Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination

Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.     

Inhibition of epoxidation of methyl farnesoate to juvenile hormone III by cockroach corpus allatum homogenates

Radiolabeled methyl farnesoate is epoxidized to juvenile hormone III by an NADPH-dependent reaction occurring in corpus allatum homogenates from the cockroach Blaberus giganteus L. Most of the enzymatically produced juvenile hormone has the 10R configuration described for previously isolated natural juvenile hormones. The unnatural 2Z geometrical isomer of methyl farnesoate is epoxidized by the above system faster than the natural 2E isomer. Several series of chemicals known to be inhibitors of mixed-function oxidases were surveyed as inhibitors of methyl farnesoate epoxidation. The anti-juvenile hormone precocene II caused negligible inhibition at 1 · 10^?4M, whereas the best inhibitor was o-bromophenoxymethyl-imidazole with an apparent I₅₀ of 4 · 10^?7M. None of the inhibitors tested were potent morphogenetic agents on Tenebrio molitor pupae, and they failed to cause precocious development of Oncopeltus fasciatus nymphs. The inhibition of in vitro juvenile hormone biosynthesis suggests the possibility of finding an anti-hormone which acts by blocking juvenile hormone biosynthesis.     

Genetic differentiation between subpopulations of Swedish mountain (Fjäll and Fjällnära) cattle

ABSTRACT

The Swedish Mountain (Fjäll) cattle and the subpopulation Fjällnära originates from northern Sweden. They are differentiated from both traditional and commercial cattle from southern Sweden. We analysed data from the GGP HD150k SNP array and investigated genetic diversity and differences between Fjäll and Fjällnära, and between different groups of Fjällnära. We found that the Fjällnära can be divided into four groups, which are differentiated from each other and from the other Fjäll cattle. We also compared allele frequencies between different groups of Fjäll and Fjällnära for some of the functional SNPs included in the SNP array. Interestingly, the B allele of Beta lactoglobulin has a very high frequency (94%) in the Fjällnära cattle, which is significantly different from the frequency in the Fjäll cattle (60%). Comparing older samples of Fjäll cattle with more recent samples, we found a significant increase in the frequency of the B allele in Fjäll cattle.     

Effect of grapefruit juice, naringin, naringenin, and bergamottin on the intestinal carrier-mediated transport of talinolol in rats

The effect of two varieties of grapefruit juice (white and ruby red) and its selected components (naringin, naringenin, and bergamottin) was investigated on the activity of the P-glycoprotein (P-gp) in male Sprague-Dawley rats. Talinolol, a nonmetabolized P-gp substrate, was used as a marker compound. The white grapefruit juice (GFJ) had a minor effect on talinolol pharmacokinetics, but the ruby red GFJ reduced the C max and the AUC (0-infinity) by 60% and 50% of the control, respectively. However, among the GFJ constituents tested, bergamottin (0.22 mg/kg) was the most potent component augmenting the C max and the AUC (0-infinity) of talinolol by 2.4- and 1.8-fold, respectively, if compared to the control group. The flavonoids naringenin (0.7 mg/kg) and naringin (2.4 and 9.4 mg/kg) had a similar effect increasing the talinolol C max and AUC (0-infinity) by 1.5- to 1.8-fold, respectively. In conclusion, the effect of GFJ on P-gp activity seems to depend on the variety, the concentration of compounds in the juice, and the composition of different ingredients.     

Large-scale copy number polymorphism in the human genome

The extent to which large duplications and deletions contribute to human genetic variation and diversity is unknown. Here, we show that large-scale copy number polymorphisms (CNPs) (about 100 kilobases and greater) contribute substantially to genomic variation between normal humans. Representational oligonucleotide microarray analysis of 20 individuals revealed a total of 221 copy number differences representing 76 unique CNPs. On average, individuals differed by 11 CNPs, and the average length of a CNP interval was 465 kilobases. We observed copy number variation of 70 different genes within CNP intervals, including genes involved in neurological function, regulation of cell growth, regulation of metabolism, and several genes known to be associated with disease.     

Enzyme activities in agricultural soils fumigated with methyl bromide alternatives

Pre-plant fumigation of agricultural soils with a combination of methyl bromide (MeBr) and chloropicrin (CP) to control nematodes, soil-borne pathogens and weeds has been a common practice in strawberry (Fragaria X ananassa Duchesne) production since the 1960s. MeBr will be phased out by 2005, but little is known about the impacts of alternative fumigants on soil microbial processes. We investigated the response of microbial biomass and enzyme activities in soils fumigated over two years with MeBr+CP and the alternatives propargyl bromide (PrBr), InLine, Midas and CP. Results were compared to control soils, which were not fumigated for the last 4-5 years for Watsonville and Oxnard, respectively, but had a 10 year history of MeBr+CP fumigation (history soils). Soil samples (0-15 cm) were taken from two sites in the coastal areas of California, USA, in Watsonville and Oxnard, at peak strawberry production after two years of repeated application. In addition to the soil enzymes, the activities of purified reference enzymes of β-glucosidase, acid phosphatase and arylsulfatase were assayed before and after fumigation with MeBr+CP and alternative biocides. At the Oxnard site, microbial respiration significantly decreased in soils fumigated with MeBr+CP (P=0.036), while microbial biomass C and N showed no response to fumigation at both sites. These results may indicate that fumigation promotes the growth of resistant species or that soil microorganisms had recovered at the time of sampling. Repeated soil fumigation with MeBr+CP significantly decreased the activities of β-glucosidase and acid phosphatase at the Watsonville site, and dehydrogenase activity at the Oxnard site. Although, enzyme activities in soils fumigated with PrBr, InLine, Midas and CP were lower compared to the control soil, effects were, in general, not significant. Fumigation with MeBr+CP and alternatives reduced the activities of purified reference enzymes by 13, 76 and 28% for acid phosphatase, β-glucosidase and arylsulfatase, respectively. Mean enzyme protein concentrations in fumigated agricultural soils were 2.93, 0.105, and 2.95 mg protein kg⁻¹ soil for acid phosphatase, β-glucosidase and arylsulfatase, respectively, all lower than in control soils. Organic matter turnover and nutrient cycling, and thus, the long-term productivity of agricultural soils seem unaffected in soils repeatedly fumigated with PrBr, InLine, Midas and CP.     

Reoxidation Behavior of Wheat and Rye Glutelin Subunits

The ability of HMW and LMW subunits of wheat glutelin to form a polymeric gluten network by intermolecular disulfide bonds is responsible for the unique rheological properties and baking quality of wheat dough. Because the mechanism of gluten formation is not fully understood, the reoxidation behavior of HMW and LMW subunits of wheat glutelin and HMW subunits of rye glutelin was studied. The subunits were isolated from wheat flour cv. Rektor (REK) and from rye flour cv. Danko (DAN) with a selective extraction and precipitation method. For reoxidation, different oxidants (KBrO₃ and KIO₃), protein concentrations (0.5, 1.0, and 2.0%), solvent compositions, pH values (2.0 and 8.0), and reaction times (0–360 min) were compared. The characterization of reoxidized products was achieved by the determination of the thiol content with the Ellman's reagent, and of the M_r distribution by gel‐permeation chromatography. The results demonstrated that both HMW and LMW subunits could be slowly reoxidized with KBrO₃ to polymers with M_r up to several millions. Yield and M_r distribution of polymers were dependent both on the protein concentration and on the molar ratio of oxidants to thiol groups. The HMW subunits of wheat glutelin (HMW‐REK) yielded slightly higher quantities of polymeric proteins than did the HMW subunits of rye (HMW‐DAN). Reoxidation with KIO₃ proceeded much faster than with KBrO₃ and led to lower proportions of polymerized proteins for HMW‐REK and HMW‐DAN. Obviously, more intra‐ and fewer intermolecular disulfide bonds were formed by reoxidation with KIO₃ compared with KBrO₃. In contrast, LMW‐REK was reoxidized with KIO₃ to higher amounts of polymeric aggregates, which indicated that LMW subunits formed intermolecular disulfide bonds with both KIO₃ and KBrO₃. Independent of the protein type and the oxidant used for reoxidation, more inter‐ and fewer intramolecular disulfide bonds were formed when the protein concentration was increased. Single subunits 5, 7, and 10 were isolated from HMW‐REK by preparative acid‐PAGE and were reoxidized with KBrO₃ for 360 min. The M_r distribution indicated that x‐type subunit 5 had a greater tendency to form polymers than x‐type subunit 7. The y‐type subunit 10 was characterized by a lower proportion of polymers after reoxidation than x‐type subunits 5 and 7.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse

Application of taste dilution analyses on freshly prepared black tea infusions revealed neither the high molecular weight thearubigen-like polyphenols nor the catechins and theaflavins, but a series of 14 flavon-3-ol glycosides as the main contributors to the astringent taste perceived upon black tea consumption. Among these glycosides, the apigenin-8-C-[alpha-l-rhamnopyranosyl-(1-->2)-O-beta-d-glucopyranoside] was identified for the first time in tea infusions. Depending on the structure, the flavon-3-ol glycosides were found to induce a velvety and mouth-coating sensation at very low threshold concentrations, which were far below those of catechins or theaflavins; for example, the threshold of 0.001 micromol/L found for quercetin-3-O-[alpha-l-rhamnopyranosyl-(1-->6)-O-beta-d-glucopyranoside] is 190000, or 16000 times below the threshold determined for epigallocatechin gallate or theaflavin, respectively. Moreover, structure/activity considerations revealed that, besides the type of flavon-3-ol aglycon, the type and the sequence of the individual monosaccharides in the glycosidic chain are key drivers for astringency perception of flavon-3-ol glycosides.