Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Positive and negative modulation of peptidases by pro-inflammatory cytokines.

The capacity of pro-inflammatory cytokines to modulate proteolysis was analyzed by liquid chromatography using human fibroblasts as cell model and enzyme source, and the immunodominant epitope gp100(280-288) (YLEPGPVTA) as substrate. The measurements made after fibroblast pre-incubation with either IL-1, TNF, or IL-6 plus its soluble receptors have been compared with those made with un-stimulated fibroblasts. The results obtained suggest an uneven association of cytokine treatment with substrate degradation, and with a prevailingly positive - but also negative - association with release of smaller peptides and free amino acids. Data obtained by separately measuring these two groups of by-products indicate that, after IL-1 cell pre-treatment, the velocity of formation of both groups of by-products increased, resulting in a net increase of substrate degradation. After TNF and IL-6 pre-treatment, the increase of one group was compensated by a decrease of the other group; specifically, the compensation was only partial for TNF, and overall substrate hydrolysis increased. In the case of IL-6, the increase of free amino acids was almost exactly compensated by a reduction of peptidic by-products, resulting in a negligible increase of substrate hydrolysis. In addition, the existence of reaction time-related modifications in the apparent velocity of substrate degradation and formation of by-products, allows hypothesizing different effects of cytokines on the enzymes degrading the substrate with different time constants. Taken together, these data can be interpreted as indicating different, positive and negative, effects of the three cytokines on the individual enzymes expressed by fibroblasts and capable of degrading peptidic substrates.

Protein 4.1 deficiency associated with an altered binding to the spectrin-actin complex of the red cell membrane skeleton.

Protein 4.1 has been defined as a major component of the subcortical skeleton of erythrocytes. It binds the spectrin--actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino acid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the molecular basis of heterozygous 4.1(-) hereditary elliptocytosis, associated with protein 4.1 partial deficiency, in nine related French families. cDNA sequencing revealed a single codon deletion (AAA) resulting in a lysine residue deletion within the 10-kD binding domain, 3' of Motif I. The mutated allele was designated allele 4.1 Aravis. In order to assess the functional effect of the codon deletion, recombinant 10-kD constructs were made and various binding assays were performed using spectrin, purified spectrin-actin complex, or red cell membranes. These experiments demonstrated that the deletion of the Lys residue clearly prevents the binding capacity. Similar results were obtained with a construct containing the Lys residue but lacking Motif I. These data strongly suggest that the binding site to the spectrin-actin complex must contain the Lys 447 (or 448), and therefore resides not only on Motif I but extends 3' of this essential motif.

Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition.

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.

Heterogeneity of purified cholera toxin.

The heterogeneity of Vibrio cholerae toxin, obtained from culture filtrates in homogeneous form by gel filtration and preparative disc gel electrophoresis has been studied. By means of disc electrophoresis on polyacrylamide gel cholera toxin was separated into three forms designated I (5%), II (15%) and III (80%). The toxic activity, amino acid content and molecular weight of the three forms were similar. The difference so far observed between the various electrophoretic fractions is a difference in net charge. Incubation of either cholera toxin II or cholera toxin III at relatively high pH leads to the formation of the more acidic forms. These forms, generated in vitro by deamidation of asparagine and/or glutamine residues, are indistinguishable from the toxins of similar electrophoretic mobilities isolated from crude culture filtrates.

Antistreptokinase platelet-activating antibodies are common and heterogeneous.

BACKGROUND: Platelet activation by antistreptokinase (SK) antibodies could impair the clinical effect of SK administration. OBJECTIVE: To better describe anti-SK antibodies with particular emphasis on procoagulant activities as a result of platelet activation. PATIENTS AND METHODS: Sera were collected from 146 patients with coronary artery disease: non-SK-treated, 95 from mainland France, 31 from French Polynesia; 20 patients from mainland in year 2 after SK treatment. Serum-induced SK-dependent platelet activation resulting in procoagulant activities was assessed with washed platelets from five donors representative of the known patterns of reactivities to IgG. RESULTS: Concentrations (2-5252 microg mL(-1)) and fibrinolytic neutralization titres (< 10 to > 1280) were found in the expected wide range and correlated (rho = 0.66, P < 0.0001). Platelet activation was detected with 145 samples, but varied in intensity and pattern (depending on the donors), although there was no systematic hierarchy; it was presumably due to IgG (inhibited by an IgG Fc receptor-blocking antibody and recovered in the IgG fraction) and only partially affected by aspirin. Marked platelet activation could be detected in samples with concentration as low as 2 microg mL(-1), and/or no detectable neutralizing titers. The way of immunization to SK was not found to influence the functional profile of antibodies. CONCLUSION: Anti-SK platelet-activating antibodies are widespread, heterogeneous, poorly predictable on the basis of their antifibrinolytic effect and strong enough to trigger procoagulant activities. Their clinical relevance should be formally assessed, using patients' own platelets for detection owing to the variation of platelet reactivity.

Lymphocyte T subset counts in children with elevated low-density lipoprotein cholesterol levels.

The aim of this study was to determine blood lymphocyte T subset counts in children with elevated levels of low-density lipoprotein cholesterol. We studied 107 children, ages 2.0 to 15.9 years, from 79 families who were referred to our Lipid Research Clinic because total cholesterol serum levels higher than 200 mg/dl had been detected in at least one child. At the time of diagnosis we analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). Children were classified according to LDL-C levels into three groups: (1) normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl, respectively); (2) at moderate risk, if levels were between the 75th and 95th percentiles (125 and 150 mg/dl, respectively); and (3) at high risk, if levels were above the 95th percentile (150 mg/dl). In children aged 2.0 to 6.9 years, all lymphocyte T subset counts were higher in the high risk group than in the normal group (P < 0.05 and P < 0.01). In children aged 11.0 to 15.9 years, the CD4 subset count was also significantly higher in the high risk group in the other two groups (P < 0.05 and P < 0.01). These results are in agreement with pathologic findings in the atheromatous plaque.

Incidence of haematological malignancies in French Polynesia between 1990 and 1995.

To determine the incidence of haematological malignancies in French Polynesia from 1990 to 1995, we collected cases from the local cancer registry, sanitary evacuation files and all the histopathology and clinical biology laboratories. All leukaemias, non Hodgkin's lymphomas, and multiple myelomas incidence was slightly lower among French Polynesians than among Maoris from New-Zealand and Hawaiians of Hawaii. Standardised Incidence Ratio (SIR) for Hodgkin's disease among females was 0.08 when comparing to Hawaiians and 0.33 when comparing to Maoris. Other salient features were a high proportion of high grade and Burkitt's lymphoma, the absence of Hodgkin's disease after 40 years of age, a low incidence of chronic lymphoid leukaemia, and a high non lymphoblastic/lymphoblastic acute leukaemia ratio in childhood. This study stresses the peculiar incidence of some haematological malignancies in this south pacific area.

Enkephalin-degrading enzymes and their inhibitors in human saliva.

The possible presence of enzymes able to hydrolyze leucine enkephalin has been investigated in human saliva. The data obtained indicate that, in the presence of saliva, Leu-enkephalin is partially hydrolyzed. The disappearance of the substrate is paired with the formation of hydrolysis byproducts whose composition indicates the presence of all three classes of enzymes known to hydrolyze enkephalins: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The presence of low molecular weight substances with inhibitory activity on proteolytic enzymes has also been detected. These substances are active on all three classes of enkephalin-degrading enzymes, although the inhibition is more evident on dipeptidylpeptidases than on aminopeptidases. Substrate degradation was found to be higher in male than in female saliva: this seems to be caused by the activities both of enzymes and low molecular weight inhibitors that are different in the two sexes.

Interindividual variability of enkephalin-degrading enzymes in human plasma.

The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis by-products has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class-as well as the expression of the single enzyme species within each class-appears to be individually determined. Interindividual variability observed seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low molecular weight plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the composition of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and constant in blood obtained from successive drawings, at least within a relatively short period of time.

Specificity of proteolysis inhibitors in rabbit plasma.

Hydrolysis and inhibition of hydrolysis of leucine enkephalin in Oryctolagus plasma were studied by kinetics and chromatographic techniques. By data obtained, in this species, enkephalins are degraded by the same enzymes active in other mammals: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. At variance with data obtained in other species, where enkephalins are hydrolyzed mostly by aminopeptidases, in Oryctolagus Leu-enkephalin hydrolysis is mainly due to dipeptidylcarboxypeptidases, whereas aminopeptidases contribution is the minimum of all three enzyme groups. Comparative analyses performed in the presence and in the absence of plasma inhibitors indicate that the ability of these substances to reduce substratum hydrolysis is very limited. On the contrary, the specific hydrolysis pattern evidenced appears to originate primarily from selective inhibition of the three groups of enzymes. Results obtained appear consistent with a role of plasma inhibitors in tuning hydrolysis to specific substrata, without appreciably modifying the amount of the substratum degraded.

Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line.

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.

Effect of differentiation on hydrolysis and association of Leu-enkephalin to K562(S) cells.

Hydrolysis of Leu-enkephalin and association to cells of the peptide-radioactive label have been studied on the K562(S) erythroleukemic cell subline. Data obtained indicate that in the presence of these cells, Leu-enkephalin is hydrolyzed, that the peptide's radioactive label is partially associated to cells, and that these phenomena are related. Hydrolysis and association are inversely modified by the cells' differentiation: hydrolysis is increased and association is decreased in differentiated compared with nondifferentiated cells. Moreover, the ratio of hydrolysis by-products is dissimilar between differentiated and nondifferentiated cells as a result of a significant modification of the soluble enzymes' release. The alterations induced by differentiation on all parameters investigated seem to indicate significant changes in the membrane structures responsible for the mechanisms controlling these phenomena.

Age-induced increase of leucine enkephalin enzyme degradation in human plasma.

Possible age-induced variations of the hydrolysis of leucine enkephalin in the presence of plasma enzymes were studied by kinetic and chromatographic techniques in a group of elderly individuals. Results obtained indicate that in elderly individuals the activity of enkephalin-degrading plasma enzymes is greater than in the controls; ANOVA analysis of these data indicates that the dependency of the variation of hydrolysis upon the two age groups is statistically significant. Increased substrate hydrolysis, and a modified hydrolysis pattern, appear to be associated with increased activity of the enzymes involved, and with different distribution of the individual enzymes within each class, as well as with severely reduced activity of the low molecular weight plasma inhibitors. The combination of these factors defines a characteristic hydrolysis pattern for the elderly individuals, different from that found in the controls.

Neuropeptide enzyme hydrolysis in allergic human saliva.

The activity of neuropeptide-degrading enzymes, and possible variations in this activity under allergic conditions, was examined in human saliva obtained from allergic volunteers and from an age- and sex-matching group of healthy controls, using leucine enkephalin as model substrate. The results obtained indicate that, under experimental conditions, the substrate was partially hydrolyzed by all three classes of enzymes known to degrade it in human saliva: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In the presence of saliva obtained from allergic donors, a large increase in the activity of aminopeptidases, and a more limited increase in the activity of dipeptidylaminopeptidases, induced an increase of substrate hydrolysis with respect to that measured in the controls. The activity of all substrate-active enzymes, the allergy-associated variations in this activity, and the amount of substrate hydrolyzed, were found to be different in male and female saliva. Specifically, in the controls the gender-related differences in substrate hydrolysis were mainly caused by the higher activity of aminopeptidases observed in male as compared to female saliva. In contrast, in allergic saliva, a greater increase in the activity of aminopeptidases in female saliva reduced the gender-related differences in the pattern of hydrolysis, which was also different from that observed in the controls.

Three new cases of dysfibrinogenemia: Poissy III, Saint-Germain I and Tahiti.

In order to identify unknown mutations, the FAMA method was used to rapidly screen the fibrinogen chain genes in individuals with dysfibrinogenemias. Chemical cleavage at mismatches on heteroduplexes DNA end-labeled with strand-specific fluorescent dyes reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. This method was successfully used for the detection of three new dysfibrinogenemias: Poissy III, Tahiti (heterozygous Aalpha Arg16His) and Saint-Germain I (heterozygous AalphaGly12Val). The mutations were confirmed by dideoxy sequencing.

Soluble proteolytic enzyme release by naive and HIV-infected cultured T-cells.

The possible hydrolysis of leucine enkephalin was measured in the presence of cell-free supernatants obtained from naive and chronically HIV-infected immunocompetent cell lines. The data obtained indicate that, under all conditions examined, leu-enkephalin was partially degraded; its disappearance was associated with the appearance of peptides whose composition is consistent with the involvement of three enzyme classes, i.e. aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In the presence of supernatants obtained from infected cells, substrate hydrolysis was less than that measured in naive controls. This appears to result from infection-associated variations in the activity of all three enzyme classes active on the substrate, variations that were different for each class. Specifically, in unfractionated supernatants, the activity of aminopeptidases was reduced, that of dipeptidylaminopeptidase was increased, and the activity of dipeptidylcarboxypeptidases was nearly unmodified. Data obtained upon chromatographic separation of the soluble supernatants allowed for the identification of features that can be interpreted as indicating the existence of infection-associated variations in the activity of single enzymes. The sum of the data shown makes it possible to advance the hypothesis that the infection-associated modifications in the release of proteolytic enzymes may contribute to the alterations in the functionality of immunocompetent cells induced by viral infection.

Neuropeptide degradation in naive and steroid-treated allergic saliva.

The hydrolysis of neuropeptides and possible variations in hydrolysis following steroidal treatment, were examined in the presence of saliva collected from allergic volunteers; data obtained were compared to those obtained with a age and sex-matching group of healthy controls. The results reported indicate the presence of a statistically significant increase in the hydrolysis of the model substrate in allergic as compared to control saliva, and a reduction of substrate hydrolysis in treated as compared to naive allergic saliva. Total enzyme activity, the relative activity of the three classes of substrate-active enzymes (aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases), the allergy-associated variations of these activities, and the variations associated to therapy were found to be different in male and female saliva. Specifically, in the controls, the lower level of hydrolysis evident in female as compared to male saliva appeared to be principally induced by lower activity of aminopeptidases. Under allergic conditions, a sex-different increase in the activity of all three classes of substrate-active enzymes modified the hydrolysis pattern differently in samples obtained from male and female donors. Finally, pharmacological treatment induced opposite effects on the enzymes present in each sex: in male saliva, the activity of all three classes of substrate-active enzymes--and, thus, of substrate hydrolysis--was reduced near to the levels measured in the controls. In female saliva, the reduction in the activity of aminopeptidases was coupled with an increase in the activity of dipeptidylaminopeptidases, causing substrate hydrolysis to remain near the levels measured in naive allergic, rather than control, saliva.

Relationship between immunoinflammatory proteins containing sialic acid and low-density lipoprotein serum concentrations.

The aim of this study was to investigate the relationship between sialoglycoproteins and the lipoprotein profile in a group of children with different levels of low-density lipoprotein cholesterol. We have studied 177 children of 132 families who were sent to our Pediatric Lipid Research Clinic because of serum cholesterol concentrations higher than 5.17 mmol/l. At the time of diagnosis, we analyzed the serum lipoprotein profile and the sialoglycoproteins: alpha 1-antitrypsin, acid alpha 1-glycoprotein, haptoglobin, alpha 2-macroglobulin, transferrin, IgA, IgG, IgM, complement C3 component and ceruloplasmin. At 7.0 to 10.9 years, alpha 1-glycoprotein serum concentrations were higher in the high risk group than in the moderate risk group (P < 0.05). At 2.0 to 6.9 years, IgA and IgM serum concentrations were higher in the moderate risk group than in the low risk group (P < 0.05 and P < 0.01, respectively), and IgG and IgM serum concentrations were also higher in the high risk group than in the low risk group (P < 0.05). Our results seems to reflect a general reaction to injury or inflammation which could be associated with the atherosclerotic process.

Effect of different parameters on the binding of two anti-inflammatory drugs to human serum albumin.

The binding to human serum albumin of two anti-inflammatory drugs, indomethacin and indoprofen, has been studied by chromatographic and spectroscopic techniques. The results shown indicate that the binding of both drugs--but more notably of indoprofen--is very sensitive to variations of the environmental conditions. The binding is also dependent upon limited modifications in the tertiary structure of the protein. The evidences shown tend to indicate that these two phenomena are related, and that the binding is permitted under conditions of a relatively open structure of the protein molecule.