Biomedical subjects

M Micheli

Publications and source records attributed to M Micheli.

10 recordsLinked to original sources

PubMed2000

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

Amides↗

PubMed1998

Nuclear sphingomyelin protects RNA from RNase action.

Chromatin phospholipidic fraction, as previously demonstrated, shows the same localization as RNA inside the nuclei. DNase and RNase treatment of nuclei removed almost totally the DNA, 63% of RNA and caused a 50% loss of phospholipids. The aim of the present investigation is to study the fraction of RNase undigested nuclear RNA and its relationship with the phospholipids still present in the nuclei. Isolated hepatocyte nuclei were treated with Triton X-100 and digested with RNase and DNase. The undigested nuclear material contained proteins (98%) and a small amount of RNA (1.7%), DNA (0.4%) and phospholipids (0.18%). The analysis of phospholipids showed the presence of two components only, namely phosphatidylcholine and sphingomyelin. In the same complex, the activity of sphingomyelin synthase, phosphatidylcholine-dependent phospholipase C and neutral sphingomyelinase has been detected. Treatment of isolated RNA with neutral sphingomyelinase modified the RNA in RNase sensitive RNA, thus suggesting that the SM may represent a bridge between two RNA strands possibly regulating transcription.

Animals↗

PubMed1998

Multiple resistance mechanisms to azole antifungals in yeast clinical isolates.

The use of antifungal agents, especially the azole class, has increased in parallel with a higher incidence of fungal infections, particularly in immunocompromised patients. This situation has favored the appearance of Candida species, prominent among them C. albicans and C. globrata, with acquired resistance to these agents. This review focuses on the latest developments in investigations of molecular mechanisms contributing to azole resistance. Multiple resistance mechanisms have been described that can coexist in resistant clinical isolates. Understanding resistance mechanisms is of value not only for the design of new antifungal agents but also the development of strategies of overcome or delay the emergence of resistance.

Journal Article↗

PubMed1996

Phospholipids and nuclear RNA.

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

Animals↗

PubMed1992

Preclinical hypogonadism in genetic hemochromatosis in the early stage of the disease: evidence of hypothalamic dysfunction.

We studied endocrine functions at baseline and after TRH and LHRH stimulation in a group of 7 young male patients with genetic hemochromatosis (HE) without liver damage (i.e. fibrosis and cirrhosis). In five patients endocrine re-evaluations after complete iron depletion was also performed. Mean basal testosterone (T), FSH, LH and PRL were significantly lower than in controls. Serum T increased normally after HCG stimulation. The normal or high increments of LH after LHRH stimulation suggest that secretion capacity of LH was intact and that hypothalamic dysfunction could be responsible for the preclinical gonadal deficiency found in our patients. The response of PRL to TRH indicates that secretion capacity of lactotrophs although present, was decreased and did not improve after phlebotomy therapy. After iron depletion the two patients with the lowest basal T levels showed the highest increments indicating that in the early stages of hypothalamic-pituitary damage gonadal dysfunction is still reversible in HE patients.

Adult↗

PubMed1989

Fibronectin levels in pregnancy and puerperium.

Fibronectin levels were serially assayed during the third trimester of pregnancy and puerperium in a group women with uncomplicated pregnancies, and two groups with mild/severe hypertensive disorders of pregnancy. The values were found increased in the complicated pregnancies, with extremely elevated levels in cases of severe preeclamptic fits.

Biomarkers↗

PubMed1984

Evidence of heterokaryosis in streptomyces coelicolor A3(2).

Hyphae from mixed cultures of complementary auxotrophs of Streptomyces coelicolor A3(2) did not grow on minimal media (MM) when fertility plasmids ( SCP1 and SCP2) were missing in both strains. The addition of one part per cent of complete medium (CM) to MM allowed growth of vigorous colonies among the tiny colonies of the parental types. The former, amounting to 1%-10% of the total population, turned out to be heterokaryons. They could be propagated on the same medium by plating of hyphal fragments. When five parts per cent of CM were added to MM, beside the heterokaryotic colonies, vigorous 'spindles' of aerial mycelium were formed whenever complementary colonies overlapped. When the SCP1 and SCP2 plasmids were present in one or both parents a clear constraint on the outburst of heterokaryotic aerial mycelium was observed.

Culture Media↗

PubMed1978

Chloramphenicol resistance in Streptomyces coelicolor A3(2): possible involvement of a transposable element.

The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1- superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1-) it was never displaced by the immigrant chromosome even when the region facing chl+ was selected for. A fraction of the original Chl- mutants presented a requirement for arginine (ArgB-). A Chl- mutant gave rise spontaneously to ArgB- derivatives at high frequency. The same ArgB- requirement come out at high frequency among Chl- derivatives from a cross NFChl- x SCP1-Chl+ in which neither parent required arginine or produced spontaneously arginine-less derivatives. It is suggested that the Chl element is a "transposable element" (Tn) presumably associated with "insertion sequences" (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.

Arginine↗

PubMed1977

A factor involved in chloramphenicol resistance in Streptomyces coelicolor A3(2): its transfer in the absence of the fertility factor.

An element controlling chloramphenicol resistance (chl) was detected in Streptomyces coelicolor A3(2). Strains sensitive to 1 microgram chloramphenicol ml-1 were obtained among dark scarlet variants. Transfer of the resistance factor was attempted in matings between chloramphenicol-resistant (Chl+) and chloramphenicol-sensitive (Chl-) strains, both of which lacked the SCP1 fertility factor. Transfer of chl was obtained at a much higher rate than that expected for chromosomal markers in SCP1- X SCP1- matings. However, in these particular crosses the latter was also several times higher than usual. All recombinants for chromosomal markers were Chl+. Attempts to locate the chl element failed to distinguish between a chromosomal and an extrachromosomal site. The observed increase in the recombination frequency for chromosomal markers suggests that the chl element may promote recombination.

Chloramphenicol↗

PubMedDate not supplied

Effects of double encapsulation and coating on synthetic seed conversion in M.26 apple rootstock.

Encapsulated vitro-derived apical buds of M.26 apple rootstock (Malus pumila Mill) can be employed for the formation of the synthetic seed. Satisfactory levels of conversion (plantlets from synthetic seed) can be achieved if there are adequate (i) rooting induction treatment, (ii) protocol of encapsulation, and (iii) nutritive and environmental conditions. For capsule manufacturing, sodium alginate is largely used; however, this is excessively permeable with loss of the nutritive substances (artificial endosperm) and/or dehydration risks during conservation and transport causing detrimental effects on the synthetic seed conversion and on the plantlet's growth. In order to overcome these problems, two experiments were carried out comparing simple encapsulation in alginate with double encapsulation, and with encapsulation-coating procedures. The presence of a second layer of alginate (double encapsulation) and of a thin external coating layer over the alginate (encapsulation-coating) did not show any detrimental effects on viability, sprouting and regrowth of the encapsulated microcuttings. Satisfactory conversion (70%) was reached with the encapsulation-coating procedure, whereas the double and simple encapsulation converted less than 40% of the synthetic seed. The effect of the addition to the capsule of an anti-microbial substance (Plant Preservative Mixture - PPM) was examined: it did not compromise the conversion of the encapsulated microcuttings sown in ex-vitro non-aseptic conditions.

Drug Compounding↗