Thymopoiesis following HSCT; a retrospective review comparing interventions for aGVHD in a pediatric cohort.

Acute graft-versus-host disease (aGVHD) complicates allogeneic hematopoietic stem cell transplantation (HSCT), and is treated with topical and/or systemic corticosteroids. Systemic corticosteroids and aGVHD damage thymic tissue. We compared thymopoietic effect of topical steroid therapy, corticosteroids and extracorporeal photopheresis (ECP) in 102 pediatric allogeneic HSCT patients. We categorized patients into 4 groups: - no aGVHD, aGVHD treated with topical or systemic steroid, or ECP. Naïve CD4+CD45RA+CD27+ T-lymphocyte values at 3, 6, 9, 12months post-HSCT were recorded: for ECP patients, values were recorded at 3, 6, 9, 12months during ECP. Differences were compared using the Kruskal-Wallis test. 41 patients had no aGVHD, 23 had aGVHD treated topically or systemically (25), 13 received ECP. Rate of thymopoiesis was significantly different between all groups at all time-points post-transplant (p=0.002, p<0.001, p<0.001, p=0.001 respectively). Even mild aGVHD impairs thymopoiesis. Worst recovery was in ECP patients. Earlier institution of ECP may speed thymic recovery.

Expression of the 3-phosphoglycerate kinase gene (pgkA) of Penicillium chrysogenum.

The sequence of the Penicillium chrysogenum pgkA gene promoter was determined up to 952 nucleotides (nt) 5' to the major transcriptional start point (position +1), and contains a 38 bp pyrimidine-rich region within which transcription initiates at this and two minor sites (-11, -23). A 21 bp segment (-99 to -79) closely matches a region which is essential for the expression of the Aspergillus nidulans pgkA gene. A further region was found with similarity to sequences in other A. nidulans promoters possibly effecting response to carbon source. The terminator region of the P. chrysogenum pgkA gene was sequenced as far as 192 nt 3' to the stop codon and three polyadenylation sites were found at 94, 103 and 107 nt from this point, the first preceded by a possible polyadenylation signal. No transcription termination signal was found but several regions potentially forming stem-loop-structures were noted. A single 1.3 kb pgkA mRNA was readily detected by Northern blot analysis of total cellular RNA. Steady-state levels of pgkA mRNA were 1.5 to 2.0 times greater in mycelium harvested at similar stages of growth from medium containing the carbon sources acetate or quinate compared to glucose. A transformed strain of P. chrysogenum containing a fusion of the pgkA promoter to the Escherichia coli lacZ reporter gene integrated at the oliC locus was constructed, and beta-galactosidase activity monitored during growth of batch cultures in defined media. The pgkA promoter activity increased during exponential growth and was 2-3 times greater and increased most rapidly in mycelium grown on quinate or acetate compared to glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme.

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensable pathways.

Disruption of the 3' phosphoglycerate kinase (pgk) gene in Aspergillus nidulans.

We report the isolation of a pgk- mutant strain of Aspergillus nidulans by means of a gene disruption strategy, and demonstrate that the pgk gene is located on chromosome VIII. The pgk- mutant conidiates poorly, will only grow on media supplemented with both a glycolytic and a gluconeogenic carbon source, and is inhibited by hexoses.

Intraorbital wood foreign body mimicking air at CT.

Computed tomography (CT) revealed a 2-cm linear area of extremely low attenuation in the left orbit of a boy who had been poked in the eye with a tree branch. The appearance and attenuation of the area suggested air, so a diagnosis of orbital emphysema was initially considered. Further research indicated that wood mimics the CT attenuation and appearance of air. A wood splinter was surgically removed from the orbit.

Differential flux through the quinate and shikimate pathways. Implications for the channelling hypothesis.

The qutC gene encoding dehydroshikimate dehydratase has been constitutively overexpressed in Aspergillus nidulans from a range of 1-30-fold over the normal wild-type level. This overexpression leads to impaired growth in minimal medium which can be alleviated by the addition of aromatic amino acids to the medium. Overexpression of the qutC gene in mutant strains lacking protocatechuic acid (PCA) oxygenase leads to the build up of PCA in the medium, which can be measured by a simple assay. Measuring the rate of production of PCA in strains overproducing dehydroshikimate dehydratase and correlating this with the level of overproduction and impaired ability to grow in minimal medium lacking aromatic amino acids leads to the conclusion that (a) the metabolites 3-dehydroquinate and dehydroshikimate leak from the AROM protein at a rate comparable with the extent of flux catalysed by the AROM protein, (b) the AROM protein has a low-level channelling function probably as a result of the close juxtaposition of five active sites and (c) this channelling function is only physiologically significant under non-optimal conditions of nutrient supply and oxygenation, when the organism is in situ in its natural environment.

Functional analysis of the expression of the 3'-phosphoglycerate kinase pgk gene in Aspergillus nidulans.

A functional analysis of the Aspergillus nidulans 3-phosphoglycerate kinase pgk promoter was undertaken using gene fusions to the lacZ gene of Escherichia coli, and introducing these into a beta-galactosidase-deficient strain of A. nidulans. Expression of a particular gene fusion in transformed strains depends upon the site of integration of the vector into the genome, and when specifically targeted to the catabolic quinate dehydrogenase qutE (selective marker) locus is directly proportional to its copy number. The analysis of transformed strains with single copies of pgk promoter deletion--lacZ fusions at the qutE locus identified three constitutive, positively acting sequence elements in the pgk gene. Sequence located between -161 and -120 nucleotides relative to the transcript start site +1, and including an element with a seven-out-of-eight nucleotide match (AAGCAAAT; -131 to -124) to the consensus eukaryotic octamer sequence ATGCAAAT, is essential for expression, and deletion of the complete 41-nucleotide sequence abolishes transcription. Sequence encompassing codons 14 to 183 and including the two introns of pgk contributes approximately one-third of the total activity, and far upstream sequence 5' to position -638 contributes approximately a further one-third total activity. In addition, sequence located -638 to -488 nucleotides, which includes an apparent consensus feature of A. nidulans glycolytic genes, affects carbon source-dependent regulation of expression. This region is required for an approximately 50% increase in pgk expression when A. nidulans is grown on gluconeogenic compared with glycolytic carbon sources.

A second gene (qutH) within the Aspergillus nidulans-quinic-acid utilisation gene cluster encodes a protein with a putative zinc-cluster motif.

A sequence of 3299 nt, contiguous with the previously sequenced quinate permease-encoding (qutD) gene and encompassing the dehydroshikimate dehydratase-encoding (qutC) gene, has been determined. Northern-blot analysis detected (i) a quinate-inducible mRNA of the expected size for the qutC gene, and (ii) a quinate-inducible mRNA of 1.45 kb divergently transcribed away from qutC towards qutD. Computer-aided sequence analysis identified an ORF of 1047 nt corresponding to the qutC gene encoding dehydroshikimate dehydratase. In addition, a genetically uncharacterized 1188-nt gene, designated qutH and containing a putative intron of 61 nt, was identified between qutC and qutD. The inferred protein sequence encoded by qutH contains a putative 'zinc cluster' motif and has a low (16%) but significant similarity with the DNA-directed DNA polymerase of hepatitis B virus. The results are interpreted as being consistent with the view that the qutH gene encodes a DNA-binding protein, possibly involved in the regulation of genes essential for the utilisation of protocatechuic acid.

Structure of the Aspergillus nidulans qut repressor-encoding gene: implications for the regulation of transcription initiation.

The nucleotide (nt) sequence of the qutR gene has been determined and shown to encode an inferred protein (QUTR) of 929 amino acids (aa). The inferred aa sequence shows a high level of similarity throughout its length with the aa sequence of the three C-terminal domains (shikimate kinase; 3-dehydroquinase; shikimate dehydrogenase) of the pentafunctional AROM protein of Aspergillus nidulans that catalyses steps 2-6 in the shikimate pathway. The inferred QUTR aa sequence has a completely conserved aa sequence motif, Gly, Xaa4, Gly, Lys, Ser, that is found in proteins that bind purine nt, suggesting that the inferred protein may have an in vivo kinase activity. The inferred QUTR protein also has a peptide sequence, DMVRLTQPAT, related to the active-site peptide in type-I 3-dehydroquinases. In active 3-dehydroquinases, the Arg (of QUTR) is replaced by Lys, which is involved in Schiff base formation as part of the reaction mechanism. The change from Lys----Arg in the inferred QUTR protein may allow the protein to bind but not metabolise the substrate for 3-dehydroquinase enzymes, namely 3-dehydroquinate. These observations are entirely consistent with the genetical model for how the QUTR protein functions, as it predicts that the protein can recognise and bind, but not metabolise, quinate, 3-dehydroquinate, and dehydroshikimate.

Spatial and biological characterisation of the complete quinic acid utilisation gene cluster in Aspergillus nidulans.

Heterologous probing of restriction digests of chromosomal DNA from Aspergillus nidulans with radioactively labelled probes encoding dehydroshikimate dehydratase (QA-4) and a repressor gene (QA1-S) from Neurospora crassa revealed a pattern of hybridisation inconsistent with an equivalent single copy of each gene in A. nidulans. Screening of size-selected and total genome A. nidulans DNA libraries allowed the isolation of four unique classes of sequence, two of which hybridised to the QA-4 probe, and two of which hybridised to the QA1-S probe. In each case, one of each pair of unique sequences was able to complement the equivalent mutations qutC (= QA-4) and qutR (= QA1-S) in A. nidulans, whereas the second of each pair was unable to complement the same mutation. The complementing sequences were physically mapped relative to the previously cloned A. nidulans QUT gene cluster, demonstrating that QUTR is distal and divergently transcribed from QUTA with approximately 3.6 kb between the ATG translational start codons, and that QUTC is transcribed in the same direction as QUTD on the other side of the cluster, approximately 1.65 kb downstream of the QUTD TAA translational stop signal. The physical and genetic maps of the QUT gene cluster correlate precisely. The non-complementing A. nidulans DNA sequences that hybridise to the N. crassa QA-4 (= QUTC) and QA1-S (= QUTR) fulfill many of the criteria characteristic of pseudogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective overexpression of the QUTE gene encoding catabolic 3-dehydroquinase in multicopy transformants of Aspergillus nidulans.

The three enzymes necessary to catabolize quinate to protocatechuate are inducible by quinic acid, and transcription of their corresponding genes is controlled by the action of a positively acting activator gene and a negatively acting repressor gene. Transformed strains of Aspergillus nidulans containing multiple copies of the activator gene (QUTA) but single copies of the other QUT genes retain normal regulation of the gene cluster and do not show any overexpression of the three quinic acid catabolic enzymes. Transformed strains containing equal multiple copies of the activator gene (QUTA) and QUTE (encoding catabolic 3-dehydroquinase), but single copies of the other QUT genes, retain normal regulation of the QUT gene cluster, but selectively overexpress the QUTE gene upon quinic acid induction. Data are presented that strongly suggested that the gene QUTG, which is physically located within the QUT gene cluster and for which no function has been identified, is not required for expression of the gene cluster and does not encode a chlorogenic acid esterase.

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans.

The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5' non-coding regions show significant homology with UASGAL and UASQA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5' sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.

Genetic regulation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans.

A large number of quinic acid non-utilizing qut mutants of Aspergillus nidulans deficient in the induction of all three quinic acid specific enzymes have been analysed. One class the qutD mutants, are all recessive and are non-inducible at pH 6.5 due to inferred deficiency in a quinate ion permease. Two regulatory genes have been identified. The QUTA gene encodes an activator protein since most qutA mutants are recessive and non-inducible although a few fully dominant mutants have been found. The QUTR gene encodes a repressor protein since recessive mutations are constitutive for all three enzyme activities. Rare dominant non-inducible mutants which revert readily to yield a high proportion of constitutive strains are inferred to be qutR mutants defective in binding the inducer. The gene cluster has been mapped in the right arm of chromosome VIII in the order: centromere - greater than 50 map units - ornB - 12 map units - qutC (dehydratase)-0.8 map units-qutD (permease), qutB (dehydrogenase), qutE (dehydroquinase), qutA (activator)-4.4 map units - qutR (repressor)-20 map units - galG. This organization differs from that of the qa gene cluster in Neurospora crassa, particularly in the displacement of qutC and qutR.

Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans.

The positively acting regulator gene QUTA from Aspergillus nidulans has been identified and located within a cluster of quinic acid utilisation (QUT) genes isolated within a recombinant phage lambda (lambda Q1). The DNA sequence of the QUTA gene reveals a single uninterrupted reading frame coding for a protein of mw 90.416 Kd. The QUTA protein sequence has a protein motif in the form of a putative "DNA finger" that shows strong homology to other such motifs in the GAL4, PPR1, ARGRII, LAC9 and QA1F regulatory gene products of S. cerevisiae, K. lactis and N. crassa. The data presented confirm the view deduced by genetical analysis that the QUTA gene of A. nidulans encodes a protein capable of interacting with QUT specific DNA sequences.

Identification and isolation of a putative permease gene in the quinic acid utilization (QUT) gene cluster of Aspergillus nidulans.

Mutations in the qutD gene of Aspergillus nidulans cause the loss of ability to grow upon quinic acid as sole carbon source in media at normal pH 6.5 and failure to induce three enzyme activities specifically required for metabolism to protochatechuic acid. All 9 qutD mutants recovered are recessive and have been found to be pH sensitive, growing strongly on quinic acid media at pH 3.5 and producing significant induced enzyme activities. These properties are consistent with the hypothesis that the QUTD gene encodes an essential component of a permease required for transport of quinate ion into mycelium at pH 6.5. The QUTD gene has been located within the cloned QUT gene cluster of A. nidulans by transformation of qutD mutants with fragments of cloned sequences from phage lambda-Q1. The QUTD locus is in a region distinct from other QUT genes and which contains sequences homologous to the QA-Y gene in the corresponding QA gene cluster of Neurospora crassa.

Transcription and processing signals in the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans.

The 3-phosphoglycerate kinase gene from Aspergillus nidulans contains two 57-bp introns and codes for a 421-amino acid (aa) protein with considerable homology to the Saccharomyces cerevisiae (68%) and mammalian (64%) proteins. Almost total conservation is found in Aspergillus of residues thought to be important to the structure and function of the yeast enzyme, and the introns fall between coding sequences for postulated structures in the N-domain. The strong codon preference found is more similar to that in other filamentous fungi than in yeast. The transcription start point (+1) has been mapped 32 bp upstream from the start codon, and the promoter region contains potential homologies for CAAT (-80 bp) and TATA (-30 bp) sequences, and certain other features common to other highly expressed genes in ascomycetes. There are three major termini 23, 83 and 115 bp beyond the stop codon and two of these are preceded by the polyadenylation consensus sequence and contain potential secondary structure.

Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans.

The Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage lambda genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23. A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.

Cloning and characterization of the three enzyme structural genes QUTB, QUTC and QUTE from the quinic acid utilization gene cluster in Aspergillus nidulans.

Heterologous DNA probes from the quinic acid gene cluster (QA) in Neurospora crassa (Schweizer 1981) have been used to isolate the corresponding gene cluster (QUT) from Aspergillus nidulans cloned in a phage lambda vector. N. crassa probes for each of the three enzyme structural genes in the cluster have been used to identify the corresponding genes within the A. nidulans cloned DNA. The three genes are in the same relative sequence [dehydrogenase (1), QA-3 = QUTB; dehydratase (3), QA-4 = QUTC; dehydroquinase (2), QA-2 = QUTE] though contained within a 3.4 kb DNA sequence in Aspergillus compared to a 5.4 kb sequence in Neurospora. The A. nidulans dehydroquinase (2) gene QUTE has been shown to complement an auxotrophic mutant aroD6 of Escherichia coli lacking biosynthetic dehydroquinase when tested for growth at 30 degrees C. A mutant of A. nidulans lacking catabolic dehydroquinase (2) and designated qutE208 has been isolated and shown to be tightly linked to the gene cluster, which maps between the ornB and fwA loci in linkage group VIII.