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Engineered yeast cells efficiently convert ergosta-5-eneol to pregnenolone and progesterone provided that endogenous pregnenolone acetylase activity is disrupted and that heterologous sterol delta7-reductase, cytochrome P450 side chain cleavage (CYP11A1) and 3beta hydroxysteroid dehydrogenase/isomerase (3beta-HSD) activities are present. CYP11A1 activity requires the expression of the mammalian NADPH-adrenodoxin reductase (Adrp) and adrenodoxin (Adxp) proteins as electron carriers. Several parameters modulate this artificial metabolic pathway: the effects of steroid products; the availability and delivery of the ergosta-5-eneol substrate to cytochrome P450; electron flux and protein localization. CYP11A1, Adxp and Adrp are usually located in contact with inner mitochondrial membranes and are directed to the outside of the mitochondria by the removal of their respective mitochondrial targeting sequences. CYP11A1 then localizes to the plasma membrane but Adrp and Adxp are detected in the endoplasmic reticulum and cytosol as expected. The electron transfer chain that involves several subcellular compartments may control side chain cleavage activity in yeast. Interestingly, Tgl1p, a potential ester hydrolase, was found to enhance steroid productivity, probably through both the availability and/or the trafficking of the CYP11A1 substrate. Thus, the observation that the highest cellular levels of free ergosta-5-eneol are found in the plasma membrane suggests that the substrate is freely available for pregnenolone synthesis.

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs. The apparent Km for pregnenolone is approximately 0.5 microm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17alpha-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

The first two steps of the steroidogenic pathway were reproduced in Saccharomyces cerevisiae. Engineering of sterol biosynthesis by disruption of the delta 22-desaturase gene and introduction of the Arabidopsis thaliana delta 7-reductase activity and coexpression of bovine side chain cleavage cytochrome P450, adrenodoxin, and adrenodoxin reductase, lead to pregnenolone biosynthesis from a simple carbon source. Following additional coexpression of human 3 beta-hydroxysteroid dehydrogenase/isomerase, pregnenolone is further metabolized to progesterone. Steroid formation appears to be coupled to yeast sterol biosynthesis.

Background: From May 1992 to June 1996 the authors have studied a group of 39 subjects with positive anti-HIV, with echo 2D color Doppler examination, to evaluate with semi-annual controls, the wide variety of cardiac complications in the various phases of clinical evolution of the illness., Methods: At the moment of recruiting, all the subjects with HIV infection were asymptomatic A1 (HIV + As). The patients whose average age was 29 +/- 5, were composed of 60% drug addicts, 17% homosexuals, 8% haemophiliacs and for the 15% heterosexual., Results: The most frequent cardiac complications are represented by hypokinesia of the left ventricle (h-aLV) and by pericardial effusion (PE); more rarely of endocardial vegetations (EV), dilatation of the left ventricle (dLV) and tricuspid insufficiency (TI). The entity of damage and the number of cases observed, are correlated with the grade of clinical severity of the illness., Conclusions: In accordance with the literature data, cardiac pathologies, particularly in the first phases of the illness, are asymptomatic or paucisymptomatic, making the clinical-instrumental observation of the patient useful also in cardiology.

Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.

In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.

The present study has been finalized to perform the content of Zn, Cu, Cr, Se, Mn, F, Mo, Ni, and B in the preterm human milk over 21 d of lactation. Trace element (TE) contents were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-MS), and median concentrations of Zn, Cu, Cr, Se, Mn, and F observed in preterm milk did not demonstrate significant differences in comparison to levels shown in term milk. A statistical significant difference (p < 0.05) has been found among Mo, Ni, and B content in preterm milk for every stage of lactation. TE content of infant blood founded concentrations of Mo in preterm babies significantly (p < 0.01) lower than in term offsprings. Similar values of other TE content were obtained in blood of preterm and term newborns. These findings point to the need for a considerable reassessment of the existing dietary recommendation for Mo content in infant feeding.

Soluble forms of low affinity receptors for the Fc portion of IgG circulate in body fluids and regulate immune functions. We describe the transfection, production and purification techniques which allow the preparation, at a laboratory scale, of milligram amounts of glycosylated recombinant mouse and human soluble Fc gamma receptors. These recombinant products bind IgG and are biologically active on immune responses, like their normal counterparts.

Studies on the identification, cloning, and biochemical characterization of natural and recombinant human and mouse low-affinity soluble Fc gamma R (sFc gamma R) have been developed using various methods. RT-PCR and/or biochemical analyses have demonstrated that low-affinity sFc gamma R (i) are generated by enzymatic cleavage of membrane-associated receptors or by an alternative splicing of the transmembrane region encoding exon and (ii) comprise only the extracellular domains or these domains plus the intracellular region of the membrane-associated molecules, respectively. Functional studies indicated that recombinant sFc gamma R bind mouse and human IgG subclasses with a binding profile identical to that of their membrane counterparts and inhibit Fc gamma R-mediated functions such as immune complex binding or ADCC. In addition, it has been demonstrated that a mouse recombinant truncated sFc gamma RII inhibits antibody responses to T-dependent antigens as well as B-cell proliferation and that a human recombinant truncated sFc gamma RIIIB blocks the Ig production by activated human peripheral blood mononuclear cells. Finally, different immunoassays devised to detect and quantitate circulating sFc gamma R showed that sFc gamma R serum levels vary in circumstances such as injections of protein antigens, in parasitic infections, in tumor-bearing mice, in patients with multiple myeloma (MM), or upon infusions of IgG or Fc gamma fragments in MM or immune thrombocytopenic purpura patients. The use of recombinant sFc gamma R, as well as the availability of monoclonal and polyclonal antibodies directed against different regions of these molecules, makes it possible to characterize further the biological effects of sFc gamma R and their biochemical and immunochemical characteristics, as well as to define their putative ligands on cell membranes.

The aim of the study was to examine the effects of oral zinc supplementation on liver ornithine transcarbamylase activity (OTC), a key enzyme in the urea cycle, in cirrhotic rats. OTC was studied in two groups of rats treated with carbon tetrachloride (CCl4): the first received zinc in the drinking water during the induction of cirrhosis; the second was the control group. Cirrhotic rats which received zinc supplementation showed an increase in liver OTC activity, positively correlated with serum and hepatic zinc content. The results suggest that zinc dietary supplementation may modify hepatic OTC activity and, therefore, plasma ammonia levels in cirrhotic rats.

Zinc deficiency is common in cirrhosis and may be involved in the alteration of ammonia metabolism. Rats with carbon tetrachloride-induced cirrhosis have high plasma ammonia and low serum and tissue zinc levels. We used this model to examine the effects of oral zinc supplementation on activities of plasma ammonia and liver ornithine transcarbamylase (a key enzyme in the urea cycle). These parameters were examined in two consecutive experiments. Each experiment included two groups of rats treated with carbon tetrachloride; one group received zinc in the drinking water during the induction of cirrhosis, and another served as a control group. Regardless of zinc supplementation, all carbon tetrachloride-treated rats exhibited similar micronodular cirrhosis, with similar histological appearance and liver function impairment. Cirrhotic rats without zinc supplementation showed high plasma ammonia and low serum and hepatic zinc levels and reduced liver ornithine transcarbamylase activity. Serum, hepatic zinc and liver ornithine transcarbamylase activity increased significantly in the zinc-supplemented group, and these rats' plasma ammonia levels became normal. Plasma ammonia level was significantly inversely correlated with liver ornithine transcarbamylase activity and positively correlated with serum and hepatic zinc content. Our results suggest that zinc deficiency may modify hepatic ornithine transcarbamylase activity and, therefore, ammonia disposal.

Soluble forms of receptors for the Fc portion of IgG (sFc gamma R) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFc gamma R increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFc gamma R levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFc gamma R. In the human, sFc gamma R were also detected in whole unstimulated saliva. Levels of sFc gamma RII and of sFc gamma RIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFc gamma R (rsFc gamma R) composed of the two extracellular domains of Fc gamma RII was produced by culture of transfected L cells in bioreactors. The purified rsFc gamma R was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5. Moreover, the i.p. injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

The production of soluble forms of low-affinity Fc gamma R by cells expressing recombinant or natural membrane Fc gamma RII, and the structural relationships between these soluble receptors and membrane Fc gamma RII are described. We show that 37-40 kD soluble Fc gamma RII, corresponding to the two N-terminal domains of Fc gamma RII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane Fc gamma RII. Moreover, we describe methods to produce and purify to homogeneity large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated Fc gamma RII cDNA. These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography. By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts. Like natural IgG-BF, the 95-99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells.

Transformation of erythronolide B to new antibiotics was attempted by feeding this compound during the fermentation of Streptomyces antibioticus ATCC31771, a blocked mutant of an oleandomycin producing strain. As a result, four new active compounds were obtained with hybrid structures between erythromycin and oleandomycin. They were identified as 3-O-oleandrosyl-5-O-desosaminyl-15-hydroxyerythronolide B (I), 3-O-oleandrosyl-5-O-desosaminylerythronolide B (II), 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxyerythronolide B (III) and 3-O-oleandrosyl-5-O-desosaminyl-(8R)-8,19-epoxyerythronolide B (IV). They were found to be less active, but more stable to acid, than erythromycin A. From their relative biogenetical relationship together with the structure elucidated some hypotheses about late stages of oleandomycin biosynthesis are inferred too.

Following the previously described semisynthetic preparation of new aglycones (8S)-8-fluoroerythronolide A (I), (8S)-8-fluoroerythronolide B (II) and the monoglycoside 3-O-mycarosyl-(8S)-8-fluoroerythronolide B (III), their conversion into new fluoroerythromycins was attempted by mutational biosynthesis. The strain Streptomyces erythraeus ATCC 31772, a mutant blocked in the biosynthesis of erythromycin, was employed in the present investigation. Four new antibiotics, (8S)-8-fluoroerythromycin A (IV), (8S)-8-fluoroerythromycin B (V), (8S)-8-fluoroerythromycin C (VI) and (8S)-8-fluoroerythromycin D (VII) were successfully derived by such an approach. The result is also discussed in terms of the substrate specificity of the enzymes involved in the biosynthesis of erythromycins. The new antibiotics exhibited promising biological properties.