain, after 3 days, as much as 4 times more proteins and 5 times more DNA than controls. As shown by high rates of 3H-thymidine incorporation, increase in DNA content was the result of active synthesis without completion of intervening mitosis according to a process that closely resembled endoreplication. Eventually, colcemidinduced MELCs presented multilobed nuclei and were arranged into discrete ploidy groups containing up to 16 N levels of DNA. Moreover, upon colcemid addition, MELCs initiated a polyploid response that was shown to continue, even in the absence of the inducer, to yield cells that became strongly positive for acetylcholinesterase (AChE) in the late stages of culture. These effects were compatible with a colcemid-induced commitment of MELCs to megakaryocyte differentiation, for which these cells seemed to be definitely programmed. The expression of megakaryocyte features in MELCs provided further evidence for the bipotentiality (erythroid/megakaryocytic) of this model.

Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells.

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts.

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 mM H2O2 for times > 60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca2+]i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H2O2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H2O2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.

Characteristic transketolase alterations in dermal fibroblasts of Alzheimer patients are modulated by culture conditions.

Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline pI, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO2 saturation in the atmosphere and with 3.7 g/liter NaHCO3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.

Acetylcholinesterase in murine erythroleukemia (Friend) cells: evidence for megakaryocyte-like expression and potential growth-regulatory role of enzyme activity.

Features of true acetylcholinesterase (AChE) regulation during growth and differentiation of Friend murine erythroleukemia cells (MELC) have been investigated with respect to other erythroid and nonerythroid murine elements. Enzyme levels of uninduced MELC were in between the very low AChE contents of erythroid cells and the huge amounts of activity exhibited by megakaryocytes and platelets. After MELC commitment to terminal division, the enzyme-specific activity increased largely, approaching values that were much closer to those of thrombocytic than of normal erythroid elements. The bulk of AChE activity in MELC, megakaryocytes, and platelets was found to be located in the cytosol as a free-soluble form. Moreover, during incubation, MELC actively released large amounts of AChE into the medium, like it occurs in murine thrombocytes. Conversely, the enzyme of the erythroid elements was mainly associated with the membranes and was not released extracellularly. Experiments with inducers showed that changes in AChE-specific activity of MELC correlated directly with the arrest of cell proliferation rather than with the activation of differentiated erythroid functions. The inverse relationship existing between MELC growth rates and AChE levels was further supported by the relative enzyme activities of the slow- and fast-growing subclones. We conclude that uninduced MELC potentially share properties of both the erythroid and megakaryoblastic phenotype. The latter might be revealed by typical regulation of AChE activity according to a thrombocytic-like program activated upon MELC commitment to terminal division. Eventually, the inhibition of MELC growth by exogenous pure bovine AChE suggested that the secreted murine enzyme might serve as a potential negative signal of cellular replication.

Enhanced proteolytic activities in cultured fibroblasts of Alzheimer patients are revealed by peculiar transketolase alterations.

Characteristic alterations of transketolase (TK) in extracts from cultured Alzheimer fibroblasts have previously been reported (Paoletti et al. (1990) Biochem. Biophys. Res. Commun., 172: 396-401). These abnormalities, encountered in 9 out of 13 Alzheimer patients, were revealed following isoelectric focusing and consisted of enzyme forms having unusually high alkaline pI values (alkaline bands). The present work has shown that immunologically detected alkaline bands were progressively expressed when Alzheimer fibroblasts were incubated for three weeks without medium changes. Full expression of the altered enzyme pattern was not linked to relative cell density in the petri dish; rather, it appeared to be dependent directly on the time elapsed since cell confluence was reached. Alkaline bands could artificially be induced also in both crude and pure TK preparations from normal cells by a treatment with commercial proteases, particularly chymotrypsin. Moreover, specific inhibitors of endogenous cysteine-proteases were capable of abolishing TK alkaline bands in Alzheimer fibroblasts thus turning a pathological into a normal enzyme pattern. Results obtained suggest that Alzheimer fibroblasts contain enhanced Ca(2+)-independent cysteine-proteolytic activities as compared to normal and other pathological cells. These enzymes, exhibiting chymotrypsin-like activity, might exert their degradative effects at the time of cell extraction using TK and probably other cell components as potential substrates. However, peculiar TK abnormalities represent so far an useful biochemical marker detectable in fibroblasts of living Alzheimer patients and closely associated to this neurological disorder.

Occurrence of transketolase abnormalities in extracts of foreskin fibroblasts from patients with Alzheimer's disease.

Foreskin fibroblast cell lines from healthy subjects and patients with Alzheimer's disease or other neurological disorders have been analyzed for transketolase, by means of isoelectrofocusing and specific immunostaining. The enzyme profile in 70% of Alzheimer cell cultures exhibits alterations which are not encountered in controls and in other neurological diseases examined. Enzyme abnormalities most frequently observed were the appearance of new transketolase forms having unusually high alkaline pI. Experiments carried out with intact cells and with cell extracts treated with and without phenylmethyl-sulphonyl fluoride, suggest that the peculiar transketolase abnormalities observed in Alzheimer fibroblasts are due to enhanced proteolysis occurring in these cells rather than to intrinsic enzyme lability.

Superoxide-driven NAD(P)H oxidation induced by EDTA-manganese complex and mercaptoethanol.

A purely chemical system for NAD(P)H oxidation to biologically active NAD(P)+ has been developed and characterized. Suitable amounts of EDTA, manganous ions and mercaptoethanol, combined at physiological pH, induce nucleotide oxidation through a chain length also involving molecular oxygen, which eventually undergoes quantitative reduction to hydrogen peroxide. Mn2+ is specifically required for activity, while both EDTA and mercaptoethanol can be replaced by analogs. Optimal molar ratios of chelator/metal ion (2:1) yield an active coordination compound which catalyzes thiol autoxidation to thiyl radical. The latter is further oxidized to disulfide by molecular oxygen whose one-electron reduction generates superoxide radical. Superoxide dismutase (SOD) inhibits both thiol oxidation and oxygen consumption as well as oxidation of NAD(P)H if present in the mixture. A tentative scheme for the chain length occurring in the system is proposed according to stoichiometry of reactions involved. Two steps appear of special importance in nucleotide oxidation: (a) the supposed transient formation of NAD(P). from the reaction between NAD(P)H and thiyl radicals; (b) the oxidation of the reduced complex by superoxide to keep thiol oxidation cycling.

Evaluation of monomethoxypolyethyleneglycol derivatized superoxide dismutase in blood and evidence for its binding to blood cells.

A method to evaluate in blood free superoxide dismutase (SOD) and SOD covalently bound to monomethoxypolyethylene glycol (MPEG-5000) is reported. The method takes advantage of gel filtration and cationic exchange chromatography to remove substances present in plasma which can interfere with the SOD enzymatic assay. Using this methodology, it was demonstrated that, contrary to free SOD, MPEG-SOD, when added to blood, was not completely recovered in plasma. Binding of MPEG-SOD to blood cell components takes place involving interactions of MPEG chains with cell membranes.

Analysis of transketolase and identification of an enzyme variant in human leukocytes.

Human leukocyte transketolase of fresh cell extracts has been analyzed by isoelectrofocusing on agarose gels (pH 3-10). The enzyme was then transblotted on nitrocellulose and detected with specific anti-transketolase IgG coupled to an avidin/biotin-immunoperoxidase system. Each sample yielded multiple enzyme forms, within a pI range of about 7.4-8.4. Transketolase profile, however, was not identical in all extracts. There are two mainly distinct patterns, showing qualitative and quantitative differences: a standard profile, which is predominant, and a variant, found in three unrelated subjects out of the two hundred and twenty. Standard and variant enzyme have similar Km values for ribose 5-P and xylulose 5-P and the same mobility on SDS-PAGE.

Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.

Changes in CuZn-superoxide dismutase during induced differentiation of murine erythroleukemia cells.

Superoxide dismutase (SOD) activity in murine erythroleukemia cells (MELC) was determined during differentiation induced by hexamethylene bisacetamide. SOD levels in hexamethylene bisacetamide-treated MELC were about twice as high as those of controls. Dose response and kinetic experiments have shown that SOD activity variations are closely related to the amount of inducer and the duration of treatment in culture. Moreover, phorbol 12-myristate 13-acetate, which inhibits hexamethylene bisacetamide-induced MELC maturation, was also effective in reducing the extent of the SOD increase. SOD changes mainly involved the CuZn form of the enzyme, having a molecular weight of about 32,000 and a striking sensitivity to cyanide inhibition. In addition, the isoelectrophoretic analysis of CuZn-SOD from both treated and untreated cells yielded an identical pattern. This suggests that quantitative rather than qualitative enzyme changes occurred during MELC terminal division. SOD levels are directly related to the degree of differentiation and particularly to the amount of cytosolic hemoglobin, whose synthesis in committed cells is paralleled by a rise in enzyme activity. The SOD increase represents a distinctive marker of erythroleukemia maturation and might tentatively be interpreted as a cellular response to oxidative stress from hemoglobin autoxidation.

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts.

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.