Determination of urinary amino acids by means of glass capillary gas—liquid chromatography with alkali-flame ionisation detection and flame ionisation detection

The determination of amino acids as their N(O)-heptafluorobutyryl-isobutyl ester derivatives by glass capillary gas—liquid chromatography has been studied. Separations of amino acids obtained from insulin hydrolysate and human urine analysed with a flame ionisation detector, an alkali-flame ionisation detector and an electron-capture detector are shown. The quantitative results of urinary amino acids of one deep-sea diver are presented.     

Identification of amino acid thiohydantoins directly by thin-layer chromatography and indirectly by gas-liquid chromatography after hydrolysis.

A method is described for the identification of amino acid thiohydantoins by two-dimensional t.l.c. An indirect method for the determination of amino acid thiohydantoins is described which, after hydrolysis, the corresponding amino acids are determined by g.l.c.     

Investigating short-term exposure to electromagnetic fields on reproductive capacity of invertebrates in the field situation

Organisms are exposed to electromagnetic fields from the introduction of wireless networks that send information all over the world. In this study we examined the impact of exposure to the fields from mobile phone base stations (GSM 900?MHz) on the reproductive capacity of small, virgin, invertebrates. A field experiment was performed exposing four different invertebrate species at different distances from a radiofrequency electromagnetic fields (RF EMF) transmitter for a 48-h period. The control groups were isolated from EMF exposure by use of Faraday cages. The response variables as measured in the laboratory were fecundity and number of offspring. Results showed that distance was not an adequate proxy to explain dose-response regressions. No significant impact of the exposure matrices, measures of central tendency and temporal variability of EMF, on reproductive endpoints was found. Finding no impact on reproductive capacity does not fully exclude the existence of EMF impact, since mechanistically models hypothesizing non-thermal-induced biological effects from RF exposure are still to be developed. The exposure to RF EMF is ubiquitous and is still increasing rapidly over large areas. We plea for more attention toward the possible impacts of EMF on biodiversity.     

Glycopeptide dendrimer colchicine conjugates targeting cancer cells

Screening of a 65,536-member one-bead-one-compound (OBOC) combinatorial library of glycopeptide dendrimers of structure ((βGal)_n + 1X⁸X⁷X⁶X⁵)₂DapX⁴X³X²X¹(β-Gal)_m (βGal = β-galactosyl-thiopropionic acid, X^8–1 = variable amino acids, Dap = l-2,3-diaminopropionic acid, n, m = 0, or 1 if X⁸ = Lys resp. X¹ = Lys) for binding of Jurkat cells to the library beads in cell culture, resynthesis and testing lead to the identification of dendrimer J1 (βGal-Gly-Arg-His-Ala)₂Dap-Thr-Arg-His-Asp-CysNH₂ and related analogues as delivery vehicles. Cell targeting is evidenced by FACS with fluorescein conjugates such as J1F. The colchicine conjugate J1C is cytotoxic with LD₅₀ = 1.5 μM. The β-galactoside groups are necessary for activity, as evidenced by the absence of cell-binding and cytotoxicity in the non-galactosylated, acetylated analogue AcJ1F and AcJ1C, respectively. The pentagalactosylated dendrimer J4 βGal₄(Lys-Arg-His-Leu)₂Dap-Thr-Tyr-His-Lys(βGal)-Cys) selectively labels Jurkat cell as the fluorescein derivative J4F, but its colchicine conjugate J4C lacks cytotoxicity. Tubulin binding assays show that the colchicine dendrimer conjugates do not bind to tubulin, implying intracellular degradation of the dendrimers releasing the active drug.

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Metabolic fingerprinting reveals differences between shoots of wild and cultivated carrot (Daucus carota L.) and suggests maternal inheritance or wild trait dominance in hybrids

Differences between the metabolic content of cultivars and their related wild species not only have implications for breeding and food quality, but also for the increasingly studied area of crop to wild introgression. Wild and cultivated western carrots belong to the same outcrossing species and hybridize under natural conditions. The metabolic fingerprinting of Dutch wild carrot and of western orange carrot cultivar shoots using (1)H NMR showed only quantitative differences in chemical content, indicating relatively low divergence after domestication. Main differences reside in the primary metabolite content and in the concentrations of chlorogenic acid and feruloyl quinic acid in the shoots of the different carrot types. Wild×cultivar hybrids cannot be distinguished from wild plants based on the metabolome, suggesting maternal, maternal environment, or dominance effects, and indicating high hybrid fitness in wild conditions. Considering these similarities, introgression is a real possibility in carrots, but understanding its consequences would require further studies using backcrosses in a multiple environments.     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

Anaerobic digestion of spent bedding from deep litter piggery housing

This paper investigates spent litter from deep litter piggery housing as a potential substrate for farm-scale anaerobic digestion. Degradability and degradation rates were evaluated under mesophilic conditions for unused, lightly soiled (used by weaner/small pigs), and heavily soiled (used by finishing/large pigs) wheat straw, barley straw, and rice husks bedding. Apparent first order hydrolysis rate coefficients varied, but were comparable across all samples analysed (<0.1 day^?1). Spent wheat straw was generally more degradable (approximately 60%) than spent barley straw, while spent barley straw was comparable to raw straw (40–50%), but with higher hydrolysis rates, indicating better accessibility. Rice husks were relatively poorly degradable (<20%), but degradability was improved by weathering in a pig shed. Digestion of spent barley and wheat straw litter was significantly faster (approximately twice the rate) at low (8% solids) than high (14% solids) solids loading. Rice husks degradation kinetics were not significantly influenced by solids concentration. Intrinsic methanogenic activity of heavily soiled spent wheat straw and rice husks bedding was initially poor, but achieved full activity after 40–60 days, indicating that reactor operation without external inoculum may be possible with care.     

Studies on sequencing of peptides from the carboxyl terminus by using the thiocyanate method.

We report on our experience in applying the thiocyanate method developed by Stark (1968) (Biochemistry 7, 1796-1807) to the sequencing of short peptides from the carboxyl end in free solution. Yields fell to very low levels after three cycles of degradation. The method was time-consuming because of the filtration and freeze-drying stages involved. To overcome these problems, peptides were attached to modified polystyrene polymers for sequential degradation in the solid phase, and a maximum of six amino acids was determined. Also, ribonuclease was attached to active-ester glass beads and sequential degradation was carried out to determine six amino acids at the C-terminal end of this protein.     

A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr²⁹³) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr²⁹³. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.