Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.

A cytoplasmically inherited genetic element in yeast, [PSI+], was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did [PSI+] inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of [PSI+]. [PSI+] altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

Derepression of ferritin messenger RNA translation by hemin in vitro.

Incubation of a 90-kilodalton ferritin repressor protein (FRP), either free or complexed with an L-ferritin transcript, with hemin or Co3+-protoporphyrin IX prevented subsequent repression of ferritin synthesis in a wheat germ extract. Neither FeCl3 in combinations with H2O2, nor Fe3+ or Fe2+ chelated with EDTA, nor Zn2+-protoporphyrin IX, nor protoporphyrin IX caused significant inactivation of FRP. FRP that had been inactivated by hemin remained chemically intact, as revealed by SDS-polyacrylamide gel electrophoresis. Inclusion of chelators of iron or free radical scavengers did not alter the inactivation produced by hemin. These and other results indicate that hemin derepresses ferritin synthesis in vitro.

Specificity of the induction of ferritin synthesis by hemin.

We have previously reported that hemin derepresses ferritin mRNA translation in vitro. As noted earlier, pre-incubation of a 90 kDa ferritin repressor protein (FRP) with hemin prevented subsequent repression of ferritin synthesis in a wheat germ extract. The significance of this observation has been investigated further. Evidence is presented here that this inactivation of FRP is temperature dependent. Neither FeCl3, Fe3+ chelated with EDTA, nor protoporphyrin IX caused significant inactivation of FRP under comparable conditions, whereas Zn2(+)-protoporphyrin IX produced an intermediate degree of inhibition. The presence of a glutathione redox buffer (GSB), which was previously shown to minimize non-specific side-effects of hemin, was not necessary for the derepression reaction. Inclusion of mannitol, a free radical scavenger, did not alter the inactivation caused by hemin. Calculation of the expected ratio of hemin monomers to dimers suggests that the active species is the monomer.

Experimental histoplasmosis in the beige mouse.

Mice carrying the beige mutation (bg/bg) on a C57Bl/6 background were challenged with Histoplasma capsulatum. bg/bg mice had higher mortality and higher lung tissue fungal counts in their lungs than either bg/+ or C57Bl/6 mice challenged with equal inocula. Immunologic studies showed that bg/bg mice developed normal delayed-type hypersensitivity (DTH) reactions to histoplasmin, but had deficient NK cell cytotoxic activity against YAC-1 target cells. Studies of macrophage killing of H. capsulatum in vitro showed that T lymphocytes of either bg/+ or bg/bg mice were able to activate fungal killing by bg/+ but not by bg/bg macrophages. These studies, while not excluding a role for the NK cell, suggest that macrophage dysfunction may be critical in the greater susceptibility of the bg/bg mouse and, by extension, that macrophage function is of major importance in host defense against H. capsulatum.

Cloning of a functional cDNA for the rabbit ferritin mRNA repressor protein. Demonstration of a tissue-specific pattern of expression.

Ferritin synthesis is controlled at the translational level in response to cellular iron status. A component of this regulatory system is the ferritin repressor protein (FRP) which binds to the iron-responsive element (IRE) located at the 5' end of all known ferritin mRNAs, thus inhibiting its translation. Antibodies against purified FRP were raised in mouse and used to isolate an FRP cDNA from a rabbit liver cDNA library cloned in the expression vector lambda gt11. The FRP cDNA encodes a 98.5-kilodalton protein which shares greater than 90% identity with IRE-binding proteins from other species. The FRP cDNA was placed under the transcriptional direction of the yeast GAL1 promoter. Yeast transformed with this gene express IRE-specific binding activity, illustrating the potential utility of yeast for the study of FRP structure/function. Analysis of FRP distribution in rabbit tissues shows that it is present in a variety of tissues. The levels of FRP differ dramatically from tissue to tissue, however. An examination of FRP mRNA levels and comparison to FRP protein suggest that synthesis of FRP is regulated transcriptionally and post-transcriptionally.

Transfer of toxin genes to alternate bacterial hosts for mosquito control.

Mosquitoes are vector of serious human and animal diseases, such as malaria, dengue, yellow fever, among others. The use of biological control agents has provide an environmentally safe and highly specific alternative to the use of chemical insecticides in the control of vector borne diseases. Bacillus thuringiensis and B. sphaericus produce toxic proteins to mosquito larvae. Great progress has been made on the biochemical and molecular characterization of such proteins and the genes encoding them. Nevertheless, the low residuality of these biological insecticides is one of the major drawbacks. This article present some interesting aspects of the mosquito larvae feeding habits and review the attempts that have been made to genetically engineer microorganisms that while are used by mosquito larvae as a food source should express the Bacillus toxin genes in order to improve the residuality and stability in the mosquito breeding ponds.

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level.

Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.

Purification of a specific repressor of ferritin mRNA translation from rabbit liver.

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.

Effect of temperature on the mycelium to yeast transformation of Paracoccidioides brasiliensis.

The influence of temperature on Paracoccidioides brasiliensis mycelium to yeast transformation was studied by sequential microscopic observations of slide cultures. When incubated at temperatures above 28 degrees C, the mycelial elements gradually produced round to oval chlamydospores and later on, exhibited multiple budding. A sizeable proportion of mycelial elements transformed at 34 degrees C; however, multiple budding was important only at 37 degrees C.

Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element.

The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5' untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with a Kd of 20-50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.

Treatment of cryptococcal meningitis in mice with fluconazole.

Fluconazole is a recently developed triazole with activity in vitro against Cryptococcus neoformans, water solubility, and excellent oral absorption. We compared fluconazole in murine cryptococcosis with ketoconazole and amphotericin B. Fluconazole was highly effective in suppressing cryptococcosis in mice challenged by the intravenous and intranasal routes, and was comparable with the other two drugs in its protective capacity. However, fluconazole was superior to ketoconazole and comparable with amphotericin B after intracerebral challenge. Fluconazole may warrant clinical evaluation in cryptococcosis.

A technique to collect and dislodge conidia produced by Paracoccidioides brasiliensis mycelial form.

Collection and separation of mycelial propagules produced by P. brasiliensis was accomplished by agitation with glass beads, centrifugation and filtration through cotton wool. The mean number of conidia liberated per plate (approximately 1 000 000) and their viability (79%), lead us to think that it is now possible to undertake experimental studies with these propagules.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin.

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3 Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Detection of histoplasmal antigens in mice undergoing experimental pulmonary histoplasmosis.

A micro-ELISA assay was developed for the quantitation of Histoplasma capsulatum antigen in lungs, bronchoalveolar lavage fluid (BALF), and serum of intranasally infected mice. As little as 0.2 ng of antigen/ml could be detected. During the course of experimental histoplasmosis, immunologically intact, thymus-containing mice (nu/+) had detectable histoplasmal antigens in their lungs, serum, and BALF within 1 day of challenge. Lung, BALF, and serum antigen concentration rose to a peak 2 wk after challenge; in nu/+ mice, antigen concentration then declined through the next 2 wk. In contrast, athymic nude mice have depressed cell-mediated immunity; their antigen concentration continued to rise throughout the course of progressive, ultimately lethal, illness. Antigen concentrations correlated with quantitative cultures of the lungs and BALF. There was little cross reactivity in mice challenged intranasally with Candida albicans or Blastomyces dermatitidis. The sensitivity of this test, and the apparently minimal cross reactivity, suggest that the micro-ELISA for histoplasmal antigen might have significant clinical application in diagnosing and monitoring the course of histoplasmosis.

Itraconazole in the treatment of paracoccidioidomycosis: a preliminary report.

The preliminary results of itraconazole therapy in 16 patients with active paracoccidioidomycosis were evaluated. The course of therapy--itraconazole administration for six months at a dose of 100 mg per day--was completed in 13 cases. This new triazole appeared as effective as ketoconazole in reducing the symptoms and arresting the progression of the mycosis. The scoring system employed to evaluate the effect of the drug showed that the condition of no patient worsened or remained the same during therapy. On the contrary, 11 (84.6%) of the 13 patients experienced major improvement and the other two (15.4%), minor improvement. No adverse reactions were reported by the patients, and there were no toxic effects on bone marrow or liver. Although experience with itraconazole is still limited, results to date indicate that this new drug is safe and effective for the treatment of paracoccidioidomycosis. Further trials with shorter periods of therapy seem warranted.

Cloning, expression and toxicity of a mosquitocidal toxin gene of Bacillus thuringiensis subsp. medellin.

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse polyclonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Polyacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot.

Experimental murine paracoccidiodomycosis induced by the inhalation of conidia.

Adult BALB/c mice of both sexes were infected intranasally with 10(6) viable P. brasiliensis conidia. Animals were sacrificed at intervals up to 6 months and studied by histopathology and organ cultures. At the time of challenge lung sections showed that instilled conidia had reached the alveoli; at 12 h such conidia were transforming into yeast cells, with multiple buds appearing by 18 h. Initially, the cellular infiltrate was composed of polymorphonuclear leukocytes; 6 days later, lymphocytes, plasmocytes and macrophages predominated. Multinucleated giant cells appeared only after 6 weeks. The rate of pulmonary infection as determined by organ culture was high (82 of the 83 mice studied). The experimental infection was progressive as indicated by increasing numbers of viable fungi with time. The results of this study demonstrate that the conidia produced by P. brasiliensis mycelial form are infectious, producing active disease in healthy animals.

Ferritin mRNA: interactions of iron regulatory element with translational regulator protein P-90 and the effect on base-paired flanking regions.

The ferritin iron regulatory element (IRE), a conserved sequence of 28 nucleotides in a hairpin loop, is a conserved mRNA-specific translational regulatory element; flanking the IRE are regions of varying sequence, which form 9-17 base pairs close to the 5' cap. P-90 is a ferritin mRNA-specific translation regulatory protein purified from animal liver and reticulocytes. To study the P-90-RNA interaction, protein nucleases (RNase S1 and T1) and chemical nucleases FeEDTA and/or 1,10-phenanthroline-Cu were used as probes of an oligonucleotide (n = 55), containing the IRE and flanking regions (FL), and natural ferritin mRNA. Footprints and "toeprints" showed that P-90 binding was confined to the stem and loop of the IRE itself. However, P-90 altered the structure of the flanking region by increasing base stacking or helicity (RNase V1 sensitivity). Comparison of the reactivity of the IRE and flanking regions in natural mRNA and the 55-mer showed that long-range interactions included protecting bulges, single-stranded, and stacked regions from protein nucleases as well as stabilizing the P-90-RNA interaction. Structural integration of the IRE with the base-paired flanking regions was indicated by common features of reactivity (periodic hypersensitivity to FeEDTA) and changes in the FL region caused by P-90. The increased secondary structure of the IRE flanking regions caused by P-90 binding to the IRE provides a likely mechanism for blocking initiation of ferritin mRNA translation, since the combined structure (IRE + FL) is so close (8-17 nucleotides) to the cap.

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3.

The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.