Screening and characterization of aldehyde dehydrogenase gene from Halomonas salina strain AS11

A population survey was made of moderately halophilic bacteria in prawn pond sediment in the Songkla region of Thailand. Twenty-two isolated halophilic bacteria capable of growing on modified ATCC culture medium 1270 for halobacterium were then assayed for aldehyde dehydrogenase (ALDH) activity which might be involved in the metabolism of xenobiotic compounds. One isolate, designated AS11, was selected based on its high amount of ALDH activity. This organism can grow at sodium chloride concentrations ranging from 2.5 to 25%, although optimum growth occurs at 5% NaCl. Phenotypic and phylogenetic studies indicated that AS11 was an isolate of Halomonas salina. The aldh gene coding for this enzyme was then cloned. The open reading frame of the aldh gene was 1521-bp long and coded for a protein of 506 amino acid residues with a calculated molecular mass of 55 kDa. The aldh gene product proved to be 76% identical to the NAD-dependent acetaldehyde dehydrogenase gene from Pseudomonase aeruginosa.     

Wheat puroindolines interact to form friabilin and control wheat grain hardness

Wheat grain is sold based upon several physiochemical characteristics, one of the most important being grain texture. Grain texture in wheat directly affects many end use qualities such as milling yield, break flour yield, and starch damage. The hardness (Ha) locus located on the short arm of chromosome 5D is known to control grain hardness in wheat. This locus contains the puroindoline A (pina) and puroindoline B (pinb) genes. All wheats to date that have mutations in pina or pinb are hard textured, while wheats possessing both the soft type pina-D1a and pinb-D1a sequences are soft. Furthermore, it has been shown that complementation of the pinb-D1b mutation in hard spring wheat can restore a soft phenotype. Here, our objective was to identify and characterize the effect the puroindoline genes have on grain texture independently and together. To accomplish this we transformed a hard red spring wheat possessing a pinb-D1b mutation with soft type pina and pinb, creating transgenic isolines that have added pina, pinb, or pina and pinb. Northern blot analysis of developing control and transgenic lines indicated that grain hardness differences were correlated with the timing of the expression of the native and transgenically added puroindoline genes. The addition of PINA decreased grain hardness less than the reduction seen with added PINB. Seeds from lines having more soft type PINB than PINA were the softest. Friabilin abundance was correlated with the presence of both soft type PINA and PINB and did not correlate well with total puroindoline abundance. The data indicates that PINA and PINB interact to form friabilin and together affect wheat grain texture.Communicated by J. Dvorak     

Determination of Optimal Sample Size for Quantification of β-Cell Area,Amyloid Area and β-Cell Apoptosis in Isolated Islets

Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive β-cell area/islet area, thioflavin S-positive amyloid area/islet area and β-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for β-cell area/islet area, amyloid area/islet area and β-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% β-cell area/islet area and 8.93% ± 1.56% β-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for β-cell area/islet area, 30% for amyloid area/islet area and 23% for β-cell apoptosis (non-transgenic: 9% for β-cell area/islet area and 45% for β-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine β cells and islet amyloid.