Preparation and antimicrobial activity of micacocidin.

Micacocidin (3), a Zn-free derivative of micacocidin A (1), was prepared to evaluate its antimicrobial activity in comparison with 1 and to obtain a starting material for chemical modification of 1. The structure of 3, quite unlike those of any previously known antimicrobial agents, was elucidated by 1-D and 2-D homonuclear and heteronuclear NMR and mass spectroscopy. Micacocidin (3) thus prepared exhibited weak or no antibacterial activity except against Mycoplasma species, i.e. 3 showed stronger activity than 1. It is noteworthy that 3 displayed high activity against fungi such as Candida, Aspergillus and Trichophyton species.

Alteration of physiological activity of activated macrophages through L-arginine metabolism.

Our aim in this study was to define the effect of L-arginine on macrophages (M phi) in relation to the decay of tumoricidal activity of activated M phi. We found that the activated M phi retained their cytotoxicity when cultured in L-arginine-deficient medium but not in conventional medium. Such a decline of tumoricidal activity was associated with increase of glucose consumption and concomitant lactate production, resulting in M phi death. Addition of glucose to the culture medium of activated M phi appeared to cause only a slight delay of the decrease of tumoricidal activity and M phi death. These events were also coincident with a decrease of electron transport activity in mitochondria. Cytological observation by electron microscopy clearly showed the structural alteration or destruction of mitochondria, which preceded the changes of other physiological and functional activities. These results demonstrate that the L-arginine-dependent cytolytic activity against tumor target cells also impairs M phi functions and ultimately induces M phi death, which is primarily mediated by the inhibition of mitochondrial activity.

Effect of L-arginine on the retention of macrophage tumoricidal activity.

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.

Macrophage-activating factor extracted from mycoplasmas.

Mycoplasmas (M. gallisepticum, chicken mycoplasmas), in concert with interferon gamma (IFN gamma), were effective in activating macrophages (M theta) to be tumoricidal. The M theta-activating capacity of mycoplasmas was maintained after treatment with heat. 0.1 M NaOH, 1 M HCl, or trypsin. M theta-activating factor was extracted from mycoplasmas with chloroform/methanol and water (Mf-B). Mf-B was also effective in activating M theta in the presence of IFN gamma. The threshold dose of Mf-B for M theta of ordinary C3H/He mice and that for those of C3H/HeJ mice, the latter being known to be low responders to bacterial lipopolysaccharide, were actually the same. This seems to indicate that the effectiveness of Mf-B was not attributable to possibly contaminating lipopolysaccharides, and that the pathway of activity of Mf-B is different from that of lipopolysaccharides. Since the M theta-activating principle was only a very small part of Mf-B, we have not yet succeeded in identifying it, but there was no evidence that it was protein, nucleic acid, sugar, or lipid. The cytotoxicity of M theta activated by Mf-B plus IFN gamma was dependent on L-arginine in the culture, suggesting that arginine metabolites are involved in M theta cytotoxicity. Mf-B induced a small amount of tumor necrosis factor in M theta, and this induction was markedly enhanced by IFN gamma.

Induction of antitumor activity in macrophages by mycoplasmas in concert with interferon.

The in vitro growth of tumor cells infected with mycoplasmas was suppressed by macrophages pretreated with interferon (IFN), but the growth of mycoplasma-free tumor cells was not suppressed. Pretreatment of macrophages with IFN plus mycoplasmas or their soluble factors either simultaneously or sequentially, IFN first and mycoplasmas second, but not in the reverse order, was effective in activating macrophages to suppress the growth of mycoplasma-free tumor cells. Macrophages from C3H/HeJ mice (which respond only slightly to lipopolysaccharide) were activated by IFN plus mycoplasmas or their soluble factor, and their action was not influenced by the addition of a lipopolysaccharide-neutralizing agent, polymyxin B. These results suggest that the macrophage-activating agent in mycoplasmas does not mimic lipopolysaccharide. The administration of mycoplasmas plus IFN to mice with ascitic or solid tumors resulted in the reduction of tumor growth. The survival rate of tumor-bearing mice was improved by the administration of mycoplasmas, and this was synergistically enhanced by the addition of IFN. These results indicate (a) that mycoplasmas can be useful as a biological response modifier, and (b) that care should be taken to prevent contamination with mycoplasmas in experiments on macrophage activation.

A morphometric study on the pars intermedia of the hypophysis during impairment of the renin-angiotensin-aldosterone system in sodium depleted mice.

Fine structural alterations were investigated in cells of the pars intermedia of the pituitary of mice treated for four weeks with (a) a sodium deficient diet, (b) a sodium deficient diet mixed with propranolol (renin-inhibitor), (c) a sodium deficient diet combined with propranolol and amino-glutethimide (corticosterone 18-hydroxylase inhibitor), and (d) a sodium deficient diet combined with propranolol, aminoglutethimide and dexamethasone. The number of secretory granules decreased from 5.0/mum2 in the normal control of 2.4/mum2 in all four experimental groups suggesting that the cells in treated groups had reached an equilibrium in the production and release of secretory granules during the chronic treatments. The number of immature Golgi granules per unit Golgi area was 0.91 in the control, while this value rose to 3.29 (3.62 fold of the control), 4.37 (4.8 fold), 4.94 (5.43 fold) and 5.16 (5.67 fold) respectively in the four experimental groups. In these groups a good correlation was observed between the number of immature granules and the percent volume of rough endoplasmic reticulum (r=0.985, p less than 0.01). The present study suggests that the pars intermedia contains an unidentified pituitary factor (or factors) essential for aldosterone biosynthesis.