A thiol protease of peritoneal macrophages in the guinea pig

Summary Proteolytic enzymes of the guinea pig peritoneal exudate macrophages were investigated using synthetic fluorogenic peptide substrates. Among several enzymes, t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide cleaving enzymes had the highest activity, and the activity in exudate macrophages was about 3 times tronger than that in resident thiol-blocking reagents, suggesting a thiol protease.We thank Dr Yoshiaki Motozato, Kumamoto University, for kind donation of Cellulofine GC-700 and valuable discussions. This work is supported in part by the Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Stimulation of the growth of murine bone marrow colonies in vitro by an acute inflammatory exudate. Comparison with colony stimulating factor CSF)]

Formation of colonies from mice bone marrow progenitors of macrophages and granulocytes in methylcellulose culture was induced by an inflammatory pleural exudate obtained from mice injected with dextran. Mitogenic activity of this acute inflammatory exudate was compared with that of colony stimulating factor (CSF). It was found that colony and cluster counts, during 10 days of culture, were similar with the two types of stimulating factors. When used at the same dose, exudate was less active than CSF. It was concluded that inflammatory exudate showed an activity similar to that of CSF but contained a smaller amount of stimulating factor. Further cytochemical studies are necessary to specify whether or not the two factors induced the same type of colonies (monocytic or granulocytic).     

Stimulation of proliferation of rat spleen cells, in vitro, by a nonspecific acute inflammatory exudate. Modulation of cell response to phytohemagglutinin (PHA)]

The effect of an acute non-specific inflammatory exudate with mitogenic activity on macrophages in culture has been tested on the spontaneous and PHA-induced DNA synthesis of spleen cells in vitro. Stimulatory effect of this exudate was observed on spontaneous DNA synthesis which was detectable over a range of 1 : 4 to 1 : 4,000 concentrations. After optimal PHA stimulation, an inhibition of mitogen-induced DNA synthesis was observed when the cells were exposed to the highest concentrations (up to 1 : 128) of the exudate. Thereafter, the phenomenon could be reversed and the stimulation was maximal at a concentration of 1 : 2,000. When a sub-optimal dose of PHA was used, the simulatory effect was more pronounced and detected from 1 : 8 up to 1 : 4,000 concentrations.     

Respiratory burst in peritoneal exudate cells in response to a modified tuftsin.

Intraperitoneal administration of tuftsin-M [Thr-Lys-Pro-Arg-NH-(CH2)2-NH-CO-C15H31] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O2-, H2O2, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O2- and H2O2 under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.     

Arginase expression in peritoneal macrophages and increase in circulating polyamine levels in mice infected with Schistosoma mansoni

We investigated the nitric oxide (NO) synthase and arginase pathways in resident peritoneal macrophages of mice infected with the tropical parasite Schistosoma mansoni. The two enzymes may have opposite effects, insofar as NO may be involved in the killing of the parasite whereas arginase may stimulate parasite growth via polyamine synthesis. We determined the effects of the infection on the expression and activity of the two enzymes in macrophages, before and after cytokine activation. Cells from infected mice expressed the hepatic type I arginase, whereas in control cells, the enzyme was expressed only after cytokine activation, as were NO synthase II and type II arginase in both groups of cells. Moreover, we found that in infected mice, arginase expression in macrophages was associated with a ten fold increase in the concentration of circulating ornithine-derived polyamines. This may be of pathological importance, since parasitic helminths are though to be dependent on their hosts for the uptake and interconversion of polyamines. Received 13 March 2001; received after revision 4 May 2001; accepted 7 June 2001     

Inhibitor of the oxidation of catechin released from the roots of corn seedlings

Summary When the specific activities of the catechin oxidases (catechin as the substrate) which were released from the roots of the seedlings of alfalfa, tomato, wheat, lettuce and corn were compared, it was found that the oxidizing activity was absent from the root exudate of corn seedlings. A 6.3 kDa protein was purified from the root exudate of corn seedlings and in the presence of this protein, the oxidation of catechin was inhibited. This inhibitor is responsible for the inability of the root exudate of corn seedlings to oxidize catechin.     

Fluid and protein clearance in the rat endometrium. Part II: Ultrastructural evidence for the presence of alternative, non-lymphatic clearance mechanisms in the rat endometrium

In the rat endometrium, resident macrophages and exudate phagocytes ensure proteolysis by means of phagocytosis, macro- and micropinocytosis. Using exogenous tracer particles no ultrastructural evidence could be obtained for the occurrence of endometrial prelymphatics. It is suggested that the free tissue fluid may be drained via the fenestrated (probably venous) blood capillaries.     

Origin of a mitogrenic factor for cultivated macrophages (IMF: Inflammatory Mitogenic Factor) found in exudates of non-specific acute inflammation]

The mitogenic activity of inflammatory exudate obtained from irradiated Rats is reduced. After transfer of bone marrow syngeneic cells into irradiated Rats this mitogenic activity is further decreased, while after transfer of thymic cells it is increased. It is postulated that the mitogenic activity of inflammatory exudate could be related to thymic cells and that T lymphocytes may be involved in non specific-inflammatory reactions.     

Fluid and protein clearance in the rat endometrium. Part II: Ultrastructural evidence for the presence of alternative,non-lymphatic clearance mechanisms in the rat endometrium

Summary In the rat endometrium, resident macrophages and exudate phagocytes ensure proteolysis by means of phagocytosis, macro-and micropinocytosis. Using exogenous tracer particles no ultrastructural evidence could be obtained for the occurrence of endometrial prelymphatics. It is suggested that the free tissue fluid may be drained via the fenestrated (probably venous) blood capillaries.Supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek-Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Belgium).     

Respiratory burst in peritoneal exudate cells in response to a modified tuftsin

Intraperitoneal administration of tuftsin-M [Thr–Lys–Pro–Arg–NH–(CH₂)₂–NH–CO–C₁₅H₃₁] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O₂ ^–, H₂O₂, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O ₂ ^– and H₂O₂ under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.     

Inflammatory factors produced by sensitized guinea-pig peripheral blood lymphocytes

Summary The supernatants obtained from stimulated tuberculin-sensitive guinea-pig peripheral blood lymphocytes contain factors that induce a cutaneous inflammatory response in normal guinea-pigs similar to the tuberculin reaction and inhibit the migration of normal guinea-pigs peritoneal exudate cells. There appears to be a correlation between the presence of in vitro migration inhibitory activity and inflammatory activity in vivo.     

Gamma-glutamyl-transpeptidase in lymphatic tissues of Mastomys natalensis during an infection with Acanthocheilonema viteae

During Acanthocheilonema viteae infection, the specific activity of gamma-glutamyltranspeptidase (gamma-GT) increased in peritoneal exudate cells and bone marrow and decreased in lymph nodes of Mastomys natalensis throughout the course of infection. However, though there was an increase in specific activity of gamma-GT in thymus and spleen during the prepatent phase of A. viteae infection, the level either returned to normal or decreased during the latent phase of infection. A close correlation was observed between the host's immune status during A. viteae infection and the level of gamma-GT in lymphoid tissues.     

Rhythmicity of aminotransferase in the cockroach,Periplaneta americana

Summary Aspartate (AAT) and alanine aminotransferase (AIAT) activities in nervous system and coxal leg muscle of the cockroach showed circadian variations with maximal activity around midnight, alternating with minimal activity at 12.00 noon of the solar day. The enhanced activity levels of the enzymes observed during night dark hours may be related to higher energy requirements during increased locomotor activity of the animals.We thank Prof. K. S. Swami, for providing facilities. The financial help rendered by UGC and CSIR, in the form of Junior (to SV) and Senior (to TPK) Research Fellowships is acknowledged.     

Effect of pretreatment with prednisolone on the phagocytic activity of mouse peritoneal macrophages in vitro

Summary The phagocytic activity on in vitro cultured mouse peritoneal macrophages derived from animals treated with 6-alpha-methyl-prednisolone was examined. The statistical evaluation of results showed an increase of phagocytic activity of macrophages derived from treated animals in comparison with controls.     

Active-site mutants of class B beta-lactamases: substrate binding and mechanistic study

Increased resistance to β-lactam antibiotics is mainly due to β-lactamases. X-ray structures of zinc β-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several β-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid. Received 28 August 2002; received after revision 22 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

Neutral proteases in the guinea-pig lymphocytes.

Partial purification of neutral proteolytic enzymes in guinea-pig lymphocytes yielded 2 enzymes. Both enzymes were heat-labile and inhibited by thiol reagents. The molecular weights were more than 200,000 and 150,000-200,000, and optimal pH around 9 and 8, respectively.     

Bioassay for hamster macrophage chemotaxis: application to study particle-lung interactions

Attraction of lung macrophages to particle deposition sites has been demonstrated in different animal species. We reported a threefold increase of the number of macrophages to occur within 40 min after polystyrene particle deposition in hamster airways [Geiser et al. (1994) Am. J. Respir. Cell Mol. Biol. 160: 594–603]. Complement-derived chemotactic activity is one of the mechanisms postulated for macrophage recruitment. It was the aim of this study to test whether complement-derived chemotactic activity is involved in the rapid recruitment of macrophages to the site of deposited polystyrene particles in hamster airways. We first developed an in vitro cell migration assay for hamster macrophages to assess complement-derived chemotaxis. Second, the bronchoalveolar lavage fluids (BALF) of four hamsters that had inhaled aerosols of polystyrene microspheres were tested for chemotactic activity by this bioassay and compared with BALF of four sham-exposed hamsters. Chemotactic response of macrophages was found toward complement-activated hamster serum, whereas macrophage migration was not increased toward BALF of particle and sham-exposed hamsters. In contrast, macrophage migration to BALF of both groups was reduced by 1.6-fold. Thus, the stimulus for macrophage recruitment to the site of deposited polystyrene particles in hamster airways could not be demonstrated using this bioassay. Received 10 September 1997; received after revision 24 November 1997; accepted 10 December 1997     

Colicin E3 enhances the oxidoreductive activity of guinea-pig leucocytes

Summary Colicin E3 increased the capacity of peritoneal exudate leucocytes to reduce iodo-nitro-tetrazolium-chloride to formazane. This effect was directly dependent on its concentration within the range 10³–10⁵ lethal units per cell. In control experiments, dectran C exerted no influence on the rate of this activity and colicin E3 did not convert INT to formazane.     

Neutral proteases in the guinea-pig lymphocytes

Summary Partial purification of neutral proteolytic enzymes in guinea-pig lymphocytes yielded 2 enzymes. Both enzymes were heat-labile and inhibited by thiol reagents. The molecular weights were more than 200,000 and 150,000–200,000, and optimal pH around 9 and 8, respectively.We thank Prof. Hideo Hayashi and Yoshimasa Morino, Kumamoto University, for valuable discussions. This work was supported in part by grants from the Ministry of Education in Japan, the Naito Research Grant, Tokyo, and the Mitsukoshi Foundation, Tokyo.