Characterization of alpha and beta tubulin genes in the dimorphic fungus Histoplasma capsulatum.

Evidence from our laboratory indicates that microtubules are involved in the differentiation of the dimorphic, pathogenic fungus Histoplasma capsulatum; therefore, we cloned the tubulin genes from a virulent strain of the organism. We report that the H. capsulatum genome contains a single alpha (TUB1) and a single beta (TUB2) tubulin gene rather than the more typical multigene family which is common in even the simplest eukaryotes. Sequence data from these genes reveal a high degree of nucleotide and protein sequence conservation relative to tubulins from other species. The coding regions of TUB1 and TUB2 contain five and eight intervening sequences, respectively. Field inversion gel electrophoresis of H. capsulatum chromosome-sized DNA fragments indicates that the TUB1 and TUB2 genes are unlinked. Potential regulatory elements common to both genes have been identified in the 5' promoter regions. These elements may direct the coordinate expression of TUB1 and TUB2 during differentiation. The cloning and characterization of alpha and beta tubulin genes from H. capsulatum provides the first description of gene structure in this widely distributed pathogenic fungus. Isolation of the tubulin genes will facilitate future studies of tubulin gene expression during the dimorphic phase transitions and clarify the role of microtubules in the differentiation process.

Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum.

Recent investigations have confirmed the presence of one alpha-tubulin gene (TUB1) and one beta-tubulin gene (TUB2) in the dimorphic fungus Histoplasma capsulatum. In the present study, Northern blot (RNA blot) analyses revealed multiple alpha-tubulin transcripts and a single beta-tubulin transcript in the yeast and mycelial phases of the high-virulence 217B strain and low-virulence Downs strain. S1 nuclease protection assays demonstrated one initiation start site and two major stop sites for the TUB1 transcripts, suggesting that variations in 3' processing generate the alpha-tubulin messages of 2.5 and 2.0 kilobases. Dot blot hybridization experiments indicated that tubulin gene expression is developmentally regulated during the dimorphic phase transitions. alpha- and beta-tubulin mRNAs increased six- to eightfold during the yeast-to-mycelium conversion and decreased two- to threefold during the reverse transition. These changes in tubulin mRNA content coincided with major morphological events associated with H. capsulatum development. Western blots (immunoblots) of H. capsulatum yeast-specific proteins resolved by two-dimensional gel electrophoresis demonstrated a single alpha- and a single beta-tubulin isoform. Multiple tubulin polypeptides expressed in mycelia are probably products of posttranslational modifications.

Case report: chylous ascites and carcinoid tumors: possible association of two rare disorders.

Three cases of chylous ascites are reported in a series of 275 patients with carcinoid tumor. All three patients had diarrhea and elevated serotonin production. The literature is reviewed and the possible causes of the chylous ascites are discussed. It is suggested that a history of clinically significant diarrhea in a patient with chylous ascites should lead to biochemical tests for carcinoid tumors.

Sulfonamide-induced pancreatitis.

While reported cases of sulfonamide-induced pancreatitis meet many criteria for an allergic drug reaction, antibody or lymphocyte recognition of the offending drug or its metabolite has yet to be demonstrated. A patient with sulfonamide-induced pancreatitis is reported in whom lymphocytes were stimulated in vitro by sulfamethoxazole, sulfapyridine, and sulfasalazine. Lymphocytes from a normal volunteer were not stimulated. This in vitro lymphocyte recognition of sulfonamides provides further evidence that sulfonamide-induced pancreatitis is an allergic drug reaction.

Characterization of an immunosuppressive factor from malignant ascites that resembles a factor induced in vitro by carcinoembryonic antigen.

Oncodevelopmental antigens may cause immunologic suppression in the host through release of suppressor molecules from the host's own immunoregulatory cells. This concept has been difficult to study until recently when carcinoembryonic antigen was shown to induce the release of such molecules from normal circulating human mononuclear cells in vitro. However, the amount of the suppressor moiety generated was too small to adequately characterize, and its presence in vivo, i.e., in the cancer-bearing host, was unknown. Therefore, we sought to isolate and characterize a similar or identical macromolecule from ascites having an elevated CEA level in patients with cancer. A single malignant ascites, when precipitated at 0 to 35% ammonium sulfate saturation, was the source of suppressive factor for purposes of isolation and standardization. Suppression was quantitated by reduction of [3H]thymidine incorporation by phytohemagglutinin-stimulated normal human peripheral blood mononuclear cells. Sephadex G-200 chromatography revealed probable aggregation of the factor in isotonic buffers; aggregation was reduced in the presence of 8 M urea. Purification was achieved by precipitation with 5% trichloroacetic acid (TCA). The suppressor factor remained soluble in TCA and demonstrated a 95-fold increase in specific activity. Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein band of 50,000 daltons. Ascites from three additional cancer patients gave identical results. Physicochemical characterization of the suppressor moiety revealed stability at 70 degrees C for 30 min and at pH 2 and pH 10 for 24 hr. Delipidation by chloroform-methanol extraction, proteolytic enzyme digestion, and protamine sulfate precipitation did not affect activity, suggesting that lipid, simple peptides, and nucleic acids were not crucial. However, periodate oxidation irreversibly destroyed suppressor activity, suggesting the importance of carbohydrate to the molecule and offering one explanation for protease resistance. Similarities in m.w. (50,000 daltons), isoelectric point (pI = 3.4), physical properties (heat and acid stability and resistance to proteases), and immunologic activity of this factor with that released from lymphocytes after in vitro exposure to carcinoembryonic antigen indicates they may be identical. Our results suggest that early aberrant events induced in the immunoregulatory network by tumor-associated antigens may be relevant and may lead to better understanding of immunosuppression in the cancer-bearing host.

Stimulatory, permeabilizing, and toxic effects of amphotericin B on L cells.

High concentrations of amphotericin B (AmB) killed mouse L cells, but low concentrations increased plating efficiency and stimulated the incorporation of labeled precursors into DNA and RNA. Thus, there were two disparate effects of AmB on L cells, stimulatory and toxic, and they occurred in distinct dose-related stages. AmB also affected the permeability of L cells. In dose-response studies, increases in cell membrane permeability, measured as the loss of K+ ions, occurred along with the stimulation of [3H]uridine incorporation into RNA. In contrast, stimulation of [3H]thymidine incorporation into DNA was only observed in cells recuperating from AmB-induced permeability changes. When the K+ concentration in the medium was lowered to 0.5 from 4.5 mM, or when 1 mM ouabain was added to the cultures, cell killing was potentiated, but the stimulatory and permeabilizing effects of subtoxic concentrations of AmB were unaffected. Furthermore, etruscomycin, a polyene antibiotic without any permeabilizing effects, nevertheless induced an enhancement of plating efficiency and of incorporation of [3H]uridine into RNA and [3H]thymidine into DNA. Our results suggest that the dose-related stimulatory, permeabilizing, and toxic effects of AmB most probably have distinct mechanisms of action and may be independent of one another.

Carcinoembryonic antigen-induced release of a suppressor factor from normal human lymphocytes in vitro.

Although it is generally accepted that tumor-bearing patients may be immunosuppressed, the mechanism for this effect is unclear. Therefore, we tested the hypothesis that a tumor-associated macromolecule, carcinoembryonic antigen (CEA), could itself suppress lymphocyte function, as quantitated by uptake of [3H]thymidine by lymphocytes stimulated with the plant lectin, phytohemagglutinin. Normal human peripheral blood mononuclear cells, after exposure to CEA for 48 hr, subsequently released a factor in vitro which markedly inhibited phytohemagglutinin-stimulated lymphocytes. In further experiments, this factor release was confirmed to be initiated by CEA and not by a contaminant, and to be induced over a broad range of CEA concentrations (0.2 to 100 ng/ml). Suppression could not be accounted for by factor-associated cytotoxicity toward indicator cells, nor was it secondary to a mixed-lymphocyte reaction, nor could CEA alone (without factor) modulate proliferation. In studies to characterize the factor, its molecular weight was greater than 10,000, its activity was partially denatured by heat and proteases, and the isoelectric point was 3.4. Polyacrylamide gel electrophoresis of an "active" fraction revealed protein bands with molecular weights of 52,000, 77,000, and 171,000. Knowledge of immunomodulatory molecules present in cancer patients may suggest new modalities for therapy.

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.

Ultrasound findings in hepatitis.

A retrospective study of ultrasound images of the liver in patients with hepatitis was undertaken. Two distinct ultrasound patterns were detected. In acute hepatitis, the predominant findings were accentuated brightness and more extensive demonstration of the portal vein radicle walls and overall decreased echogenicity of the liver. Chronic hepatitis primarily revealed decreased brightness and number of portal vein radicle walls and verall increased liver echogenicity. In addition, the pathological severity closely paralleled these ultrasound patterns. A prospective study confirmed the same acute hepatitis ultrasound findings with close correlation to the clinical severity. These distinct ultrasound patterns will help to evaluate patients with suspected acute and chronic hepatitis and more accurately define intrahepatic causes of jaundice.

Amphotericin B-induced sensitivity to actinomycin D in drug-resistant Hela cells.

HeLa cells, which were selected for resistance to actinomycin D on the basis of decreaded penetration of the antibiotic into the cells, were treated with nontoxic concentrations of the polyene antibiotic amphotericin B. In the presence of amphotericin B, the cells became sensitive to the effects of actinomycin D, as demonstrated by loss of cell viability, typical morphological changes, and effects on the pattern of RNA synthesis. Furthermore, we were able to demonstrate that amphotericin B increased the amount of (3H)actinomycin D incorporated into HeLa cells as determined by both radioactive counts and radioautography. Amphotericin B might be useful in overcoming the resistance is based on decreased entry of the agents into cells.

In vitro aggregation of mixed embryonic kidney and nerve cells. Influence on macromolecular synthesis.

The possible role of nerve on growth of embryonic parenchymal organs such as kidney was explored by measuring macromolecular synthesis (DNA, RNA, and protein and three enzymes) in aggregates of mixed suspensions of cells from dissociated chick embryo kidney and nerve tissue. One and one-half to threefold increments in net synthesis of the three different types of macromolecules were observed in the mixed aggregates of kidney and nerve cells as compared with those of single organs or mixtures of kidney with nonneural cells. The addition of nerve-growth factor (NGF) did not significantly affect the results. Increased incorporation of label was paralleled by increases in chemically measured DNA and protein, suggesting an increase in growth in the mixed kidney-nerve aggregates compared with those of single tissues. Measurements of survival rate did not indicate increased cell stability in the mixed aggregates. The activities of three enzymes, acid phosphatase, alkaline phosphatase, and lactic dehydrogenase, were also enhanced two to four times in cultures of kidney plus nerve cells. Morphologic studies indicated a high degree of reorganization of tubular structures within the reaggregates of kidney cells alone or in those mixed with nerve. In addition, radioautographs of thymidine-(3)H-labeled cells in the aggregates showed a high level of DNA synthesis in the reformed tubular cells. Electron micrographs revealed the presence of large numbers of nerve fibers containing microtubules in the mixed cell aggregates. The data suggest a significant role for nerve in the growth processes of embryonic parenchymal organs.