Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology.

Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donors, CMV was detected in 0 (0%) of the samples extracted with a QIAamp blood kit (QIAGEN), 1 (1%) of the samples extracted with an Amplicor whole-blood specimen preparation kit (Roche), and 8 (8%) of the samples extracted with an Isoquick nucleic acid extraction kit (modified by the addition of carrier tRNA) (Microprobe). CMV DNA was not detected in samples from seronegative blood donors (n = 13). Nested PCR of selected samples confirmed the detection of CMV in the sane eight samples extracted with the modified Isoquick nucleic acid extraction kit and detected an additional nine CMV-positive samples (n = 50). Samples from volunteer blood donors contain low copy numbers of CMV DNA. PCR amplification of such specimens can result in analytical sampling errors, giving results similar to the variations in titers recognized during determinations of the 50% tissue culture infective dose. The detection of CMV in blood samples from volunteer blood donors by PCR is a function of sample preparation, amplification conditions, and detection methodology. Accurate assessments of the clinical utility of CMV DNA detection by nucleic acid amplification for blood product screening and patients will require highly standardized and quantitative methodology.

Immunoassay of triiodothyronine in serum by time-resolved fluorometric measurement of europium-chelate complexes in solution.

We describe the development of a competitive immunoassay for triiodothyronine (T3) in serum. The assay combines immobilized antigens in microtitration wells with a biotinylated monoclonal anti-T3 antibody and a streptavidin-based universal detection reagent labeled with the Eu3+ chelator 4,7-bis(chloro-sulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid (BCPDA). In the assay, T3 released from binding proteins by thimerosal competes with immobilized antigen for binding to a limited amount of antibody. The bound biotinylated antibody is identified by a subsequent reaction with the detection reagent, and fluorescence of the final complex is then quantified in solution after it has been dissociated from the solid support by the addition of a detergent solution. Evaluation of the method demonstrated good overall precision and appropriate detection limit (0.2 nmol/L) and dynamic range. Analytical recovery averaged 99.9%, and results of dilution experiments were in agreement with the expected values. Measurements by the present method correlated well with those by a commonly used radioimmunoassay.

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.

Phosphatidylcholine and 4-methylumbelliferyl phosphorylcholine hydrolysis by purified placental sphingomyelinase.

We present evidence which indicates that highly purified placental acid sphingomyelinase hydrolyses [14C]phosphatidylcholine [( 14C]PC) and the synthetic phosphodiester 4-methylumbelliferyl phosphorylcholine (4-MUPC). Hydrolysis was achieved by phospholipase C phosphodiesterase action. Of the several detergents tested, sodium taurocholate alone was necessary for PC hydrolysis, while 4-MUPC was hydrolysed independent of any detergent requirement. The pH optima for the reactions were 4.6-4.8 for PC hydrolysis and 4.8-5.0 for 4-MUPC hydrolysis. As with sphingomyelin hydrolysis, degradation of both PC and 4-MUPC was inhibited by 5'-, 3'-, and 2'-AMP, 5'-AMP being the most effective of the three. Furthermore, the phosphodiesterase activity against PC and 4-MUPC copurified with sphingomyelinase from human placenta and cross-reacted with a specific anti-sphingomyelinase monoclonal antibody, strongly indicating identity of the phosphodiesterases. This explains phospholipase C deficiency in sphingomyelinase-deficient Niemann-Pick disease cells.

Studies on the activation of sphingomyelinase activity in Niemann-Pick type A, B, and C fibroblasts: enzymological differentiation of types A and B.

Cultured skin fibroblast homogenates from patients with Niemann-Pick disease Type C, were able to degrade sphingomyelin liposomes at a normal rate. Fibroblasts from patients with Niemann-Pick disease Types A and B were less active (0.08-0.55 versus 0.96-2.93 nmol/h/mg). When fibroblasts were maintained in synthetic media (MCDB-104) devoid of fetal calf serum for a period of 21 days, sphingomyelinase activity measured at pH 3.8 increased in control and Niemann-Pick Type C (up to 15-fold) and in Niemann-Pick Type B (up to 3-fold) while Niemann-Pick Type A showed no significant increase in sphingomyelinase activity. Addition of a protein activator isolated from the spleen of a Type I Gaucher's disease patient stimulated a 2-7.5-fold increase in sphingomyelinase activity in normal, Niemann-Pick Type B and C fibroblasts, while under the same conditions the Niemann-Pick Type A fibroblast enzyme responded poorly. Our data show that the residual sphingomyelinase activity in Niemann-Pick Type A can be differentiated from that present in other phenotypic forms by its lack of response to the Gaucher activator. Furthermore, we can find no evidence to support the view that Niemann-Pick Type C sphingomyelinase differs from the normal enzyme in its response to Gaucher activator.

Studies on the activation of the enzymatic hydrolysis of sphingomyelin liposomes.

Two pH optima were observed for the hydrolysis of sphingomyelin liposomes by brain and fibroblast extracts; one at pH 4.2-4.5, the other at pH 7-8. The proportion of the acidic activity in fibroblasts was affected greatly by the culturing conditions. Both the acidic and neutral enzyme activities were deficient in Niemann-Pick Type A fibroblasts, suggesting that both were genetically related. Partially purified activators from normal as well as Gaucher disease spleen stimulated the hydrolysis of sphingomyelin, at both pH values, by fibroblast and brain extracts. After further purification by DE-52 and Sephacryl 200 column chromatography the Gaucher activator retained its ability to stimulate sphingomyelinase and was active as well towards beta-glucocerebrosidase and beta-galactocerebrosidase.

Monoclonal antibodies against human placental sphingomyelinase.

Monoclonal antibodies against human placental acid sphingomyelinase have been raised. The antibodies are all of the IgG1 type, and are quite specific for the enzyme. One of the antibodies has been used to demonstrate a common antigenic identity of three polypeptides (mol.wts. 90,000, 72,000, and 48,000) of a purified sphingomyelinase preparation.

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.

The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups.

The natural substrate for sphingomyelinase contains hydrophobic and polar moieties. In this study, we have employed pH rate studies and examined hydrophobic compounds and phosphorylated esters for their effect on sphingomyelinase activity in an attempt to determine some of the kinetic properties of this enzyme. Sphingomyelinase, purified from human placentae, undergoes noncompetitive inhibition by octylglucoside and Nonidet P-40, two nonionic detergents containing terminal octyl groups. The effect of these detergents at the hydrophobic binding site is somewhat different from that of Triton X-100, which contains an isooctyl terminal group, and this may serve to identify a structural basis for the effects. Sphingomyelinase activity is also modulated by several nucleotides. Inhibition by 5'-adenosine monophosphate (5'-AMP) is also noncompetitive. Other nucleotide monophosphates (such as 5'-uridine monophosphate (5'-UMP), 5'-cytidine monophosphate (5'-CMP), 2'-adenosine monophosphate (2'-AMP), and 3'-adenosine monophosphate (3'-AMP) and phosphorylated intermediates (such as phosphorylcholine, phosphorylethanolamine and hexose phosphates) have a lower inhibitory effect. The data suggest that the inhibition by 5'-AMP involves the combined effect of the phosphate group and the purine ring, structural requirements which may also be satisfied by bis(4-methylumbelliferyl)phosphate, a synthetic enzyme substrate. Studies of pH rate indicate that the maximal velocity for the hydrolysis of sphingomyelin is independent of pH over the range 3.5-6.2 while the Km value shows a pH dependence. The Km value is lowest from pH 4.0-5.2 and rises at pH values outside this range. The log Vmax/Km and pKm relationships, when plotted as a function of pH, have been used to identify the dissociation constants for the binding of sphingomyelin by the enzyme. These occur at pK values of 4.1 and 5.5. The activity of sphingomyelinase is also reduced when the enzyme is photooxidized in the presence of methylene blue or rose bengal and carbamylated by diethylpyrocarbonate (DEPC). These results are interpreted to show that 1). the enzyme contains a hydrophobic binding site which involves linear aliphatic moieties containing at least eight carbon atoms; 2) two ionic groups are involved in formation of the enzyme substrate complex, one of which is presumed to be the carboxylate group of aspartate or glutamate (represented by pK 4.1) and the second may be the protonated imidazolium group of histidine (represented by pK 5.5); and 3) since the maximal velocity shows no pH dependence, the interactions involving the hydrophobic and ionic groups affect only the binding of the substrate to the enzyme and formation of the enzyme-substrate complex.

Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography.

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.

Studies on the hydrophobic properties of sphingomyelinase.

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.

Sphingomyelinases in human tissues. IV. Purification of sphingomyelinase from human placenta and effect of Triton X-100.

Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A-Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 degrees C. The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.