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We demonstrated previously that expression of a single trans-membrane region of the Δ(12) -desaturase gene of Synechocystis sp. PCC 6803 in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) altered the membrane physical state of this pathogen, induced a significant change in the pattern of mRNA transcription of major heat shock genes, and inhibited pathogen growth inside murine macrophages. In this study, we demonstrate that injection of the modified Salmonella strain [Stm(pBAD200)] into C57Bl6j mice is safe. Survival of mice was associated with bacterial clearance, an increased number of splenic leukocytes, and high levels of interleukin-12, interferon γ and tumor necrosis factor α in spleens as well as in sera. Furthermore, Stm(pBAD200)-injected mice developed a Salmonella-specific antibody and Th1-like responses. Mice challenged with Stm(pBAD200) are protected from systemic infection with Salmonella wild-type. Similarly, mice infected with Stm(pBAD200) by the oral route survived when challenged with an oral lethal dose of Salmonella wild-type. The avirulent Stm(pBAD200) phenotype is associated with a remarkable change in the expression of the hilC, hilD, hilA, invF and phoP genes, among others, whose products are required for invasion and replication of Salmonella inside phagocytic cells. These data demonstrate the use of trans-membrane peptides to generate attenuated strains, providing a potential novel strategy to develop vaccines for both animal and human use.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the “Membrane Sensor Hypothesis” which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Transcriptional response of desaturase genes to low temperature was investigated in the dimorphic fungus Mucor rouxii. The two morphological forms of M. rouxii, yeast-like and mycelial cells containing different fatty acid profiles were shifted from 30 to 10°C. Both cultures exhibited significantly altered fatty acid composition, whose content in polyunsaturated fatty acids increased as consequence of the temperature shift and was accompanied by a reduction of C18:1Δ(9) about 2 h after the temperature shift. These changes were particularly significant in phosphatidylcholine and phosphatidylethanolamine fractions. Moreover, the fatty acid profiles of monoacylglycerol and diacylglycerol were also modulated in response to the lower temperature of incubation. The changes of membrane lipids of M. rouxii were due to the cold-induced expression of Δ(9)-, Δ(12)- and Δ(6)-desaturase genes. Although the mRNA levels of the three desaturases were transiently induced by lowering the temperature, the pre-existing composition of fatty acid profiles of mycelial and yeast-like forms of M. rouxii may have lead to different expression profiles of desaturase genes that modified their membrane physical state under cold shock. While expression of Δ(12)-desaturase gene contributed mainly to cold acclimation of mycelia, Δ(9)-desaturase expression was the main transcript identified in the yeast-like culture after temperature shift.

The ability of intracellular pathogens to cause infection is related to their capacity to survive and grow inside macrophages or in other cell types. Candida albicans latent virulence is likely to be related to a similar mechanism of avoiding killing by specialized cells and to the resulting ability to grow in such hostile environments. Using a differential display reverse transcription polymerase chain reaction technique, we have identified seven genes induced in C. albicans during macrophage phagocytosis. Sequence analyses and database searches revealed that these cDNAs coded for proteins homologous to yeast metabolic proteins. Interestingly, four of them are putative peroxisomal proteins, and two are involved in environmental signal sensing and transduction. Among the seven genes induced by C. albicans, six represent new information that were not described in other infection models.

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.

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.

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 Δ9-fatty acid desaturase gene (Ole1) ofS. cerevisiaeunder the control of desaturase promoters derived either from wild type or mutant strains of the dimorphic fungusH. capsulatum.

International audience; Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol μ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds.

OBJECTIVES: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC50 = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

Chionodraco hamatus and Trematomus bernacchii are perciforms, members of the fish suborder Notothenioidei that live in the Antarctic Ocean and experience very cold and persistent environmental temperature. These fish have biochemical and molecular features that allow them to live at these extreme cold temperatures. Fine tuning of the level of unsaturated fatty acids content in membrane is a key mechanism of living organisms to adapt to cold and high temperatures. Desaturases are key enzymes that synthesize unsaturated fatty acyl-CoAs from saturated fatty acids. We cloned and sequenced a Δ(9)-desaturase gene and its cDNA of C. hamatus, and the cDNA of T. bernacchii. The coded proteins are virtually identical and share homology to other Δ(9)-desaturase fish sequences. These proteins contain, in the first trans-membrane domain, two cysteine residues that may form a disulfur bond present in the corresponding membrane region of Δ(9)-desaturase proteins of other Antarctic fish but not in Eleginops maclovinus that experiences higher environmental temperatures and in all other Δ(9)-desaturase genes of mammals present in data bases. C. hamatus Δ(9)-desaturase gene complements a Saccharomyces cerevisiae mutant lacking Δ(9)-desaturase (Ole1) gene. Analysis of sequence homology of the trans-membrane domains of Δ(9)-desaturase and the cytoplasmic region of the same proteins of Antarctic fish, non-Antarctic fish and mammals suggest that the significant differences found in the homologous sequences of the first trans-membrane domain may be due to the specific lipid content of their membrane.

We have isolated and characterized the Δ9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains ofHistoplasma 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°C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, arevirtually identical and analogous to the Δ9-desaturase gene ofSaccharomyces 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°C (yeast-to-mycelia transition). Nevertheless, Δ9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25°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 Δ9-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.

Summary We showed previously that insertion of Synechocystis Δ12-desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ12-desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α-antimicrobial peptide (α-AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA-Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α-AMPs to a specific MLD generating live non-virulent bacterial strains.

New arylthioindoles (ATIs) were obtained by replacing the 2-alkoxycarbonyl group with a bioisosteric 5-membered heterocycle nucleus. The new ATIs 5, 8, and 10 inhibited tubulin polymerization, reduced cell growth of a panel of human transformed cell lines, and showed higher metabolic stability than the reference ester 3. These compounds induced mitotic arrest and apoptosis at a similar level as combretastatin A-4 and vinblastine and triggered caspase-3 expression in a significant fraction of cells in both p53-proficient and p53-defective cell lines. Importantly, ATIs 5, 8, and 10 were more effective than vinorelbine, vinblastine, and paclitaxel as growth inhibitors of the P-glycoprotein-overexpressing cell line NCI/ADR-RES. Compound 5 was shown to have medium metabolic stability in both human and mouse liver microsomes, in contrast to the rapidly degraded reference ester 3, and a pharmacokinetic profile in the mouse characterized by a low systemic clearance and excellent oral bioavailability.

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation ofSalmonella entericaserovar Typhimurium LT2 (SalmonellaTyphimurium) with a heterologous Δ12-desaturase (or with itstrans-membrane regions) causes major changes in the pathogen's membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature inSalmonellais strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.

We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions. During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37 degrees C in the temperature tolerant and mouse-virulent G222B strain, while 34 degrees C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)'s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

In addition to high temperature, other stresses and clinical conditions such as cancer and diabetes can lead to the alteration of heat-shock protein (HSP) levels in cells. Moreover, HSPs can associate with either specific lipids or with areas of special membrane topology (such as lipid rafts), and changes in the physical state of cellular membranes can alter hsp gene expression. We propose that membrane microheterogeneity is important for regulating the HSP response. In support of this hypothesis, when particular membrane intercalating compounds are used to alter membrane properties, the simultaneous normalization of dysregulated expression of HSPs causes beneficial responses to disease states. Therefore, these compounds (such as hydroxylamine derivatives) have the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy'.

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes – and which are so essential to their function.

Promoter study was performed to understand the transcriptional control of delta9-desaturase gene of Mucor rouxii. Several putative cis-elements involved in lipid metabolism were mapped by computational analysis. 5' deletion analysis shows the presence of elements with repressing activity, especially in 122 bp located upstream of the transcription start site. Truncation of these repressor domains showed that the promoter of M. rouxii is functional in Saccharomyces cerevisiae without additional components and is insensitive to nutritional depletion. The promoter also drove effectively the expression of a M. rouxii delta12-desaturase gene, and the linoleic acid content increased with the age of the yeast culture in parallel with the promoter activity. This approach provides a genetic tool for programming heterologous protein production in the yeast.

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.

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.

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.

The purity and integrity of isolated RNA are essential prerequisites for a successful analysis of differential gene expression by DDRT-PCR. Thus extreme care should be taken during all the steps of RNA preparation.

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.

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.

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 °C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium-to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 °C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 °C proceeds at rates comparable to those of the virulent strains.Key words: heat shock, thermotolerance, ATPase, 70-kilodalton heat-shock protein, fungal morphogenesis.

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp 60 and hsp 70-like proteins have been proposed in Saccharomyces cerevisiae . hsp 70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp 60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 °C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the CI-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 °C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.

A cysteine dioxygenase, cysteine oxidase (EC 1.13.11.20), has been purified from the cytosolic fraction of yeast phase cells of the dimorphic fungus Histoplasma capsulatum. The cysteine oxidase is an iron-containing dioxygenase with a molecular weight of 10500 (±1500) and is present only in the yeast phase of the fungus. The enzyme is highly specific for l-cysteine, with a Km of 2 × 10−5 M in vitro. The product of cysteine oxidation is cysteinesulfinic acid, as analyzed by thin-layer chromatography and mass spectroscopy. To our knowledge, this is the first cysteine oxidase isolated from a fungus, and it probably plays an important role in the mycelial to yeast phase transition of H. capsulatum during which redox potential and cysteine levels are crucial factors. © 1983, American Chemical Society. All rights reserved.

Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.

1. The oxidative phosphorylation and the temperature dependence of mitochondria prepared from the outer compact layer and the inner spongy layer of adult tuna heart ventricle have been examined. 2. Mitochondria of the inner layer show higher succinoxidase and NADH-oxidase activities as compared with those of the outer layer. 3. Arrhenius plots for succinate oxidation by phosphorylating mitochondria show that the temperature dependence of the inner layer is higher than that of the outer layer. 4. Experiments performed with disrupted non-phosphorylating mitochondria demonstrate that this difference in temperature dependence of the two cardiac compartments depends on the integrity of the mitochondrial membranes. 5. These findings are discussed in relation to the physiology of the fish.

Three stages can be distinguished in the temperature-induced mycelial-to-yeast phase transition of Histoplasma capsulatum. Stage one is characterized by a progressive decrease in the respiration rate and in the intracellular concentrations of cysteine and other amino acids. By stage two, respiration has ceased completely and free cysteine has fallen to low levels. Exogenous cysteine is required during the second stage for activation of mitochondrial respiration (stage three) and completion of the morphological transition. Mitochondria isolated from cells in the second stage show no respiration with NADH, succinate, or other substrates unless they are first incubated with cysteine. In addition, a novel, cytosolic cysteine oxidase appears during the latter part of the second stage. In stage three, the respiration rate rises, intracellular concentrations of free cysteine and other amino acids increase to levels characteristic of yeast, and the morphological transition is completed. The results support the idea that alterations in cysteine metabolism play a key role in this differentiation process.

1. 1. The restriction patterns of three different Antarctic fish DNA ( P. bernacchi, N. rossi and C. kathleene ) have been analysed. 2. 2. The restricted DNAs have been probed for the presence of the heat shock 70 sequence (hsp70) by using the corresponding Drosophila sequence. The three patterns are closely related for the presence of several bands in common, though a closer arrangement for the hsp70 gene is present in P. bernacchi and N. rossi . The analysis with different stringency conditions has also suggested that in these fishes more than one copy of hsp70 per genome is present. 3. 3. Poly-A RNA analysis with Northern blot have shown that the hsp70 gene is transcribed, upon temperature increase, between 5 and 12°C with a maximum of expression at 8°C.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.

Plasma membrane vesicles from the yeast and mycelial phases of Histoplasma capsulatum have been purified and characterized. The method of purification involved differential centrifugation of ballistically fractured cells followed by sedimentation through discontinuous sucrose density gradient and equilibrium centrifugation. Purity of the preparation was assessed by electron microscopy. The protein composition of the membrane preparations from the yeast and mycelial phases of the fungus was analyzed by polyacrylamide gels. A comparison of the two morphologic phases revealed quantitative and qualitative differences in the expressions of several membrane-specific proteins. Physical differences in the appearance of the membranes were also observed by electron micrography of membrane preparations. Alteration in membrane fluidity may be one of the many causes for differences in the appearance of membrane vesicles in the two phases.

Respiration in the yeast and mycelial phases of Histoplasma capsulatum proceeds via a cytochrome system and an alternate oxidase, both present constitutively. The mycelial cytochrome system is distinguished by an additional partial shunt around the antimycin-sensitive site.