Microbiological and densitometric TLC analyses for peptides in liposomes.

Quantitative determination of Leucinostatins and/or of similar peptides, such as Peptaibols, is sometimes quite difficult to perform especially when they are entrapped in vectors, i.e. liposomes, whose components display UV absorbances that may obscure those of the active principle. Therefore, in these cases, it is useful to find alternative ways, especially when high pressure liquid chromatography (HPLC) is difficult to perform or needs long procedure times. In the present paper, the use of microbiological and densitometric methods for quantitative analysis of Leucinostatin A (Leu-A) are described and the results compared with those from HPLC analyses. The use of microbiological and densitometric assays, furnished results comparable with those obtained by HPLC. Of the two methods used, the microbiological procedure appeared to be less accurate and precise.

Structure activity studies on chemically modified homologues of the antibiotic phytotoxic leucinostatin A.

The synthesis and a conformational study of a number of homologues of the well known antibiotic, phytotoxic leucinostatin A are reported. The circular dichroism of all the compounds are discussed. Some conclusions on the SAR of these compounds are drawn. The influence of the alpha-helical conformation and/or the increased lipophile character on their interesting biological activities is emphasized.

Studies on annelated [1,4]benzothiazines and [1,5]benzothiazepines, V. Synthesis and biological activity of N-2 alkylamino derivatives of 4,5-dihydro-s-triazolo[3,4-d]-1,5-benzothiazepine.

The synthesis of a new series of N-2 alkylamino derivatives of 4,5-dihydro-s-triazolo[3,4-d]-1,5-benzothiazepine has been accomplished starting from 2,3-dihydro-1,5-benzothiazepin-4(5H)ones and their 2-methyl and 2-aryl derivatives. All the compounds were tested in vitro for their antimicrobial activity, but none of them showed remarkable activity. The tricyclic compounds 7a-j, 8a-j, 9a-j, 10a-j, and 11a-j were also screened for their CNS activity in mice and several of them showed interesting activity.

Evaluation of the mutagenic activity of leucinostatins, a novel class of antibiotic peptides produced by Paecilomyces marquandii, in the modul Aspergillus nidulans.

Leucinostatins A, B, C, D, E, G, H, and K were thoroughly investigated for their genotoxic activity using the modul Aspergillus nidulans as the test organism. The results of assays for gene mutation (8-azaguanine resistance and methionine suppressors), gene conversion, mitotic crossing-over and mitotic aneuploidy induction suggest that these peptide antibiotics lack significant mutagenicity and that non-genotoxic mechanism(s) underlie their cytotoxic properties.

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

[Experimental pathogenicity of Cryptococcus cereanus in mice]. / Patogenicita' sperimentale del Cryptococcus cereanus in topini.

Pathogenicity studies in mice with Cryptococcus cereanus. Cr.cereanus, a yeast which showing a characteristic ability to grow above 40 degrees C, was found to induce pathogenicity when i.p. inoculated into mice after 6-7 repeated inoculations. Infected mice were sacrificed after 4, 8, 24 h and 3, 7, 14, 21 days following i.v. inoculation (5 X 10(7) cells); and microbiological, histopathological and blood-clinical tests were performed. The time course of C.F.U. in kidneys and brains (Fig.1) the yeasts colonization in heart and kidney tissues (Fig.5-3) and the characteristic "soap's balls" in brains (Fig.4) were confirmed by the modified serum levels of CPK, LDH, GOT, urea and creatinine. For the first time experimental pathogenicity of Cr. cereanus has been demonstrated.

[Experimental pathogenicity of Candida lusitaniae in mice]. / Patogenicità sperimentate di Candida lusitaniae su topini.

Experimental pathogenicity was for the first time demonstrated in a strain of C. lusitaniae of clinical origin by the use of concentrated inocula (5.10(7) Y-shaped cells) and the administration of 4 mg of methylprednisolone acetate i.m. injected 4 days before and 3 days after the inoculation. Counts on malt-agar (fig. 1) in kidney and brain showed a decrease during the first 12 hours, followed by a definite increment in cell number between the first and second day. Clinical tests showed a strong proteinuria, high levels of blood urea and creatinine, and a low GOT increase. Glycemia, GPT, and total serum protein values were normal. Histological examinations of kidney showed a delayed penetration with discrete clumps of Y-shaped cells, inflammatory focuses, and compromised tubules and glomerula (Fig. 2). After 2 weeks of study accumulations of cells followed by degenerative phenomena were seen in the various structures examined. Reparation processes were seldom observed (Fig. 3-4).

[Rapid method for identifying Candida of medical importance]. / Metodo rapido di identificazione di candide di interesse medico.

A rapid identification method of Candida yeasts of medical interest is proposed. It calls for morphological examinations (germ-tube and chlamydospore-test included), and reduced fermentation as well as assimilation tests, performed at defined temperature and inoculum concentration values.