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.

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.

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.

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.

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.

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.

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.

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)

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.

Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans.

Genetic analysis of 119 acetate non-utilizing (acu) mutants in Aspergillus nidulans revealed ten new loci affecting acetate metabolism in addition to the three previously recognized on the basis of resistance to fluoroacetate and acetate non-utilization. The enzyme lesions associated with mutations at seven of the acu loci are described. These are: facA (= acuA), acetyl-CoA synthase; acuD, isocitrate lyase; acuE, malate synthase; acuF, phosphoenolpyruvate carboxykinase; acuG, fructose 1,6-diphosphatase; acuK and acuM, malic enzyme. The acu loci have been mapped and are widely distributed over the genome of A. nidulans. Close linkage has only been found between acuA and acuD (less than 1% recombination). There is no evidence for any pleiotropic mutation in that region affecting the expression of both these genes. Poor induction of the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in mutants lacking acetyl-CoA synthase, and also in the other two classes of fluoroacetate-resistant mutants, indicates that the inducer, acetate, may be metabolized to a true metabolic inducer, perhaps acetyl-CoA, to effect formation of the enzymes. There is no evidence of any other class of pleiotropic recessive acu mutations affecting the expression of the acuD and acuE genes, which are therefore thought to be subject to negative rather than positive control.

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.

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.

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.