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T J Leonard

Publications and source records attributed to T J Leonard.

At least 19 recordsLinked to original sources
PubMed1996

Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans.

Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.

Aflatoxins↗
PubMed1996

Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus.

Under limiting growth conditions, Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to the Aspergillus flavus and Aspergillus parasiticus aflatoxin regulatory gene aflR. Deletion of the A. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. Because A. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined by A. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of the A. flavus aflR gene in A. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved between Aspergillus spp. and that aflR expression is sufficient to activate genes in the ST pathway.

Aflatoxins↗
PubMed1995

Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase.

A filamentous fungus, Aspergillus nidulans, produces the carcinogenic mycotoxin sterigmatocystin (ST), which is a polyketide-derived secondary metabolite. A gene (pksST) encoding the ST polyketide synthase (PKSst) in A. nidulans was cloned, sequenced, and characterized. Large induced deletion mutants, which did not make ST or any ST intermediates, were used to identify genes associated with ST biosynthesis. Among the transcripts detected within the deletion region, which showed developmental expression with ST production, was a 7.2-kb transcript. Functional inactivation of the gene encoding the 7.2-kb transcript blocked production of ST and all ST intermediate substrates but did not affect transcription of the pathway genes, indicating that this gene was involved in a very early step of ST biosynthesis. These results also indicate that PKSst was not associated with activation of other ST genes. Sequencing of the region spanning this gene revealed that it encoded a polypeptide with a deduced length of 2,181 amino acids that had high levels of similarity to many of the known polyketide synthases and FASs. This gene, pksST, encodes a multifunctional novel type I polyketide synthase which has as active sites a beta-ketoacyl acyl carrier protein synthase, an acyltransferase, duplicated acyl carrier proteins, and a thioesterase, all of these catalytic sites may be multiply used. In addition, a 1.9-kb transcript, which also showed developmental expression, was mapped adjacent to pksST, and the sequence of this gene revealed that it encoded a cytochrome P-450 monooxygenase-like peptide.

Amino Acid Sequence↗
PubMed1995

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus.

Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCR-derived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A)+ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, beta-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.

Aflatoxins↗
PubMed1992

Variation in regulation of aflatoxin biosynthesis among isolates of Aspergillus flavus.

Two new phenotypes of Aspergillus flavus which exhibit novel patterns of aflatoxin production have been identified and characterized. In one of the new variants of A. flavus, aflatoxin is made in the absence of carbohydrate and concomitantly with growth, without a lag period. A second variant did not produce aflatoxin in the presence or absence of carbohydrate. Chemical mutagenesis of this nonaflatoxigenic strain resulted in mutant strains which produced aflatoxin on carbohydrate-containing media. The aflatoxin production pattern observed in these mutants resembled the typical production scheme, with a lag period through log phase growth.

Aflatoxins↗
PubMed1992

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.

Aflatoxins↗
PubMed1989

Ptosis and supranuclear downgaze paralysis.

A patient developed the unusual combination of a supranuclear downward gaze paralysis and bilateral ptosis. It was caused by a single midbrain glioma. Other ocular motor functions were intact. The neuropathologic examination showed a tumor growing mainly around the third ventricle and the aqueduct. The findings agree with recent experimental evidence that a network of neural elements involved in eyelid control lies in the supraoculomotor area immediately dorsal to the oculomotor nucleus.

Aged↗
PubMed1987

Essential fatty acids in the plasma phospholipids of patients with atopic cataracts.

Abnormal levels of the essential fatty acids (EFA) in plasma phospholipids have been reported in atopic dermatitis. We have compared EFA levels in 10 patients with atopic cataracts and 29 atopic dermatitis patients without cataracts and found no significant differences, with the exception that higher levels of adrenic acid occurred in those with cataracts.

Adult↗
PubMed1987

Corneal thickness, astigmatism, and atopy.

Keratometry and regional pachometry were performed on atopic patients with no history or signs of atopic keratoconjunctivitis. In a series of 80 eyes there was a skewed distribution towards thinner corneas (p less than 0.01) and a preponderance of against-the-rule astigmatism. The maternal age at birth of this group of patients was higher than that in the general population. These findings are discussed in the context of the known association of atopy with keratoconus.

Adult↗
PubMed1984

Graves' disease presenting with bilateral acute painful proptosis, ptosis, ophthalmoplegia, and visual loss.

Two middle-aged women presented with bilateral acute painful proptosis, ptosis, ophthalmoplegia, and visual loss. In both an initial diagnosis of orbital cellulitis was made, but they did not respond to systemic antibiotics. Orbital computerised tomographic (CT) scans were thus done within 36 h of admission and they showed grossly enlarged extraocular muscles in each case suggestive of dysthyroid eye disease. Clinical examination was otherwise normal. When high doses of systemic steroids were substituted for the antibiotics the physical signs resolved rapidly, accompanied by a dramatic reduction in the size of extraocular muscles on CT scanning. One patient subsequently became clinically hypothyroid, while the other showed clinical and biochemical evidence of thyroid overactivity. These case-reports suggest that patients with bilateral acute painful proptosis should have an early CT scan to exclude atypical dysthyroid disease. Delay in giving systemic steroids may allow the development of unnecessary visual loss due to optic nerve damage.

Acute Disease↗
PubMed1984

Ophthalmoplegia in carotid cavernous sinus fistula.

The aetiology of ophthalmoplegia in 15 patients with carotid-cavernous sinus fistula is discussed, and the clinical findings are correlated with angiographic and orbital CT appearances. After closure of the fistula the majority of patients with generalised ophthalmoplegia recovered full ocular movements rapidly, while patients with an isolated abduction weakness required much longer to return to normal. Orbital CT studies showed enlarged extraocular muscles in the patients with generalised ophthalmoplegia but muscles of normal size in those with abduction failure alone. After closure of the fistula repeat CT studies of patients with enlarged extraocular muscles showed a diminution in muscle size. We suggest that generalised ophthalmoplegia in carotid cavernous sinus fistula is due to hypoxic, congested extraocular muscles. Isolated abduction weakness is due to a sixth nerve palsy, which probably occurs either in the cavernous sinus or more posteriorly near the inferior petrosal sinus. A combination of these 2 mechanisms may be found in some patients.

Adult↗
PubMed1984

Deliberate alteration of the visual evoked potential.

Seven out of 12 normal subjects could deliberately produce abnormal pattern-reversal visual evoked potentials (VEPs) which simulated disorders of the anterior visual pathways without detection. In six the mechanism was near-point accommodation and in one eccentric fixation. If voluntary suppression of the VEPs is suspected various modifications to the recording technique may be of value. These include the use of a large check size and stimulus field and binocular stimulation.

Adult↗
PubMed1984

Intracerebral haemorrhage complicating dural arteriovenous fistula: a report of two cases.

Two patients presented with unilateral peri-orbital pain, proptosis, chemosis and external ophthalmoplegia. They were shown to have dural arteriovenous fistulae related to the cavernous sinus. Intracerebral haemorrhage occurred in both patients within 18 months of presentation; this gave rise to focal seizures and signs of unilateral hemisphere dysfunction. The haematomas were in the region drained by the superficial middle cerebral vein ipsilateral to the shunt and are presumed to have been the result of locally raised venous pressure.

Aged↗