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B Maresca

Publications and source records attributed to B Maresca.

At least 19 recordsLinked to original sources
PubMed2000

Host response and Histoplasma capsulatum/macrophage molecular interactions.

Histoplasma capsulatum is the etiological agent of histoplasmosis, a chronic respiratory infection that is generally asymptomatic in healthy individuals, but severe or fatal in patients who are immunosuppressed or otherwise debilitated. H. capsulatum is found as a mould in soil and becomes a pathogenic yeast in the mammalian host. The first line of defense that H. capsulatum faces during host invasion is the attack of polymorphonuclear neutrophils and resident macrophages. In animal models, once phagocytosed, H. capsulatum is not killed by fusion of the phago-lysosomes, instead it multiplies within non-activated macrophages and destroys them. Upon induction of cell-mediated immunity, cytokines activate macrophages and destroy the yeast cells. Some aspects of the fungus-macrophage interaction have been elucidated, and it is clear that some of the mechanisms by which H. capsulatum escapes the lethal effects of this very hostile environment, involve the regulation of specific genes. Recently, using the differential display reverse transcriptase polymerase chain reaction technique, a number of H. capsulatum genes that are induced after the yeasts are ingested by macrophages have been identified. However, the mechanisms that underlie the capacity of H. capsulatum to adapt to the new environmental conditions present in macrophages remain to be clarified.

Animals↗
PubMed1999

An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

Amino Acid Sequence↗
PubMed1999

Mucor rouxii delta9-desaturase gene is transcriptionally regulated during cell growth and by low temperature.

Unsaturated fatty acids are essential lipid components of Mucor rouxii. Gamma-linolenic acid (GLA) is synthesized via the desaturase enzymes: delta9-desaturase catalyzes mono-unsaturated fatty acids that are utilized as substrate for GLA biosynthesis. We cloned and characterized a M. rouxii gene highly homologous to delta9-desaturase genes. This sequence encodes for a protein of 452 amino acids and contains two introns of 60 and 61 nucleotides. Delta9-desaturase of M. rouxii is expressed during cell growth when cells are subjected to temperature shifts. At 30 degrees C, the mRNA level of late log phase is about 6.4-fold higher than that of early log phase. A shift from 30 to 15 degrees C induced transcription of delta9-desaturase gene in both early and late log phases. However, the pattern of increased transcription by cold induction varied depending on growth conditions: transcription of late log phase is higher than that of early log phase. These results indicate that cell growth and low temperature influence the expression of delta9-desaturase gene and fatty acid composition of M. rouxii.

Amino Acid Sequence↗
PubMed1998

Identification and isolation by DDRT-PCR of genes differentially expressed by Histoplasma capsulatum during macrophages infection.

Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of genes in both parasite and host. In order to identify and isolate relevant genes of the fungus, Histoplasma capsulatum, in which expression is specifically induced during its interaction with murine macrophages (Mphi), we performed a comparative analysis of the pattern of gene expression of the fungus before and after exposure to, and internalization into Mphi by using differential display reverse transcriptase-PCR (DDRT-PCR). Using a limited set of primer combinations, six cDNA fragments of H. capsulatum were identified and isolated; five representing fungal genes in which expressions were enhanced during Mphi infection, whereas one mRNA fragment was down-regulated. Slot blots followed by Northern blot analyses confirmed that the transcripts detected with cDNA clones were over expressed after 1 h of Mphi infection, whereas no transcripts were detected with mRNA purified from H. capsulatum before infection. Sequence analyses and database searches revealed no significant homology to any known sequence for five of these clones. One of the clones showed homology to the rat p105 kD protein, and to the p100 kD co-activator proteins of human and Caenorhabditis elegans. To our knowledge, this is the first experimental evidence that specific genes are differentially expressed by a fungal pathogen when it is exposed to, and phagocytosed by Mphi. Furthermore, these results show that the DDRT-PCR procedure has adequate sensitivity to detect fungal genes induced during parasite-host interaction to identify potential new targets that can be used to develop new antifungal drugs.

Amino Acid Sequence↗
PubMed1998

Does the membrane's physical state control the expression of heat shock and other genes?

Membranes provide the structural framework that divides cells from their environment and that, in eukaryotic cells, permits compartmentation. They are not simply passive barriers that are liable to be damaged during environmental challenge or pathological states, but are involved in cellular responses and in modulating intracellular signalling. Recent data show that the expression of several genes, particularly those that respond to changes in temperature, ageing or disease, is influenced and/or controlled by the membrane's physical state.

Animals↗
PubMed1998

Stress proteins in fungal diseases.

Heat shock proteins (hsps) are ubiquitous families of proteins, found in all organisms studied so far. They are highly conserved across the species barrier and serve fundamental functions in cell physiology. The term 'heat shock' was adopted because of the early observation of the heat-inducible nature of these proteins, although, as it is now realized that they can be induced by a variety of stressful stimuli, it is probably more appropriate to call them 'stress proteins'. The nomenclature of many hsps, for example hsp90, hsp70 and hsp60, reflects the approximate molecular mass of hsps within each of these families. For many bacterial and parasitic infections, hsps were first recognized as immunodominant antigens on immunoblots of extracts from the organism probed with immune sera, or in T-cell proliferation assays. They have now been identified in a range of fungal pathogens, again often linked to an immune response. In this symposium, we review the association of hsps with humoral immunity to candidosis and aspergillosis, cellular immunity to histoplasmosis, and the identification of hsp70 in another dimorphic fungus, Paracoccidioides brasiliensis. Finally, the crucial role of the membrane in setting the temperature of the heat shock response in yeasts is discussed.

Animals↗
PubMed1997

Highly increased TNF sensitivity of tumor cells expressing the yeast delta 9-desaturase gene.

L929 and WEHI tumor cell lines were genetically modified to constitutively express the Saccharomyces cerevisiae Ole 1 gene, coding for the delta 9-desaturase enzyme. These cells exhibit an increased ratio of monounsaturated fatty acids in their membrane phospholipids paralleled by an overall decrease in the membrane molecular order and a highly increased tumor necrosis factor-alpha (TNF) sensitivity. The TNF-alpha signaling cascade involves events, like receptor clustering and cleavage of membrane constituent lipid molecules by phospholipases, which are influenced by the physical state of cellular membranes. We discuss the possible involvement of non-bilayer forming lipids in the control of signaling mechanisms leading to TNF cytotoxicity.

Animals↗
PubMed1997

Effects of membrane fatty acids on thermal and oxidative injury in the human premonocytic line U937.

Heat shock (HS) proteins (HSP) function as molecular chaperones and protect cells from thermal and oxidative injury. The signals leading to HSP synthesis, i.e. the "cellular thermometer(s)," are still a matter of debate. In the human premonocytic line U937, we investigated the effects of specific modification of membrane fatty acid (FA) composition by incubation with various saturated and unsaturated fatty acids (UFA) on the HS response and on hydrogen peroxide (H2O2)-induced cell death. FA readily incorporated into U937 cell membranes. UFA did not modulate the HS response but potentiated H2O2-mediated damage, while pre-exposure to HS protected the UFA-treated cells from this increased H2O2 toxicity.

Calcium↗
PubMed1997

Modulation of lipid unsaturation and membrane fluid state in mammalian cells by stable transformation with the delta9-desaturase gene of Saccharomyces cerevisiae.

The composition and physical state of membrane lipids determine the dynamic nature of membranes, which in turn, could directly be linked to the activity of various membrane-associated cellular functions. To better understand the molecular basis of different membrane-related phenomena we established a novel strategy to alter unsaturation of mammalian cell membranes with an identical genetic background. We transfected L929 mouse fibroblastoid cells with DNA constructs containing the Delta9-fatty acid desaturase gene (Ole1) of S. cerevisiae under the control of desaturase promoters derived either from wild type or mutant strains of the dimorphic fungus H. capsulatum.

Animals↗
PubMed1997

An AP1 element is involved in transcriptional regulation of delta9-desaturase gene of Histoplasma capsulatum.

We have characterized a region of the promoter of a cloned delta9-desaturase gene (Ole1) of Histoplasma capsulatum, a dimorphic pathogenic fungus of humans. The product of the delta9-desaturase gene is involved in regulating membrane fluid state in animal cells and microorganisms. To identify sequences critical for Ole1 expression in both the saprobic mycelial and parasitic yeast phases of this organism, we performed a deletion analysis. Evidence is presented that a 240 nt region of the proximal promoter is involved in a phase-specific binding in vitro. By sequence analysis we have identified one likely regulatory element that coincides with an AP1 binding site (TGACTAA) that is located at -740 nt of 5'-upstream from the ATG. Using gel mobility shift assays, we show that this cis-acting element binds nuclear proteins extracted from the yeast and mycelial phases of H. capsulatum that may participate in control of expression of the delta9-desaturase gene.

Base Sequence↗
PubMed1997

Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects.

Preservation of the chemical architecture of a cell or of an organism under changing and perhaps stressful conditions is termed homeostasis. An integral feature of homeostasis is the rapid expression of genes whose products are specifically dedicated to protect cellular functions against stress. One of the best known mechanisms protecting cells from various stresses is the heat-shock response which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). A large body of information supports that stress proteins--many of them molecular chaperones--are crucial for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions, and thus can be considered "homeostatic proteins." Recently emphasis is being placed on the potential use of these proteins in preventing and/or treating diseases. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce the accumulation of HSPs in patients with chronic disorders such as diabetes mellitus, heart disease or kidney failure. Here we show that a novel cytoprotective hydroxylamine derivative, [2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoil-chloride maleate, Bimoclomol, facilitates the formation of chaperone molecules in eukaryotic cells by inducing or amplifying expression of heat-shock genes. The cytoprotective effects observed under several experimental conditions, including a murine model of ischemia and wound healing in the diabetic rat, are likely mediated by the coordinate expression of all major HSPs. This nontoxic drug, which is under Phase II clinical trials, has enormous potential therapeutic applications.

Animals↗
PubMed1996

Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast.

Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.

Blotting, Northern↗
PubMed1996

Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

Amino Acid Sequence↗
PubMed1996

Presence of heat shock protein in chronic periapical pathology of pulpal origin.

In order to establish whether tissues damaged by Chronic Periapical Pathology (CPP) of endodontic origin produced heat shock protein (HSP) capable of attracting reactive lymphocytes, paraffin sections of samples from the oral cavity of 10 patients with CPP were incubated with commercially available anti-HSP monoclonal antibodies. Antibodies were evidenced employing the alkaline phosphatase immunoenzymatic method (DAKO). HSP was found in the lamina propria infiltrated by lymphocytes, in six of the ten samples. These results suggest that HSP may be one of the lymphocyte recruiting factors in the damaged area and opens new possibilities for further research.

Adolescent↗
PubMed1995

A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

Amino Acid Sequence↗
PubMed1995

Unraveling the secrets of Histoplasma capsulatum. A model to study morphogenic adaptation during parasite host/host interaction.

Early in the developmental period of microbiology, Pasteur first observed the phenomenon of dimorphism in fungi when he noticed that the bread mold Mucor grew as a filamentous mold aerobically on the surface of broth cultures but at the bottom of the flask where the environment was anaerobic it reproduced as budding yeast cells. Several infectious fungal pathogens of humans, namely Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenkii, and Coccidioides immitis change from a multicellular filamentous form to an unicellular morphology when they invade tissues. The ability of pathogenic fungi to assume a different shape is referred to as dimorphism. This phenomenon has intrigued clinicians, and medical mycologists since its discovery at the turn of the century. The ability of pathogens to initiate infection, invade host tissues and survive in mammalian hosts is critically linked to the induction of specific gene products. In dimorphic fungi, developmentally regulated gene expression is particularly important, since they may exist in phylogenetically distinct hosts with different body temperatures. Using Histoplasma capsulatum as a model to study parasite-host interactions at the biochemical and molecular level, my laboratory has attempted to relate the clinical spectrum of disease to natural variations in the characteristics of this organism and to adaptations it must make as a saprobe and a parasite. Histoplasma capsulatum is the etiologic agent of histoplasmosis, a respiratory infection that is world-wide in distribution. As a saprobe in soil it is mycelial, but it becomes a budding yeast as a parasite in susceptible hosts. These morphological phases can be reversibly reproduced in vitro by shifting the temperature from 25 degrees C, at which it is mycelial, to 37 degrees C, when it becomes a budding yeast. The process of mycelial-to-yeast conversion is of particular interest since it is triggered by an increase in temperature and conversion to virulence. Viable mycelial fragments and conidia become airborne and enter the pulmonary tract by inhalation after which the fungus rapidly disseminates to other organs. Progressive disseminated histoplasmosis along with candidiasis, cryptococcosis, and invasive aspergillosis are opportunistic fungal infections in patients who are immunosuppressed or otherwise debilitated. Importantly, they are diagnostic hallmarks of acquired immunodeficiency disease syndrome (AIDS). The clinical features of these infections and the genetic characteristics of the etiologic agents present unique parasite-host interactions that make them valuable research models to study. In the infected host, Histoplasma capsulatum encounters various environmental stresses to which it adapts by regulating the expression of specific genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed1994

Hsp70 in parasites: as an inducible protective protein and as an antigen.

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with the hsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.

Animals↗