Sensory perception is related to the rate of change of volatile concentration in-nose during eating of model gels.

The relationship between perceived aroma and the volatile concentration measured in-nose was investigated during eating of a model food. Sensory ranking and time-intensity analysis (TI) were used to measure perceived aroma, while in-nose volatile concentration was monitored by atmospheric pressure ionization mass spectrometry, which produced time release data. A gelatine-sucrose gel with a range of gelatine concentrations (2-8% w/w) and flavoured with furfuryl acetate was used as the model food. Sensory scaling showed decreased flavour intensities and TI showed a decrease in the flavour perceived over time, as the gelatine concentration increased. Studies in model systems and in people demonstrated that the different rates of release observed for different gelatine concentrations were not due to binding of volatile to protein in the gel, nor to mucous membranes, but were due to different rates of gel breakdown in-mouth. There were no significant differences in the maximum in-nose volatile concentrations for the different gelatine concentrations, so the amount of volatile present did not correlate well with the sensory analysis. However, the rates of volatile release were different for the different gels and showed a good correlation with sensory data.     

Influence of harvest date and light integral on the development of strawberry flavour compounds

Strawberries cv. Elsanta were grown in peat bags in a glasshouse and subjected to three shading levels (0%, 25%, 47%) for 2 weeks, commencing 1 week prior to first fruit ripening. Fruit was harvested at five intervals and analysed using Atmospheric Pressure Chemical Ionization (APCI) and direct liquid-mass spectrometry techniques. Thirteen volatiles implicated in strawberry flavour and three non-volatiles, sucrose, glucose and citric acid, were measured. Highly significant differences in volatile and non-volatile concentrations existed between harvest dates. Shading had a significant effect on hexanal, hexenal, ethyl methyl butyrate, and methyl butyrate concentrations at some harvests. In general, at each harvest the higher the level of shading the lower the level of the volatile in the fruit. Sucrose concentration showed a decrease throughout the harvest period, whereas glucose and citric acid showed less clear trends. Shading had a significant effect on glucose and sucrose concentrations. Some possible reasons for the variability in strawberry flavour are discussed.     

Volatile release from liquids: a comparison of in vivo APCI-MS, in-mouth headspace trapping and in vitro mouth model data

In-mouth volatile release from flavoured water was followed using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) or using a hand-held, computer-controlled device based on sequential trapping of flavours on Tenax traps. The present results verify recent in vitro data obtained with a sophisticated, fully computerized mouth model apparatus and confirm its validity for the simulation of in-mouth dynamic volatile release. In-nose APCI-MS measurements showed considerable person-to-person variability in non-trained individuals during drinking due to subconscious control of muscles during swallowing and subsequent breathing. Data showed a 'swallow breath' volume reaching the nasal cavity from the throat, not from the mouth cavity. Flavour enriched air from the mouth was shown to be transported to the nose (via exhalation) immediately after the swallowing event, but the dynamic process of volatile equilibration between residuals of the swallowed liquid and the exhaled air predominantly determined volatile in-nose concentration. Owing to its dynamic character, the process of volatile equilibration and release in the throat upon exhalation should be similar to the in-mouth process studied in the present work. A full mechanical simulation of retronasal volatile transport, however, will remain difficult.     

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.     

Pheomelanin as a Binding Site for Drugs and Chemicals

Certain drugs and chemicals, such as chloroquine, chlorpromazine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are bound to melanin and retained in pigment cells for long periods. This specific retention in pigmented tissues can cause adverse effects in the skin, eye, inner ear, and pigmented nerve cells of the substantia nigra of the brain. To date, all studies have been focused on eu- and neuromelanin. In the present study, we show that chloroquine, chlorpromazine, chlomipramine, paraquat, acridine orange, and nickel, which are bound to eumelanin, also bind to synthetic pheomelanin, but the binding to pheomelanin is lower. The binding varied with the cysteine content and pH, and the results indicate that the binding is complex and includes ionic interactions. In addition, we have shown that these substances also bind to synthetic thiourea-containing melanin, but to quite a low extent. We also present a microautoradiographic study on the binding of ¹⁴C-chloroquine to natural pheomelanin in vivo in yellow mice C57BL (A^y/a). Black (C57/BL) and albino (NMRI) mice were used as controls. The autoradiography demonstrated a pronounced uptake of chloroquine in the hair follicles and the dermal melanocytes in the ear of yellow mice, which was comparable to the corresponding accumulation of label in black mice. In the albino mouse, the uptake was lower and more homogeneously distributed in the skin. These results suggest that the toxicologi-cal risks of melanin-related adverse effects are applicable to persons with a high content of pheomelanin in the skin and hair.     

SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation

SV40 large T antigen has been reported to be modified with several different sugars including N-acetylglucosamine, galactose, and mannose. In this report we have reexamined the glycosylation of T antigen and found that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein. Surprisingly, even though [3H]galactose could be metabolically incorporated into the protein, analysis showed that all of the radioactivity in T antigen had been converted to other species. The N-acetylglucosamine was demonstrated to be linked to the protein in the form of O-linked N- acetylglucosamine, the best characterized form of nuclear and cytoplasmic glycosylation in mammalian systems. We have localized the major site of glycosylation to the amino terminal portion of the molecule. Analysis of mutated T antigen where serines 111/112 were substituted with alanine suggest that these residues constitute a glycosylation site on the protein. These two serines fall within a typical O-linked N-acetylglucosamine glycosylation site (PSS) and are also known to be phosphorylated. Thus, it is likely that competition between phosphorylation and glycosylation occurs at this site.      

In vivo measurement of flavour release from mixed phase gels

Flavour release was investigated from pure gelatin, pure agarose and mixed gelatin-agarose gels, all containing 25% sucrose and flavoured with p-cymene, ethyl butyrate, pyrazine and ethanol. Gels were characterised by optical microscopy, and rheological techniques to determine phase separation, elastic modulus and melting temperature. Volatile release was measured by monitoring the four volatiles in the expired air from one individual eating the gels, using Atmospheric Pressure Chemical Ionisation-Mass Spectrometry. The release pattern of p-cymene was not affected by gel type. The release of ethanol, ethyl butyrate and pyrazine was affected to different extents by the matrix suggesting that both the properties of the volatile and the matrix determine volatile release in vivo.     

Docking studies on novel analogues of 8 methoxy fluoroquinolones against GyrA mutants of Mycobacterium tuberculosis

Background

Modeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues.

Results

Using sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rβ and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus.

Conclusions

We succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.