Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily. (1/26)

This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.  (+info)

Delayed-type hypersensitivity responses to a cell wall fraction of the mycelial phase of Coccidioides immitis. (2/26)

A skin test-active fraction was isolated from the mycelial-phase cell walls of Coccidioides immitis. This alkali-soluble, water-soluble antigen (C-ASWS) elicited positive reactions in 22 of 24 (92%) of the Coccidioides-sensitized guinea pigs whereas only 14 (54%) of the same guinea pigs reacted to commercial coccidioidin (BioCox). None of the 21 Histoplasma-sensitized guinea pigs cross-reacted with the C-ASWS antigen. Footpad tests in mice actively infected with Coccidioides further established the efficacy of the C-ASWS antigen in eliciting a delayed-type hypersensitivity response. One-microgram doses of C-ASWS produced reactions comparable to 100-mug doses of nondialyzable coccidioidin (Smith's lot 64 D4). The C-ASWS fractions isolated from three different C. immitis strains showed similar reactivity in terms of the number of positive reactions produced in Coccidioides-sensitized guinea pigs. However, the induration responses (diameter in millimeters) elicited by the C-ASWS fraction of one strain were significantly less than those elicited by the C-ASWS fractions of the other two C. immitis strains.  (+info)

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins. (3/26)

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)  (+info)

An immunoreactive apoglycoprotein purified from Coccidioides immitis. (4/26)

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and greater than 200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.  (+info)

Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis. (5/26)

The cytokine tumor necrosis factor alpha (TNF-alpha) functions as an immunomodulatory protein and as a mediator of cachexia. We report that viable or Formalin-killed spherules of Coccidioides immitis induced the secretion of TNF-alpha by peritoneal-exudate cells from BALB/c mice. The identification of the cytokine as TNF-alpha was based on its lytic activity against the TNF-alpha-sensitive LS murine fibrosarcoma cell line but not the TNF-alpha-resistant LR cell line, its neutralization by rabbit anti-TNF-alpha, and its secretion by peritoneal cells having characteristics of macrophages. The induction of TNF-alpha was to spherules and not to contaminating lipopolysaccharide (endotoxin), as evidenced by the finding that polymyxin B, a reagent that blocks the TNF-alpha-inducing component of lipopolysaccharide, did not negate the production of TNF-alpha in response to spherules, whereas pretreatment of spherules with hyperimmune goat antiserum to spherulin neutralized the induction of TNF-alpha by these cells. The demonstration that C. immitis activates macrophages to secrete TNF-alpha in vitro is a new finding and warrants studies to determine whether this cytokine is produced during active coccidioidomycosis.  (+info)

Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody. (6/26)

Detection of immunoglobulin M (IgM) precipitin antibody to coccidioidin, the autolysate of mycelial-phase cells of Coccidioides immitis, is an important serologic aid in establishing a diagnosis of primary coccidioidomycosis. In the present study, the component of coccidioidin that reacts with IgM precipitin antibody was isolated by a combination of immunoaffinity and anion-exchange chromatography. Antigenic analysis of the purified antigen in two-dimensional immunoelectrophoresis against goat anti-coccidioidin revealed a precipitinogen characterized by a complete cathodal leg and a partial anodal leg. The reactivity of this incomplete precipitating antigen with anti-C. immitis IgM was established by serologic assays and by the adsorption of reference IgM precipitin antibody on solid-phase immunosorbents containing the purified precipitinogen. The isolation of the coccidioidin component that reacts with IgM precipitin antibody and the production of monospecific antibody will provide the necessary reagents for the development of a sensitive immunoassay for detecting this serodiagnostic response.  (+info)

Symptoms and routine laboratory abnormalities associated with coccidioidomycosis. (7/26)

To assess the relationships of various symptoms and other early findings to the diagnosis of primary coccidioidomycosis, we devised a 40-question survey that was completed by 556 college students seeking medical care for illness possibly due to Coccidioides immitis. The results of routine laboratory studies on these patients were also compiled. Of 269 who had coccidioidal antibody determinations and other diagnostic tests, coccidioidomycosis was diagnosed in 36 (13%). By logistic regression procedures, an elevated erythrocyte sedimentation rate, male gender, "red lumps on shins," recent arrival to an endemic area, acuteness of symptoms, and decreased total peripheral blood lymphocyte counts were independent factors positively associated with infection (P less than .05). Relative risk analysis indicated that 60% of patients with four or more of these factors were found to have coccidioidomycosis. Other significantly but not independently associated factors were an increased total leukocyte count, chest pain with breathing, fever, an absence of hoarseness, and an abnormal chest roentgenogram.  (+info)

Induction and expression of cell-mediated immune responses in inbred mice infected with Coccidioides immitis. (8/26)

Comparisons of the course of coccidioidomycosis in two strains of inbred mice established that BALB/c mice are significantly more susceptible to pulmonary infection with Coccidioides immitis than are DBA/2 mice. The susceptibility of BALB/c mice does not reside in their inability to mount a delayed-type hypersensitivity response to C. immitis antigen. That is, BALB/c mice manifested footpad hypersensitivity to coccidioidin early during the course of disease, to a level comparable to that of DBA/2 mice. In contrast to the more resistant DBA/2 mouse strain, however, BALB/c mice developed anergy by day 15 postinfection. Suppression of the delayed-type hypersensitivity response was not specific for C. immitis antigen, as evidenced by the finding that BALB/c mice immunized with mycobacterial purified protein derivative prior to infection with C. immitis were suppressed in their footpad response to mycobacterial antigen at day 15 postinfection. Taken together, these results establish that genetically determined susceptibility to this fungus is associated with an acquired suppression of cell-mediated immune reactivity.  (+info)

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