Expression, purification and mutagenesis of recombinant class 1 Aldehyde dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University /

Aldehyde dehydrogenase (AIDH) catalyses the conversion of aldehydes, for example acetaldehyde and retinal, to carboxylic acids in an NAD+- dependent reaction involved in the detoxification of aldehydes and alcohols. There are several isoenzymes, class 1 being the cytosolic form. Three over-expression and purification systems have been tested, in order to gain a high yield of active, pure class 1 aldehyde dehydrogenase. The traditional method, using T7 polymerase-driven expression in E. coli followed by ion-exchange and p-hydroxyacetophenone-affinity chromatography, gave 3 mg/L human aldehyde dehydrogenase with a high specific activity of 1.2 units/mg. Human Class 1 AIDH has been over-expressed and purified using the GST Gene Fusion System (Pharmacia Biotech), avoiding the need for AIDH-affinity chromatography and therefore allowing straight-forward purification of mutated enzymes. The GST fusion system produced 2.6 mg/L pure AIDH with a specific activity of 0.39 units/mg. The methylotrophic yeast Pichia pastoris was chosen for its high yields, in the region of grams/litre. Preparatory work was carried out with the construction of the expression plasmids and screening of the Pichia transformants. A highly conserved lysine residue (Lys-272) may be involved in acid-base catalysis of aldehyde oxidation, as well as of the esterase reaction also catalysed by AIDH. Preliminary work has been carried out on the generation of the K272A, K272R, K272H and K272L altered enzymes. The resultant activity level, kinetic behaviour and active site structure of these modified enzymes should help to elucidate further the mechanism of action of AIDH

New sub-millimeter limits on dust in the 55 Cancri planetary system

We present new, high-sensitivity sub-millimeter observations towards 55 Cancri, a nearby G8 star with one, or possibly two, known planetary companion(s). Our 850 $\mu$m map, obtained with the SCUBA instrument on the James Clerk Maxwell Telescope, shows three peaks of emission at the 2.5 mJy level in the vicinity of the star's position. However, the observed peaks are 25\arcsec--40\arcsec away from the star and a deep $R$-band optical image reveals faint point sources that coincide with two of the sub-millimeter peaks. Thus, we do not find evidence for dust emission spatially associated with 55 Cancri. The excess 60 $\mu$m emission detected with ISO may originate from one or more of the 850 $\mu$m peaks that we attribute to background sources. Our new results, together with the HST/NICMOS coronographic images in the near-infrared, place stringent limits on the amount of dust in this planetary system, and argue against the existence of a detectable circumstellar dust disk around 55 Cnc.Comment: 11 pages, 2 PostScript figures, to appear in The Astrophysical Journal Letter

The Evaluation and Followup of Children Referred to Pediatric Endocrinologists for Short Stature

Objective. To characterize the pediatric endocrinologists' evaluation and followup of short-statured patients. Study Design. Observational study of 21,548 short-statured children (April 1996 to December 1999). Baseline demographics, laboratory testing, height standard deviation score (SDS), target height, and height relative to target height were analyzed at initial and return visits with the specialist. Patients were scheduled for at least one return visit and no recombinant human growth hormone therapy was administered. Results. Mean patient age was 8.6 years with a mean height SDS of −2.1. Patients were predominantly male (69%), prepubertal (73%), and white (76%). Few screening tests were obtained during initial evaluation. Nearly 40% of children did not return for their second scheduled visit. The follow-up rate was unrelated to demographics or degree of short stature. Conclusions. Low return rates limit specialists' ability to monitor growth or obtain laboratory testing over time. Further studies are needed to determine which tests should be obtained at the initial clinic visit as well as the basis for the low return rate in this group of children

Possible liquid immiscibility textures in high-magnesia basalts from the Ventersdorp Supergroup, South Africa

The lowermost succession of lavas in the Proterozoic Ventersdorp Supergroup contains light weathering ocelli up to 15 cm in diameter which occur in layers of a darker weathering volcanic material. Some ocelli appear to merge, and discrete light weathering layers may be the ultimate end-stage of this coalescence. Alternatively, coexisting magmas in the neck of the volcano may have been erupted in varying proportions, and turbulence during flow caused spalling of large drops of the lighter weathering material into the other. Several lines of field evidence suggest that two distinct liquids coexisted and were rapidly quenched after eruption. Chemical data for ocelli and matrix are consistent with the hypothesis of liquid immiscibility. The differences in compositions between the coexisting pairs of liquids are small and it is suggested that the original magmas must have been close to the consulute composition

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns.

BACKGROUND: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. METHODS: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. RESULTS: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. CONCLUSIONS: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns.

BACKGROUND: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. METHODS: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. RESULTS: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. CONCLUSIONS: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE