AAT Expression Research Articles

Aroma Production in Climacteric Fruit: A Comparison of Tomato, Apple, and Banana

Volatile aroma compounds produced by apple, banana, and tomato are produced throughout development, however, those associated with ripening and edible quality are dependent upon ethylene action. In apple and banana, characteristic aroma is, in large part, dependent upon the formation of volatile esters. In tomato, many of the characteristic aromas are dependent upon tissue disruption and result from aldehydes and alcohols following lipid degradation. For apple and banana, the enzyme alcohol acyl-CoA transferase (AAT, EC 2.3.1.84) is the enzyme responsible for the final reaction in the pathway for ester formation and catalyzes the union of an alcohol and the CoA derivative of fatty acids. In both tissues, AAT gene expression was detected prior to the onset of ester production. In apple, AAT expression was found to be closely tied with the onset of autocatalytic ethylene synthesis. In banana, ethylene synthesis peaked and began to decline well before ester synthesis began. However, the expression of AAT increased as ester production increased for both tissues. Tomato fruit, like apple and banana, produced characteristic aromas following the onset of the ethylene climacteric, suggesting changes in the activity of various components of the lipoxygenase pathway. In all three tissue types, there are continuous, significant shifts in the aroma profile as fruit ripen age, suggesting shifts in specific metabolic pathways associated with precursor synthesis or degradation.

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells.

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.

The penicillin regulator PENR1 of Aspergillus nidulans is a HAP-like transcriptional complex.

In Aspergillus nidulans, a DNA-binding complex, PENR1, was shown to bind to two CCAAT-box-containing DNA elements located in the promoter regions of the bidirectionally oriented penicillin biosynthesis genes acvA and ipnA, and of the aat promoter. Here, partial purification of PENR1 and western blotting using anti-HAPC sera indicated that the previously identified HAPC protein, which was suggested to be part of the CCAAT-binding complex AnCF, is also part of PENR1. This was confirmed by band shift assays using protein extracts of a delta hapC strain which exhibited no PENR1 DNA-binding activity. Supershift assays and immunoprecipitation analysis using anti-HAPC sera provided evidence that HAPC is part of the PENR1 complex. In delta hapC strains, penicillin production was reduced, as was expression of both an ipnA-lacZ and aat-lacZ gene fusion. Hence, HAPC-containing PENR1 appears to act as an activator on ipnA and aat expression. However, deletion of hapC had little effect on acvA expression during a fermentation run in fermentation medium. Previous results which had shown that specific deletion of the PENR1-binding site between acvA and ipnA resulted in a strong increase of expression of an acvA-uidA gene fusion, together with the present data, suggest the possibility of the existence of a repressor protein that binds close to or overlaps the PENR1-binding site. It is also shown that binding of PENR1 induced bending of a DNA fragment spanning the PENR1-binding site between acvA and ipnA in vitro.

Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full-length open reading frame. The deduced amino acid sequence included 317 residues, with 81–86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles.

Analysis of the regulation of the Aspergillus nidulans penicillin biosynthesis gene aat (penDE), which encodes acyl coenzyme A:6-aminopenicillanic acid acyltransferase.

The regulation of the Aspergillus nidulans penicillin biosynthesis gene aat (penDE), which encodes acyl coenzyme A:6-aminopenicillanic acid acyltransferase (AAT), was analysed. Major transcriptional start sites map within 100 nucleotides upstream from the aat initiation codon. To study the regulation of aat expression, various aat-lacZ gene fusions were constructed, in which the aat promoter region was fused in frame with the Escherichia coli lacZ reporter gene. A. nidulans strains carrying recombinant plasmids integrated as single copies at the chromosomal argB locus were identified. In both fermentation and minimal media, aat-lacZ expression was maximal during the first 24 h of a fermentation run. Compared with minimal medium, aat-lacZ expression was increased two-fold in fermentation medium. Although AAT specific activity was reduced in mycelia grown on glucose instead of lactose, expression of aat-lacZ gene fusions was not repressed on glucose, suggesting that the glucose effect is mediated posttranscriptionally. The effect of glucose on AAT activity was reversed by further incubation of glucose-grown mycelia on lactose. Neither the inclusion of the first intron of the aat gene in the aat-lacZ fusion integrated at the chromosomal argB locus, nor the disruption of the acvA gene had any regulatory effect on aat-lacZ expression. In the heterologous, nonpenicillin producer A. niger, basal expression of aat-lacZ gene fusions was observed at about the same level as in A. nidulans.

An evaluation of the prognostic significance of alpha-1-antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis

Expression of alpha-1-antitrypsin (AAT) in tumour cells of 102 surgically resected lung adenocarcinomas was examined by immunohistochemical method using anti-AAT antiserum. While only 13 cases (13%) were negative for AAT expression, 89 cases (87%) contained AAT at varying degrees. The degree of AAT-positive tumour cells was significantly higher in advanced cases than in early cases. Clinical follow-up study of the patients, particularly in stage I, showed that strongly AAT-positive cases have poor prognosis than weak-to-moderately AAT-positive or AAT-negative cases. Thus, AAT expression status in tumour cells of lung adenocarcinoma may be a biological marker of prognostic significance in regard to tumour growth.

Use of alpha-1-antitrypsin staining in the diagnosis of nodular regenerative hyperplasia of the liver

Nodular regenerative hyperplasia (NRH) is a disorder characterized by regenerative nodules scattered diffusely throughout the liver without associated fibrosis. It is most often recognized at autopsy when the entire liver is available for inspection. The diagnosis of NRH by needle biopsy is much more subtle. Since alpha-1-antitrypsin (AAT) expression by hepatocytes in a variety of liver diseases has suggested that it may represent a marker for regenerative or damaged hepatocytes, we elected to study the expression of AAT by immunohistochemical staining of paraffin-embedded tissue of biopsy material as a possible marker of this diagnosis. Seventeen biopsies of the liver showing histologic features consistent with NRH were selected and compared with 20 biopsies of the liver without features of NRH. Eight of the NRH cases showed periportal granular AAT staining as opposed to only one of the non-NRH biopsies (P less than .01; Fisher exact test). These results indicate that AAT expression is increased in the regenerating compartment (as opposed to the presumably damaged atrophic portion) of the liver in NRH and suggest that AAT staining may be useful in confirming the biopsy diagnosis of NRH.

Tissue-specific expression of the human alpha 1-antitrypsin gene is controlled by multiple cis-regulatory elements.

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.