Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation.

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

Histological and chemical damage induced by microcystin-LR and microcystin-RR on land snail Helix aspersa tissues after acute exposure.

Microcystins (MCs) are the most common cyanotoxins with more than 200 variants. Among these cyanotoxins, microcystin-LR (MC-LR) and microcystin-RR (MC-RR) are the most studied congeners due to their high toxicity and frequent occurrence in surface waters. MC-LR has been detected in more than 75% of natural cyanobacteria bloom, along with other toxic and less toxic congeners. Accumulation of several microcystins variants (MC-LR and MC-RR) has been confirmed in aquatic snails exposed naturally or in the laboratory to toxic blooms. Thus, this paper aims to compare the biochemical and histological impact of both toxic variants (microcystin-LR and microcystin-RR) and their mixed form on a bioindicator, the land snail Helix aspersa. During experiments, snails were gavaged with a single acute dose (0.5 µg/g) of purified MC-LR, MC-RR, or mixed MC-LR + MC-RR (0.25 + 0.25 µg/g). After 96 h of exposure, effects on the hepatopancreas, kidney, intestine and lungs were assessed by histological observations and analysis of oxidative stress biomarkers. The results show that a small dose of MCs variants can increase the non-enzymatic antioxidant glutathione (GSH), inhibit glutathione-s-transferase (GST) level and trigger a defense system by activating glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Microcystin-RR causes serious anomalies in the hepatopancreas and kidney than Microcystin-LR. The organ most affected is the kidney. The damage caused by MC-LR + MC-RR is greater than that caused by single variants.

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

[Risk factors of osteonecrosis of the femoral head following slipped capital femoral epiphysis]. / Nécrose de la tête fémorale chez les enfants traités pour épiphysiolyse fémorale supérieure. Quels sont les facteurs de risque?

UNLABELLED: Osteonecrosis is a serious complication of the treatment of slipped capital femoral epiphysis. The purpose of this study was to identify factors predisposing to the development of this complication. We reviewed retrospectively 127 patients (150 hips) treated for slipped capital femoral epiphysis in our institution between 1980 and 2004. Clinical and radiological data were analyzed before and after treatment, and at consecutive follow-up examination. Osteonecrosis was defined in the basis of radiological criteria. The risk of development of osteonecrosis was correlated with multiple clinical and radiographic parameters. RESULTS: 12 hips in 11 patients (8%) had development of osteonecrosis. Ten of them had an unstable slip. From 130 stable hips, regardless of grade, two had development of osteonecrosis. In patients who had presented with an unstable hip, the risk of osteonecrosis increased with the grade of the slip. Osteonecrosis was more likely to develop in hips that had been treated with multiple screws than in those who had been treated with a single screw. In conclusion, partial or complete reduction of an unstable slipped capital femoral epiphysis increases the risk of development of osteonecrosis. Pinning in situ without reduction with a single screw is the method of choice of the treatment of a slipped capital femoral epiphysis.

Neurotization of isolated axillary nerve palsy in a teenage patient.

BACKGROUND AND AIM: The aim of this article was to study isolated axillary nerve injury, his etiologies, symptomatology and treatment via nerve transfer or neurotization. METHODS: We describe the procedure of long head triceps radial branch transfer to the axillary nerve motor branch in adolescent patient with right deltoid muscle palsy and shoulder anesthesia following a motorcycle crush six months ago. RESULTS: Total recovery of the shoulder sensibility, abduction and extension at one-year follow-up, and patient returned progressively to his normal live and sports activities without any functional effect on the donor muscle. CONCLUSION: The advantages of the axillary nerve transfer are demonstrated through many publications. It is a good therapeutic option if it concerned a young patient and practiced at early time followed by adequate rehabilitation.

[Supra and intercondylar elbow fractures in children]. / Les fractures sus- et intercondyliennes de l'humerus chez l'enfant.

Supra- and intercondylar elbow fractures are rare in children. The treatment of these fractures is controversial. The purpose of this report is to discuss diagnosis and treatment of this unusual injury. Our study looked at nine patients between six and 15 years old (average age: 11.5). In four patients, the fracture was T-condylar and in five patients, the fracture was epiphyseal-diaphyseal. Communition was noted in five cases. All fractures are secondary to high-energy trauma. All patients were treated by open reduction and internal fixation. Three patients had associated nervous lesions. Patients were reviewed at an average of 30 months follow-up and the results evaluated according to the criteria of Flynn. The results were good or excellent in six patients, fair in two patients and poor in one. Cubitus varus and limitation of elbow motion were the main complication. Supra- and intercondylar elbow fractures should be differentiated from the more common supracondylar humerus fractures. We recommended open reduction with internal fixation. Complications are due to the severity of the initial trauma and sometimes to defective treatment especially in the case of complex fracture.

IL-1alpha, IL-1beta, and IFN-gamma mark beta cells for Fas-dependent destruction by diabetogenic CD4(+) T lymphocytes.

Cytokines such as IL-1alpha, IL-1beta, and IFN-gamma have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4(+) T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, beta cell-specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1alpha-, IL-1beta-, and IFN-gamma-treated beta cells from NOD mice. Untreated NOD beta cells and cytokine-treated beta cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR-transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4(+) T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect beta cell-specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1alpha, IL-1beta, and IFN-gamma in autoimmune diabetes, whereby these cytokines mark beta cells for Fas-dependent lysis by autoreactive CD4(+) T cells.

Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.

Autoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic beta cells by T lymphocytes. It is believed that CD8(+) cytotoxic T lymphocytes (CTLs) effect the initial beta-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of beta cell-reactive CD8(+) T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8(+) CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2Kd complex on beta cells, to effect beta-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide-pulsed non-beta-cell targets via both perforin and Fas, but they killed NOD beta cells via Fas exclusively. Perforin-deficient 8.3-TCR-transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8(+) CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill beta cells in a Fas-dependent and perforin-independent manner.

Marked genetic differences in the regulation of blood glucose under immune and restraint stress in mice reveals a wide range of corticosensitivity.

Male and female mice from different control strains (C57BL/6, DBA/2, BALB/c) and the nonobese diabetic (NOD) strain, a spontaneous model of type 1 diabetes, were subjected to various stressors (restraint, lipopolysaccharide or interleukin-1 injection). Significant differences were measured among strains in blood glucose, insulin and corticosterone levels and, for restraint, IL-6. Addition of dexamethasone, a glucocorticoid receptor agonist, to inhibit the expression of several proteins by LPS-stimulated bone marrow-derived macrophages in vitro showed a gradient among control strains: C57BL/6>DBA/2>BALB/c corroborating the pattern of corticosensitivity suggested by their stress-induced glucose responses at the systemic level.

Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation.

Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities).

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings.

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

Localization and characterization of interleukin-1 receptors in the islets of Langerhans from control and nonobese diabetic mice.

Numerous in vivo and in vitro studies have shown the effects of interleukin-1 (IL-1) on insulin and glucagon secretion. To understand the mechanism of these effects, we performed localization and characterization of IL-1 receptors (IL-1R) in pancreas using a quantitative autoradiography method and recombinant human (rh) [125I]IL-1 alpha as a ligand. Frozen sections of pancreas were studied in control (C3H/He) and nonobese diabetic (NOD) mice (a model of autoimmune type I diabetes). Compared to splenic IL-1R, a very high density of specific IL-1R (> 4-fold that in spleen) was found on the islets of Langerhans in both strains. In C3H/He mice, competition experiments demonstrated the presence of one high affinity binding site (Ki = 3.4 and 3.2 x 10(-10) M; binding capacity, 137 and 122 fmol/mg protein for rhIL-1 alpha and rhIL-1 beta, respectively), comparable to type I IL-1R described on T-lymphocytes. In prediabetic NOD mice, these IL-1R were expressed with the same density, affinity, and specificity as in the control strain. Before the onset of diabetes, the expression of IL-1R protein on the islet cells appears to be entirely normal. In contrast, in diabetic NOD mice, IL-1R are sharply decreased, correlating with the intensity of islet destruction. In conclusion, the localization and high density of IL-1R on the mouse islets of Langerhans complement previous studies showing the presence of messenger RNA for type I IL-1R on the islets of Langerhans. These results support a direct physiological effect of IL-1 on pancreatic hormones, such as insulin and glucagon, and a potential role of IL-1R in the pathogenesis of type I diabetes.

Glucose homeostasis in the nonobese diabetic mouse at the prediabetic stage.

Because few data were available on glucose homeostasis at the early prediabetic stage in the nonobese diabetic (NOD) mouse, we investigated glycemia, insulinemia, and pancreatic insulin content under basal conditions in both sexes of 4-, 6-, and 8-week-old fed NOD mice, compared with sex- and age-matched fed C57BL/6 mice. We also investigated glucose tolerance in both sexes of fasting 8-week-old NOD and C57BL/6 mice. The main results obtained under basal fed conditions, when comparing both strains, were lower glycemia and higher insulinemia in NOD females at all ages investigated and in NOD males (particularly at 6 weeks of age). Glucose tolerance tests showed that: 1) the blood glucose response to 1 g/kg i.p. glucose was less sustained in both sexes of 8-week-old NOD mice than in their control counterparts; 2) the blood insulin response to glucose (1 g/kg i.p.) appeared earlier in both sexes of NOD mice than in sex-matched C57BL/6 mice; 3) an unusual sexual dimorphism existed in NOD mice, compared with controls, with females secreting, in response to glucose, twice as much insulin as males; 4) dose-response studies (1-6 g/kg glucose) confirmed the lower increase in blood glucose levels in both sexes of NOD mice and their unusual sexual dimorphism in insulin secretion; and 5) glucose tolerance tests in 4- to 8-week-old NOD mice showed that although the sexual dimorphism in insulin secretion was not observed in 4-week-old mice, it was particularly striking at 6 weeks of age. Taken together, these results suggest that beta-cell hyperactivity exists in the NOD mouse at the early prediabetic stage, especially in NOD females.

Cardiac weight in hypertension induced by nitric oxide synthase blockade.

Wistar rats given a nitric oxide synthase inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME), for 4 weeks develop time- and dose-dependent hypertension without cardiac hypertrophy. This initial study of the relation between left ventricular weight and L-NAME-induced hypertension has now been extended by giving 50 mg/kg per day L-NAME to Wistar rats (n = 30) for 8 weeks and comparing results with those from control rats (n = 10) and two-kidney, one clip rats (n = 14). Although L-NAME rats and two-kidney, one clip rats had increased systolic blood pressures during the last 3 weeks of the experiment (202 +/- 24 and 224 +/- 16 mm Hg, respectively), the ratio of left ventricular weight to body weight of L-NAME rats (2.12 +/- 0.32 mg/g) was not statistically different from that of control rats (1.93 +/- 0.13 mg/g), whereas that of two-kidney, one clip rats was increased (2.85 +/- 0.20 mg/g). The plasma renin activity of L-NAME rats was not significantly different from that of control rats. Two L-NAME rat subgroups were defined according to the presence of left ventricular hypertrophy (ratio of left ventricular weight to body weight > 2.19 mg/g, control mean +2 SD) (6 of 25) or its absence (19 of 25). Systolic blood pressure, plasma renin activity, and cardiac angiotensin converting enzyme activity of L-NAME rats with left ventricular hypertrophy were significantly higher than those of the subgroup without.(ABSTRACT TRUNCATED AT 250 WORDS)

The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria.

Stachydrine (proline betaine) can be used by Sinorhizobium meliloti as a source of carbon and nitrogen. Catabolism depends on an initial N-demethylation, after which the resultant N-methyl proline enters general metabolism. Deletion and insertion mutagenesis demonstrated that the information necessary for catabolism is carried on the symbiotic plasmid (pSym) distal to nodD2 and the nod-nif cluster. Sequencing of an 8.5kb fragment spanning this region revealed four open reading frames with functional homology to known proteins, including a putative monooxygenase and a putative NADPH-FMN-reductase, which were shown by insertional and frame-shift mutagenesis to be necessary for stachydrine catabolism. Other open reading frames, encoding a putative flavoprotein and a repressor, were judged not to be required for stachydrine catabolism, since they were not included in a fragment capable of complementing a deletion of the entire stc region. Sequence and mutagenesis data suggest that stachydrine is demethylated by an iron-sulfur monooxygenase of the Rieske type with a requirement for a specific reductase. The stc catabolic cluster, therefore, resembles xenobiotic degradation in other bacteria and recalls rhizopine catabolism in S. meliloti. Stachydrine appears to have multiple roles in osmoprotection, nutrition and nodulation. Genes involved in stachydrine catabolism are also necessary for carnitine degradation; thus, they could be important in the catabolism of a variety of root exudates and mediate other relationships.

Interleukin-1 effect on glycemia in the non-obese diabetic mouse at the pre-diabetic stage.

Cytokines, particularly interleukin 1 (IL-1) and tumor necrosis factor, are known to induce hypoglycemia in normal rodents or different experimental models of type II diabetes. We investigated, at the pre-diabetic stage, the effect of short-term administration of murine recombinant interleukin-1 alpha (mrIL-1 alpha) on the levels of glucose, insulin and corticosterone in the non-obese diabetic (NOD) mouse, a spontaneous model of type I diabetes. Two-month-old, pre-diabetic NOD mice of both sexes were insensitive to mrIL-1 alpha (12.5 and 50 micrograms/kg) 2 h after administration, the time at which the maximal decrease (around 50%) was observed in the C57BL/6 mouse strain. Kinetic studies however showed that mrIL-1 alpha lowered glycemia in both sexes of NOD mice, but the effect was limited and delayed. In the NOD and C57BL/6 strains, mrIL-1 alpha had no influence on insulin levels in females, but significantly increased them in males (P < 0.0001). Castration of NOD males abrogated the stimulatory effect of mrIL-1 alpha on insulin secretion. Corticosterone secretion was stimulated by mrIL-1 alpha in both sexes of NOD and C57BL/6 mice, and this effect was faster and greater in NOD females than in C57BL/6 females. The incomplete hypoglycemic response to mrIL-1 alpha in females may be attributed to the anti-insulin effect of glucocorticoids, an effect which can be demonstrated when mrIL-1 alpha is administered to adrenalectomized animals or when mrIL-1 alpha is administered together with the glucocorticoid antagonist RU38486. In NOD males, in contrast, glucocorticoids did not play a major role in the limited hypoglycemic response to mrIL-1 alpha, since RU38486 and adrenalectomy were not able to unmask a hypoglycemic effect. Moreover, NOD mice of both sexes were less sensitive than C57BL/6 mice to the hypoglycemic effect of insulin (2.5 U/kg), which suggests some degree of insulin-resistance in NOD mice. With regard to the effect of IL-1 on NOD mouse glycemia, therefore, these results suggest that glucocorticoids and/or androgens, according to the animal's sex, may induce a state of insulin-resistance.

Atrial natriuretic factor influences in vivo plasma, lung and aortic wall cGMP concentrations differently.

Atrial natriuretic factor (ANF) promotes natriuresis and diuresis, increases vascular permeability and may induce peripheral vasodilatation. Endothelium-derived relaxing factor (EDRF), which is nitric oxide (NO), promotes local vasodilatation. ANF and EDRF-NO both cause vascular relaxation by generating cGMP via the activation of the particulate and soluble guanylate cyclases, respectively. This study examines the in vivo effect of exogenous ANF administration in normal Wistar rats, and of increased endogenous ANF in an experimental model of heart failure, on plasma and tissue cGMP concentrations. Low-dose ANF increased plasma and pulmonary cGMP concentrations, whereas 10-fold higher doses were necessary to increase aorta cGMP concentrations. Rats with a myocardial infarction had increased plasma ANF and cGMP and pulmonary cGMP concentrations, but aorta cGMP concentration remained similar to that of sham-operated rats. NG nitro L-arginine methyl ester (L-NAME) was administered chronically to sham-operated and myocardial infarction rats to block NO-synthase: soluble guanylate cyclase activity. L-NAME did not lower the increase in plasma ANF concentration or in urinary, plasma or pulmonary cGMP concentration. In contrast, L-NAME reduced the aorta cGMP concentration 6-fold, despite an increased level of circulating ANF. In summary, the pathophysiological range of plasma ANF concentrations greatly increases plasma and pulmonary cGMP concentrations (by activating particulate guanylate cyclase), but has little influence on the aorta cGMP concentration (which remains mainly dependent on NO-synthase: soluble guanylate cyclase activity).

Carica papaya latex is a rich source of a class II chitinase.

A class II chitinase is present in the latex of the tropical species Carica papaya. The enzyme may be readily purified by using a combination of hydrophobic interaction- and cation-exchange chromatography. This enzyme preparation is homogeneous with respect to the three physico-chemical criteria of charge, M(r) (28,000) and hydrophobicity. It is also completely free of any proteolytic and bacteriolytic activities. The enzyme was classified as a class II chitinase on the basis of its N-terminal amino acid sequence up to the 30th residue. In agreement with this classification, the enzyme preparation hydrolyses chitinase substrates only very slowly and several free thiol functions are present in the polypeptide chain. These free thiol functions are buried, and to be available for titration with 2,2'-dipyridyldisulphide, the enzyme must be denatured. Unfolding of papaya chitinase requires particularly drastic conditions, not less than 4 M guanidinium hydrochloride at 25 degrees and pH 6.8.