Serena Traniello

Rabbit liver fructose 1,6-diphosphatase. Properties of the native enzyme and their modification by subtilisin☆

Abstract A simple two-step procedure is described for the purification of fructose 1, 6-diphosphatase from rabbit liver. The enzyme shows activity at neutral pH, in contrast to the previously described “alkaline” fructose diphosphatase. The neutral fructose diphosphatase has a higher molecular weight, 140,000, as compared with 130,000 for the alkaline enzyme and a somewhat different amino acid composition. Most significant is the presence of one residue of tryptophan per subunit; this amino acid is not present in the alkaline fructose diphosphatase. The subunit molecular weight of the neutral enzyme is 36,000, and it contains 4 moles of COOH-terminal alanine. Digestion with subtilisin converts the neutral enzyme to one with properties resembling those of the alkaline form. The pH optimum is shifted to pH 9 and the sensitivity toward inhibition by AMP is decreased. The molecular weight of the modified enzyme is 120,000. The results suggest that the changes in catalytic properties are associated with the removal of a tryptophan-containing peptide or peptides with molecular weights totaling approximately 6000 per subunit, and that the alkaline enzyme previously studied in this and other laboratories is a modified form of the native neutral fructose diphosphatase.

Fructose 1, 6-diphosphatase from liver: Isolation of the native form with optimal activity at neutral pH

A new procedure has been developed for the purification of rabbit liver fructose 1,6-diphosphatase which yields homogeneous enzyme preparations with optimum activity at neutral pH. The properties of this protein differ from those of previous preparations, which show alkaline pH optima. The neutral fructose diphosphatase has a higher molecular weight and yields only one species of subunit in SDS disc-gel electrophoresis. The Km values for substrate and divalent cations were not significantly different from those of the alkaline fructose diphosphatase. It is, however, more sensitive to AMP inhibition, and there are also differences in the reactivity of some sulfhydryl groups with p-mercuribenzoate. It appears to represent a native form of fructose diphosphatase.

Effect of interferon-α therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals

The majority of hepatitis C virus (HCV)‐infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV‐specific CTL responses directed to different HCV‐derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA‐A2‐presented, HCV‐derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon‐α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4‐derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV‐specific CTL responses and therapy‐induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3‐derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN‐α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3‐derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV‐derived epitopes with a dominant response to the NS3‐derived peptide antigen. This suggests that CTL responses directed to this NS3‐derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposis sarcoma (KS), primary effusion lymphoma and Castelmans disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.

Two For-Met–Leu–Phe-OMe analogues trigger selective neutrophil responses: A differential effect on cytosolic free Ca2+

For-Thp-Leu-Ain-OMe and for-Met-delta(z)Leu-Phe-OMe are two conformationally restricted fMLP-OMe analogues able to discriminate between different biological responses of human neutrophils. In this paper, we demonstrate that the former peptide, which evokes only chemotaxis, does not alter human neutrophil Ca2+ levels. In contrast, for-Met-delta(z)Leu-Phe-OMe, which induces superoxide anion release and degranulation but not chemotaxis, significantly increases the cation concentration. The chelation of Ca2+ in both extracellular and intracellular media abolishes O2- production triggered by for-Met-delta(z)Leu-Phe-OMe, while the same procedure does not affect neutrophil chemotaxis towards for-Thp-Leu-Ain-OMe. We therefore suggest that chemotaxis, unlike superoxide anion release, is independent of Ca2+ enhancement in human neutrophils.

Modulation of neutrophil functions by activated platelet release factors.

Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.

Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues

The N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-OMe (1) analogues for-Thp-Leu-Ain-OMe (2), for-Thp-Leu-Phe-OMe (3), for-Met-Leu-Ain-OMe (4), for-Met-Delta(z)Leu-Phe-OMe (5), for-Met-Lys-Phe-For-Met-Lys-Phe (6), for-Met-Leu-Pheol-COMe (7), and for-Nle-Leu-Phe-OMe (8) have been studied. Some of these have been found selective towards the activation of different biological responses of human neutrophils. In particular, peptides 2 and 3, which evoke only chemotaxis, are ineffective in enhancing inositol phosphate, as well as cyclic AMP (cAMP) levels. On the contrary, analogues 5 and 7, which induce superoxide anion production and degranulation, but not chemotaxis, significantly increase the levels of the two intracellular messengers, as is the case of the full agonists 1 and 6. The Ca(2+) ionophore A23187 also activates phospholipase C (PLC) and increases the nucleotide levels; when tested in combination with peptide 1 or 5, a supra-additive enhancement of cAMP concentration is obtained. The PLC blocker, U-73122, inhibits the formylpeptide-induced inositol phosphate formation, as well as cAMP increase. Moreover, this drug drastically reduces superoxide anion release triggered by 1 or 5, whereas it inhibits to a much lesser extent neutrophil chemotaxis induced by 1 or 2. Our results suggest that: (i) PLC stimulation is involved in cAMP enhancement by formylpeptides; (ii) the activation of PLC by formylpeptides, in conditions of increased Ca(2+) influx, induces a supra-additive enhancement of the nucleotide; (iii) the inability of pure chemoattractants to significantly alter the PLC activity or cAMP level, differently from full agonists or peptides specific in inducing superoxide anion release, appears as a general property. Thus, the activation of neutrophil PLC seems essential for superoxide anion release, but less involved in the chemotactic response.

Rabbit liver and rabbit kidney fructose diphosphatases: Catalytic properties of enzymes activated by coenzyme A and acyl carrier protein

Abstract The catalytic properties of rabbit liver and rabbit kidney fructose diphosphatases are altered when these enzymes are treated with CoA or acyl carrier protein from Escherichia coli . The activity in the neutral pH range is increased several fold, and the pH optima are shifted from pH 8.8 to pH 7.5 in the presence of MgCl 2 , and from pH 9.1 to pH 8.2 when MnCl 2 is the cofactor. Maximum activity requires the presence of a chelating agent such as EDTA, histidine, or glycine. The untreated enzymes are inhibited by excess fructose-1,6- P 2 , whereas the activated enzymes are not, although the K m for this substrate is increased by approximately 10-fold. The modified enzymes are also more sensitive to inhibition by AMP. The reactions with CoA or acyl carrier protein are prevented by the addition of high concentrations of substrate, but not by AMP. In the activated enzyme approximately two sulfhydryl groups appear to be blocked, and the changes in catalytic properties are reversed by treatment with sulhydryl compounds such as cysteine or glutathione. This preliminary evidence indicates that activation involves the formation of disulfide linkages between CoA or acyl carrier protein and the enzyme. Activation by CoA or an ACP-like protein may represent a physiological mechanism for the reciprocal control of gluconeogenesis and fatty acid synthesis.

Polymorphonuclear neutrophils pulsed with synthetic peptides efficiently activate memory cytotoxic T lymphocytes

Polymorphonuclear neutrophils (PMNs), traditionally considered effector cells in the inflammatory response, have recently been regarded as potential regulators of the immune response. In the present study we investigate whether PMNs are efficient antigen‐presenting cells for reactivation of memory cytotoxic T lymphocytes (CTLs). PMNs were pulsed with synthetic peptides derived from Epstein‐Barr virus (EBV) antigens. We have used the IVTDFSVIK (IVT) peptide derived from the Epstein‐Barr virus—encoded nuclear antigen 4 protein, corresponding to the immunodominant epitope of HLA‐A11–restricted CTL responses, and the CLGGLLTMV (CLG) peptide derived from the latent membrane protein 2 antigen, representing a subdominant epitope of HLA‐A2–restricted CTL responses. The data indicate that peptide‐pulsed PMNs selectively activate specific CTL responses to both immunodominant and subdominant epitopes. The efficiency of CTL induction by PMNs was comparable to that observed with the conventional method of EBV‐specific CTL reactivation with the autologous lymphoblastoid cell line, as well as with peptide‐pulsed monocyte‐enriched adherent cells. On the contrary, unactivated peptide‐pulsed lymphocytes failed to induce an epitope‐specific CTL response. These results demonstrate that PMNs efficiently present antigens to memory virus‐specific CTLs and suggest that they may have a role as antigen‐presenting cells. J. Leukoc. Biol. 60: 207–213; 1996.

Inhibition of human leucocytes locomotion by anti-inflammatory drugs

In this study we have demonstrated that acidic non-steroidal anti-inflammatory drugs in low concentrations inhibited human PMN locomotion in vitro. A speculative mechanism of action is proposed.