Changes in tubulin mRNAs during differentiation of a parasitic protozoan Leishmania mexicana.
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Animals↗
Biomedical subjects
D Fong
Publications and source records attributed to D Fong.
Distribution of ascorbate in the retina, subretinal fluid and pigment epithelium.
The posterior segment of the eye was divided into four compartments: retinal cytosol (R), subretinal fluid on the retinal surface (S/R), retinal pigment epithelial (RPE) cytosol, and subretinal fluid on the RPE surface (S/RPE). The volume of each compartment was estimated from the dilution of creatinine (in the extraction buffer) by the endogenous tissue fluid. The ascorbate concentrations in R, S/R, S/RPE, and RPE were 20.6, 12.3, 3.7, and 5.8 mg/dl respectively. Dehydroascorbate was observed only in the RPE and S/RPE. The decreasing ascorbate concentration from the retina to RPE, and the distribution of dehydroascorbate suggest a movement of ascorbate from the vitreous cavity into the subretinal space. The permeability of retinal cell layers to ascorbate was confirmed by the high radioactivity observed in the subretinal space after an intravitreal injection of C14-ascorbate. The occurrence of dehydroascorbate in the RPE and the S/RPE indicates the presence of oxidative reaction of ascorbate in these compartments, where light induced free radicals are located.
Animals↗
Measurement of e+e---> micro+ micro-: A test of electroweak theories.
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Journal Article↗
Measurement of the strong-coupling constant alpha s to second order for 22 <= sqrt s <= 46.78 GeV.
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Journal Article↗
Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.
The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.
Animals↗
Cell biology of host-parasite membrane interactions in leishmaniasis.
Molecular interactions at the host-parasite interface are crucial for the outcome of microbial infection, particularly in infection by intracellular parasites, such as Leishmania donovani and Leishmania mexicana, whose natural transmission begins with the delivery of the promastigote stage by the sandfly vector into the susceptible host. The ensuing event is intracellular parasitism of macrophages in the host by the amastigote stage. The establishment of this event in leishmaniasis must follow the sequence: (1) Leishmania-macrophage attachment; (2) entry of Leishmania species into macrophages; (3) intra-macrophage survival and differentiation of Leishmania species; and (4) intracellular multiplication of Leishmania species. This sequence precedes all clinical symptoms and pathological consequences in different forms of the disease. Study of these cellular events in Leishmania-macrophage systems in vitro indicates that host-parasite membrane interactions dictate many of the cellular events. Some morphological and functional changes of macrophages in response to leishmanial infection are related to their membrane activities, i.e. endocytosis and exocytosis. Leishmania parasites undergo profound plasma membrane-related changes, on entry into macrophages, at the morphological, antigenic and molecular levels. Most of these changes probably reflect necessary steps for the transition of Leishmania species from an extracellular to an intracellular life. The remarkable ability of Leishmania species subsequently to live in the secondary lysosome of the macrophage may also be due to certain intrinsic structures and dynamic properties of the parasite plasma membrane. Further analysis of leishmanial surface molecules and their interactions with macrophages is essential in any attempt to understand the pathogenic mechanism in leishmaniasis.
Animals↗
Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta.
A 1,942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage beta and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a molecular weight of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. We infer that initiation of translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.
Amino Acid Sequence↗
Post-transcriptional control of tubulin biosynthesis during leishmanial differentiation.
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Animals↗
The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts.
Proteins of IMR-90 fibroblasts incorporating [35S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.
Arginine↗
Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.
The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.
Animals↗
Increased arabinitol levels in experimental candidiasis in rats: arabinitol appearance rates, arabinitol/creatinine ratios, and severity of infection.
Renal colony counts, total arabinitol appearance, and the arabinitol/creatinine ratios in the serum and urine of rats with lethal candidiasis were measured. Total arabinitol appearance and the arabinitol/creatinine ratios increased significantly after infection. The serum arabinitol/creatinine ratio was high (2 SD above the mean value in normal animals) in five of six infected rats at 24 hr, in eight of eight at 48 hr, and in none of the control animals. The urinary arabinitol/creatinine ratio was high in 14 of 19 infected rats by 24 hr, in 11 of 11 by 48 hr, and in none of the control animals. Total arabinitol appearance and the arabinitol/creatinine ratios were highly correlated with renal colony counts. Arabinitol production in vitro was sufficient to account for the increased arabinitol appearance observed in vivo in 12 of 14 rats. Thus it is concluded that the excess arabinitol that appeared in vivo was produced by the infecting fungus and that arabinitol is a quantitative marker substance for candidiasis.
Animals↗
The arabinitol appearance rate in laboratory animals and humans: estimation from the arabinitol/creatine ratio and relevance to the diagnosis of candidiasis.
The effects of renal function on serum concentrations of the candidal metabolite arabinitol were studied by examining the accumulation and elimination of arabinitol in animals and humans. Serum concentrations of arabinitol rose sharply and in direct proportion to creatine concentrations after nephrectomy in rats. The serum half-life of exogenous arabinitol was 20.8 hr in anephric rats but only 0.62 hr in sham-nephrectomized control animals. The mean +/- SD apparent space of distribution of arabinitol was 419 +/- 26 ml/kg. The fraction of exogenously administered arabinitol recovered in the urine was 0.73 +/- 0.13 in uremic rats, 0.85 +/- 0.28 in control rats, and 0.95 +/- 0.10 in normal dogs. The arabinitol/creatinine clearance ratio was 0.99 +/- 0.08 in normal dogs and 0.99 +/- 0.25 in 22 critically ill patients with cancer. Thus arabinitol is eliminated by nearly quantitative urinary excretion and its cleared at virtually the same rate as creatinine. Therefore, the rate of arabinitol appearance in the body from any source equals the urinary arabinitol excretion rate and is directly proportional to the concentration ratio of arabinitol to creatinine in serum or urine.
Animals↗
Antigenic changes during intracellular differentiation of Leishmania mexicana in cultured macrophages.
Antigenic changes during the intracellular transformation of Leishmania mexicana subsp. amazonensis from promastigotes to amastigotes in macrophages of J774G8 line were noted mostly among protein bands of 24 to 68 kilodaltons in apparent molecular weight. In this region, six were identified as common antigens of both stages, six to seven were identified as promastigote specific, and three to five were identified as amastigote specific. At the higher-molecular-weight region (greater than 68 kilodalton) were two bands, one being predominant in amastigotes and the other in promastigotes. There may be a transformation-specific band (apparent molecular weight = 20 kilodaltons). The transition of these stage-specific antigens varies considerably with different protein species and may play important roles in intracellular leishmanial differentiation.
Animals↗
Argon laser trabecular surgery as an alternative to carbonic anhydrase therapy.
Thirty-four eyes (25 patients) with diagnosed chronic open angle glaucoma on maximal medical therapy were studied to determine whether argon laser trabecular surgery was a desirable alternative to carbonic anhydrase therapy. Ninety-four per cent of patients responded favourably to argon laser trabecular surgery as an alternative to carbonic anhydrase therapy. The average pressure drop was 9.9 mm Hg. Interestingly, there was a mean drop of 4.2 mm Hg in the contralateral eye. There was minimal, if any, iris response although there was a small but probably not clinically significant decrease in corneal endothelial cell count. Blanching of the trabecular wall was used as the endpoint to help minimize post-laser pressure rise. Blacks and aphakic patients responded satisfactorily. Laser surgery is best reserved for those patients with a pretreatment intraocular pressure no higher than the 30 to 35 mm Hg level. Trabeculectomy, in spite of its known complications, offers a better prognosis in those cases where the pretreatment pressure exceeds the 30 to 35 mm Hg level.
Aged↗
Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages.
Cytodifferentiation in the transition cycle of the parasitic protozoan Leishmania mexicana amazonensis was studied in vitro. The flagellated motile promastigotes transform into the nonmotile amastigotes in 7 days at 35 degrees C intracellularly in the murine macrophage line J774G8. In medium 199 plus fetal bovine serum, the reverse transformation occurs extracellularly at 27 degrees C in 2 days. Slab gel electrophoresis of leishmanias labeled with [35S]methionine during transformation revealed changes in protein banding patterns. The intensity of two protein species with apparent molecular weights of approximately equal to 55,000 increased in the amastigote-to-promastigote differentiation and decreased during the reverse transformation. These two protein species comigrated approximately with alpha- and beta-tubulin of Chlamydomonas flagella in two-dimensional gel electrophoresis. The lower band was further identified as beta-tubulin by immunoprecipitation using rabbit antiserum specific to the beta-tubulin of Chlamydomonas axonemes. The biosynthetic change of tubulin was found to correlate with the morphological change of microtubules is leishmanial flagella and cytoskeleton during transformation.
Animals↗
Bone and gallium scanning in the evaluation of disseminated coccidioidomycosis.
Bone and gallium scans were performed on 79 patients to determine the presence and extent of disseminated coccidioidomycosis. Bone scans appeared to be more sensitive than skeletal radiographs or gallium scans in determining the extent of coccidioidal bone involvement. The gallium scan was useful only in the identification of coccidioidal tissue abscesses. All patients with clinical evidence suggestive of dissemination and all patients with poor prognostic factors who are to receive amphotericin B therapy should be evaluated with a bone scan.
Adolescent↗
Proteases in cellular slime mold development: evidence for their involvement.
Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation.
Amino Acids↗
Protease activity during cell differentiation of the cellular slime mold Dictyostelium discoideum.
Individual aggregates, migrating pseudoplasmodia, and sorocarps of Dictyostelium discoideum were assayed for proteolytic activities by colorimetric and fluorometric techniques. Cathepsin D-like and cathepsin B-like acid protease activities were found to decrease throughout development, but the patterns of decrease were different for the two enzymes. A gradual decrease was found for cathepsin D, whereas a sharp decrease between aggregates and migrating pseudoplasmodia was detected for cathepsin B. By using microdissection techniques and fluorometric assays for amino acids and peptides, prestalk cells and prespore cells exhibited no difference in cathepsin D activity, whereas cathepsin B activity was higher in the prestalk cells. Similarly, stalk cells and spores in the sorocarps showed no difference in cathepsin D activity, but showed a fivefold higher cathepsin B activity in the stalk cells. This finding suggests a possible role for cathepsin B in stalk cell differentiation.
Cathepsins↗