Structural features of a polypeptide carrier promoting secretion of a β-lactamase fusion protein in yeast

Escherichia coli β-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150δ-carrier. The hsp150δ-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150δ-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150δ-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.     

Trichloroethylene uptake into fruits and vegetables: three-year field monitoring study

Trichloroethylene (TCE) contaminated groundwater migrating into communities surrounding Hill Air Force Base (HAFB) in northern Utah prompted a multiyear monitoring program (2001-2003) to examine the extent of TCE uptake and transfer into edible fruits. During the initial sampling in fall 2001, TCE was detected in a small fraction of the 167 fruit and tree core samples collected from 17 private residences. Samples were analyzed using headspace gas chromatography (GC) with electron capture detection (ECD) with limited confirmation by mass spectrometry (MS) in selected ion monitoring mode. In fall 2002, over 300 samples were collected from the same general locations sampled in 2001. No TCE was found in any of the fruit or vegetable samples above the method detection limit (MDL) for the headspace GC/MS method (approximately 0.1 microg/ kg fresh weight, depending on sample size), but TCE was again detected in several fruit tree trunk core samples. The detection of TCE in fruit in 2001, but not in 2002, may have been due to improvements in the analytical procedure or changes in the environmental conditions impacting transfer to fruit. The 2003 monitoring focused on repeated sampling over several months at five locations that were selected to represent the range of exposure scenarios evaluated during the previous years. No TCE was identified in any of the fruit above the MDL during 2003, however TCE was again found in tree core samples as observed in 2001 and 2002.     

Detection of nematodes in cod (Gadus morhua) fillets by imaging spectroscopy

ABSTRACT:  A promising method for detection of parasites in whitefish fillets has been developed. By use of imaging spectroscopy it is possible to record both spectral and spatial information from an object. In this work it is shown that by applying a white light transmission setup and imaging spectroscopy to cod ( Gadus morhua ) fillets, it is possible to make spectral images containing information to differentiate between fish muscle and parasites. The spectral images are analyzed by discriminant partial least square regression as well as image-filtering techniques. The method identifies parasites on the surface of the fillets as well as embedded parasites. One parasite was detected at 0.8 cm below the fillet surface, which is 2 to 3 mm deeper than what can be found by manual inspection of fish fillets. The method is nonintrusive and should thus be feasible for industrial purposes.     

Coupled Fe(II)-Fe(III) electron and atom exchange as a mechanism for Fe isotope fractionation during dissimilatory iron oxide reduction

Microbial dissimilatory iron reduction (DIR) is an important pathway for carbon oxidation in anoxic sediments, and iron isotopes may distinguish between iron produced by DIR and other sources of aqueous Fe(II). Previous studies have shown that aqueous Fe(II) produced during the earliest stages of DIR has delta56Fe values that are 0.5-2.0%o lowerthan the initial Fe(III) substrate. The new experiments reported here suggest that this fractionation is controlled by coupled electron and Fe atom exchange between Fe(II) and Fe(III) at iron oxide surfaces. In hematite and goethite reduction experiments with Geobacter sulfurreducens, the 56Fe/54Fe isotopic fractionation between aqueous Fe(II) and the outermost layers of Fe(III) on the oxide surface is approximately -3%o and can be explained by equilibrium Fe isotope partitioning between reactive Fe(II) and Fe(III) pools that coexist during DIR. The results indicate that sorption of Fe(II) to Fe(III) substrates cannot account for production of low-delta56Fe values for aqueous Fe(II) during DIR.     

Elimination of experimentally induced bovine intramammary infection assessed by multiplex real-time PCR and bacterial culture

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.     

Effect of gamma-irradiation on flavour 5′-nucleotides,tyrosine, and phenylalanine in mushrooms (Agaricus bisporus)

The impact of gamma-irradiation on 5′-nucleotides and on the free amino acids tyrosine and phenylalanine in fresh mushrooms was studied. After irradiation the samples were freeze-dried to avoid enzyme induced chemical changes. Three 5′-nucleotides could be detected using HPLC–UV and LC–ESI-MS: adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP) and guanosine 5′-diphosphate (GDP). Irradiation significantly reduced (p = 0.05) the GDP concentration (22%). AMP showed a marked reduction (46%) only at 5 kGy. GMP, tyrosine, and phenylalanine were not affected by gamma-irradiation.     

Combinatorial synthesis and characterization of mixed metal oxides for soot combustion

A polymerizable-complex method (PCM) was used with automated solution deposition to generate combinatorial libraries of oxide powders that were evaluated for their ability to catalyze the combustion of soot. Chemical compositions were examined for catalytic activity in the soot combustion reaction in parallel using infrared thermography and serially using automated thermogravimetric analysis. Although infrared analysis is much faster, it is not easily suited to analyzing soot combustion in the configuration employed in the present study. Instead, the slower automated thermogravimetric analysis (TGA) was found to be much more reliable and reproducible. The results of characterizing simple oxide systems (Ce–K–O, Cu–K–O, Ce–Na–O and Cu–Na–O) are reported.