bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum
Authors: Faneca H. , Simões S. , Pedroso de Lima M. C.
Published: March 2004

Background

The successful application of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Such efficacy is strongly related to key parameters including serum resistance and protection of DNA.

Methods

The complexes were tested in terms of their biological activity, in the absence or presence of serum, by following transfection activity. Interaction with plasma proteins was evaluated by immunoblotting, while cytotoxicity was assessed by the Alamar Blue assay. Extent of DNA protection was determined both by using ethidium bromide intercalation and DNase I digestion assays.

Results

Our results show that, depending on the charge ratio and on the lipid composition, albumin and protamine can be used (either individually or co‐associated) to generate cationic liposome/DNA complexes fulfilling in vivo requirements, while exhibiting high levels of transfection activity. In the present work a novel cationic lipid was tested. It was demonstrated that 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes constitute a very promising carrier for gene delivery as illustrated by their enhancing effect on transfection, as compared with DOTAP‐containing liposomes. Moreover, the biological activity of EPOPC‐containing complexes is significantly improved upon association of albumin, even in the presence of 60% serum (namely for the 4/1 lipid/DNA charge ratio). Nevertheless, our studies also show that transfection activity mediated by DOTAP‐containing complexes can be significantly enhanced upon pre‐condensation of DNA with protamine.

Conclusions

Co‐association of HSA and protamine to lipoplexes ensures a high degree of DNA protection and results in high levels of transfection activity even in the presence of serum. Copyright © 2004 John Wiley & Sons, Ltd.
Gene therapy with novel adeno‐associated virus vectors substantially diminishes atherosclerosis in a murine model of familial hypercholesterolemia
Authors: Lebherz Corinna , Gao Guangping , Louboutin Jean‐Pierre , Millar John , Rader Daniel , Wilson James M.
Published: March 2004

Background

Familial hypercholesterolemia is an inherited disease caused by mutations in the LDL receptor gene leading to severe hypercholesterolemia and atherosclerosis. The LDL receptor is predominantly expressed in the liver, making it a preferred target organ for somatic gene therapy. We recently isolated a new family of vectors based on adeno‐associated viruses (AAVs) isolated from nonhuman primates, which enable efficient and stable transgene expression following in vivo gene delivery to liver.

Methods

Traditional vectors based on AAV serotype 2 and two novel AAVs from nonhuman primates, serotypes AAV7 and AAV8, were produced encoding for the human LDL receptor. Vectors were injected into the portal veins of LDL receptor deficient mice that were fed a high‐fat diet to achieve severe pretreatment hypercholesterolemia.

Results

Animals receiving the novel AAV vectors realized nearly complete normalization of serum lipids and failed to develop the severe atherosclerosis that characterized the untreated animals; the AAV2 vector constructs demonstrated partial lipid correction and only a modest improvement in atherosclerosis.

Conclusions

Using vectors based on novel nonhuman primate AAVs, which provide advantages in terms of efficiency, we were able to achieve a long‐term correction of the metabolic defect in LDL receptor deficient mice. Copyright © 2004 John Wiley & Sons, Ltd.
Optimization of an siRNA‐expression system with an improved hairpin and its significant suppressive effects in mammalian cells
Authors: Miyagishi Makoto , Sumimoto Hidetoshi , Miyoshi Hiroyuki , Kawakami Yutaka , Taira Kazunari
Published: March 2004

Background

RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double‐stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector‐based siRNA‐expression systems that can induce RNAi in living cells.

Methods

We describe here a comparative analysis of various siRNA‐expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors.

Results

As a result of the comparative analysis, we determined the following optimized siRNA‐expression system: U6 promoter‐driven hairpin‐type dsRNA with 21‐nt stem length, three to four mutations in the sense strand only, and the optimized 9‐nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA‐expression system with a tetracycline‐regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector‐mediated transfer of an siRNA‐expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five.

Conclusion

The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi. Copyright © 2004 John Wiley & Sons, Ltd.
Enhancer‐deleted retroviral vectors restore high levels of superoxide generation in a mouse model of CGD
Authors: Schwickerath Oliver , Brouns Gaby , Thrasher Adrian , Kinnon Christine , Roes Jürgen , Casimir Colin
Published: March 2004

Background

Retroviral vectors possess many advantages for use in gene therapy protocols, especially within the haematopoietic system. A number of attendant problems, however, still limit their safety in clinical application. The effects of the enhancer present in the retroviral long terminal repeat (LTR) are a major concern for the clinical usage of such vectors, as they can exert a powerful regulatory influence on the genes that surround them.

Methods

To improve the safety and widen the applicability of retroviral vectors for use in gene therapy we have developed an enhancer‐deleted (Δ‐LTR) retroviral vector that retained high titre and demonstrated transcriptional activity in myeloid cells.

Results

When used to correct a mouse model of autosomal recessive chronic granulomatous disease, the Δ‐LTR vectors gave acceptable levels of gene transfer to mouse bone marrow cells. Evidence for a slight preferential expression in myeloid cells was obtained with all the vectors studied. Nitroblue tetrazolium assay of superoxide generation in mouse bone marrow derived haematopoietic colonies revealed that transduction with Δ‐LTR vectors could restore functional NADPH oxidase to cells from these animals. Superoxide assay of peripheral blood confirmed that, although relatively low numbers of cells were transduced, the Δ‐LTR vector was capable of reconstituting very high levels of oxidase activity, comparable to that obtained from normal cells.

Conclusions

The Δ‐LTR vector described here could provide the basis for a new generation of retroviral vectors with improved safety. Copyright © 2004 John Wiley & Sons, Ltd.
Synthesis of [uniformly ring‐14C]‐labelled 4‐hydroxybenzaldehyde, vanillin, and protocatechualdehyde
Authors: Ji Rong , Schäffer Andreas
Published: March 2004

[Uniformly ring‐14C]‐labelled 4‐hydroxybenzaldehyde, vanillin, and protocatechualdehyde were synthesized from [14C]‐labelled phenol, guaiacol, and catechol with methyl dichloromethyl sulfide (CH3SCHCl2) under Friedel–Crafts alkylation conditions in dichloromethane at −78°C for 5 min (in the case of phenol and guaiacol) or at −20°C for 1 min (in the case of catechol), by rapid addition of SnCl4 to mixtures of the phenolic compound and CH3SCHCl2, followed by hydrolysis with HCl. Regioselective formylation (para to the –OH group) was achieved. The conversion rates were 96, 81, and 88% for 4‐hydroxybenzaldehyde, vanillin, and protocatechualdehyde, respectively, and the yields of the recovered products after work‐up amounted to 88, 75, and 83%, respectively. In the case of guaiacol, 17% of isovanillin was obtained as by‐product. It was found that the presence of water or ethyl acetate in the reaction mixture, at a molar ratio of 60:1 (water:guaiacol) or 120:1 (ethyl acetate:guaiacol), had little influence on the yields under the reaction conditions. Factors influencing the yields are discussed in the study. Copyright © 2004 John Wiley & Sons, Ltd.
Substitution‐reduction: an alternative process for the [18F]N‐(2‐fluoroethylation) of anilines
Authors: Briard Emmanuelle , Pike Victor W.
Published: March 2004

Substitution of a halo atom (chloro or bromo) in easily prepared N‐haloacetyl‐anilines with no‐carrier added (NCA) cyclotron‐produced [18F]fluoride ion (18F, t1/2= 109.8 min; β+=96.9%), followed by reduction with borane–tetrahydrofuran (BH3–THF), provides an alternative route to NCA [18F]N‐(2‐fluoroethyl)‐anilines. This two‐step and one‐pot process is rapid (∼50 min) and moderately high yielding (∼40% decay‐corrected radiochemical yield (RCY) overall). In the nucleophilic substitution reaction, 18‐crown‐6 is preferred to Kryptofix® 222 as complexing agent for the solubilization of the counter‐ion (K+), derived from an added metal salt, in acetonitrile. Weakly basic potassium bicarbonate is preferred as the added metal salt. Inclusion of a small amount of water, equating to 4–5 molar equivalents relative to 18‐crown‐6, base or precursor (held in equimolar ratio), is beneficial in preventing the adsorption of radioactivity onto the wall of the glass reaction vessel and for achieving high RCY in the nucleophilic substitution reaction. BH3–THF is effective for the rapid reduction of the generated [18F]N‐fluoroacetyl‐aniline to the [18F]N‐(2‐fluoroethyl)‐aniline. Copyright © 2004 John Wiley & Sons, Ltd.
Dibenzocyclooctadiene lignans from Schisandra chinensis protect primary cultures of rat cortical cells from glutamate‐induced toxicity
Authors: Kim So Ra , Lee Mi Kyeong , Koo Kyung Ah , Kim Seung Hyun , Sung Sang Hyun , Lee Na Gyong , Markelonis George J. , Oh Tae H. , Yang Jae Ho , Kim Young Choong
Published: March 2004

A methanolic extract of dried Schisandra fruit (Schisandra chinensis Baill.; Schisandraceae) significantly attenuated the neurotoxicity induced by L‐glutamate in primary cultures of rat cortical cells. Five dibenzocyclooctadiene lignans (deoxyschisandrin, gomisin N, gomisin A, schisandrin, and wuweizisu C) were isolated from the methanolic extract; their protective effects against glutamate‐induced neurotoxicity were then evaluated. Among the five lignans, deoxyschisandrin, gomisin N, and wuweizisu C significantly attenuated glutamate‐induced neurotoxicity as measured by 1) an inhibition in the increase of intracellular [Ca2+]; 2) an improvement in the glutathione defense system, the level of glutathione, and the activity of glutathione peroxidase; and 3) an inhibition in the formation of cellular peroxide. These results suggest that dibenzocyclooctadiene lignans from Schisandra chinensis may possess therapeutic potential against oxidative neuronal damage induced by excitotoxin. © 2004 Wiley‐Liss, Inc.
Characteristics of fruit and oil of terebinth (Pistacia terebinthus L) growing wild in Turkey
Authors: Özcan M
Published: March 2004

Nutrients and physical properties were determined in terebinth (Pistacia terebinthus L) fruits from İçel, Turkey to investigate their potential uses. Ripe fruits were evaluated for moisture, crude protein, crude oil, crude fibre, crude energy, ash, HCl‐insoluble ash, essential oil yield, dimethyl sulphite, weight and width/length ratio. Relative density, refractive index, free fatty acids, peroxide value, iodine value, saponification number, unsaponifiable matter and carotenoid content were determined in the fruit oil. The main fatty acids identified by gas chromatography were oleic (52.3%), palmitic (21.3%) and linoleic (19.7%) acids. Contents of Na, K, P, Ca, Fe, Mg, Zn, Cu, Mn, Li, Ni, Pb, S, Se, Cd, Co, Cr, Sr, Ti, V, Ag, Al, As, B, Ba and Bi were also determined in the fruits. The fruits were found to be rich in protein, oil, fibre, unsaturated fatty acids and minerals, suggesting that they may be valuable for food uses. The data may also be useful for the evaluation of nutritional information. Copyright © 2004 Society of Chemical Industry
Ensilage as a means of reducing the concentration of cyanogenic glycosides in the pods of Acacia sieberiana and the effect of additives on silage quality
Authors: Ngwa Thomas A , Nsahlai Ignatius V , Iji Paul A
Published: March 2004

Cyanogenic glycosides are anti‐nutritional components capable of liberating hydrogen cyanide, a respiratory poison that has been reported to be responsible for the death of livestock that browse some Acacia species containing these compounds. The objective of the study was to improve the nutritive value of the pods of Acacia sieberiana by reducing their contents of cyanogenic glycosides through ensiling, and to examine the influence of various levels of added molasses and urea on the aerobic stability of the silage. In experiment 1, laboratory silages, prepared by mixing ground pods with water in two ratios (60:40 and 40:60 w/w pod material:water), were put into 250 ml polythene bottles, ensiled for 4, 7, 14, 21, 28 or 35 days and analyzed for cyanide. In experiment 2, silages prepared as described above but using only one of the ratios (40:60), and with the addition of molasses (0, 30 or 60 g kg−1) and urea (0, 2.5 or 50 g kg−1) were included in a 3 × 3 factorial design, to study the effects of these additives on the stability of the silage. The period of ensilage was 14 days. In experiment 3, the concentrations of molasses and urea were modified to 0, 45 or 90 g kg−1 and 0, 7.5 or 15 g kg−1, respectively, and the period of ensilage was extended to 45 days. The parent material contained 130.6 mg CN kg−1 DM and ensiling the material for 35 days reduced the concentration to 18.1 mg CN kg−1 DM. Moisture content at ensiling significantly (P < 0.001) affected the concentration of cyanide in the stored silages. Silages opened after 14 days were unstable irrespective of the use of additives. However those with combined urea and molasses exhibited a slow rate of deterioration and low fungal counts. Extending the period of ensiling to 45 days improved the aerobic stability of all the silages. The addition of urea significantly (P < 0.001) increased the pH, crude protein content, gross energy and fermentation acids while molasses treatment increased (P < 0.001) the lactic acid and ash content of the silages. Ensiling ground pods for 45 days was enough to reduce the cyanide content to non‐toxic levels and produced a silage which was aerobically stable, while the inclusion of additives further improved the quality of the silages. Copyright © 2004 Society of Chemical Industry
Off‐flavour development in soybeans: comparative role of some antioxidants and related enzymes
Authors: Dahuja Anil , Madaan Tilak R
Published: March 2004

Off‐flavour development is the major impediment in the effective use of soybeans as food ingredients. In the present study, the levels of vitamin E and vitamin C along with those of antioxidant enzymes, like superoxide dismutase, peroxidase and catalase, were estimated in sixteen soybean varieties to explore their effectiveness against off‐flavour generation. Thiobarbituric acid (TBA) number, an indicator of lipid peroxidation, was used to measure the quality with respect to off‐flavour. Data on activities of various antioxidant enzymes indicated that the off‐flavour is mainly generated enzymatically by the action of lipoxygenase on polyunsaturated fatty acids. Furthermore, varieties PK 416 and JS 7105 had the highest level of vitamin E while their TBA number was the lowest indicating the inverse relationship between the two parameters. It was further observed that the peroxidative effect of higher vitamin C concentrations is controlled by vitamin E. Among the varieties analyzed, PK 416 and JS 7105 appeared to have greater potential as most acceptable food ingredients. Copyright © 2004 Society of Chemical Industry
Effect of flaxseed processing on its true metabolizable energy values for adult chicken
Authors: Shen Yingran , Feng Dingyuan , Chavez Eduardo R
Published: March 2004

The objective of the present experiment was to study the effect of flaxseed processing on nitrogen corrected true metabolizable energy (TMEn) values for adult roosters. Flaxseed was processed as pelleted, autoclaved or microwave roasted. Leghorn roosters were used for the TME determination procedure. The TMEn value of flaxseed batch A, 14.48 MJ kg−1 DM, was significantly (P < 0.05) increased to 17.89 MJ kg−1 DM by three‐time repeat‐pelleting, or to 18.07 MJ kg−1 dry matter (DM) by autoclaving, respectively. Microwave roasting also significantly (P < 0.05) increased the TMEn value of flaxseed batch B by 22%. The TMEn improvement observed due to processing was accompanied by increased ether extract utilization. The apparent ether extract digestibility of flaxseed batch A, 61.2%, was very significantly (P < 0.01) increased to 81.5 and 83.2% by processing as repeat‐pelleting and autoclaving, respectively. Microwave roasting also significantly (P < 0.05) increased the apparent digestibility of ether extract for flaxseed batch B from 49.1 to 64.4%. Proper flaxseed processing as pelleting, autoclaving and microwave roasting led to higher TMEn values for Leghorn roosters, mainly as the result of increased ether extract utilization. Copyright © 2004 Society of Chemical Industry
Effects of stage II and postharvest deficit irrigation on peach quality during maturation and after cold storage
Authors: Gelly M , Recasens I , Girona J , Mata M , Arbones A , Rufat J , Marsal J
Published: March 2004

Effects of regulated deficit irrigation (RDI) on the quality of a mid–late maturing peach (Prunus persica L Batsch cv Andross) were determined at commercial harvest as well as 1 week before (early pick‐up) and after 1 week of cold storage. Respiration and ethylene evolution were measured during 96 h on fruits sampled at early pick‐up and at harvest. Fruits from a well‐watered irrigation treatment (Control) were compared with a treatment of RDI during stage II of fruit growth (RDI‐SII) and with RDI applied postharvest (RDI‐PH). A fourth treatment received a combination of deficit irrigation applied during stage II and postharvest (RDI‐SII‐PH). Higher soluble solids content (SSC) amd SSC/titratable acidity (TA) ratio for RDI‐SII were already noted at early pick‐up and these differences were enhanced after cold storage (RDI fruits having higher SSC, lower TA and higher SSC/TA ratio than Control fruits). This improvement in quality (high SSC/TA ratio) was also correlated with a more reddish colouration in the fruit skin. Moreover, ethylene production indicated different behaviour of the physiological evolution for fruits from the different treatments. Copyright © 2004 Society of Chemical Industry
Application of a polymerase chain reaction (PCR) method as a complementary tool to microscopic analysis for the detection of bones and other animal tissues in home‐made animal meals
Authors: Prado Marta , Casqueiro José , Iglesias Yolanda , Cepeda Alberto , Barros‐Velázquez Jorge
Published: March 2004

A polymerase chain reaction (PCR) method was compared with a variation of the official microscopic technique (Directive 98/88/EC) for the detection in animal meals of cereals (wheat and corn) and animal parts (bone, feathers, meat, liver, fat and blood). Microscopy successfully detected animal bones in raw feeds with a sensitivity of 1 g kg−1, while the sensitivity of the PCR method was in the range of 5–10 g kg−1. Microscopy also allowed the detection of animal bones and feathers in feeds processed at 115 and 133 °C but failed to detect other animal materials. The PCR method successfully detected cereals (wheat and corn) as well as meat, bone, liver, fat and feathers after processing at 115 °C for 20 min. Heating at 133 °C under overpressure (autoclave) conditions resulted in more intense DNA fragmentation and lower DNA extractability. Nevertheless, bone and liver, as well as wheat and corn in home‐made animal meals, were successfully detected even after heating at 133 °C for 20 min. Copyright © 2004 Society of Chemical Industry
The effect of germination on seed trypsin inhibitors in Vicia faba and Cicer arietinum
Authors: Muzquiz Mercedes , Welham Tracey , Altares Pilar , Goyoaga Carmen , Cuadrado Carmen , Romero Concepción , Guillamon Eva , Domoney Claire
Published: March 2004

During germination, trypsin inhibitor (TI) content increased relative to total protein and dry weight in cotyledons from two varieties of Vicia faba. These data, together with changes in total protein profiles and amounts, suggest that TI proteins are not hydrolysed in parallel with other seed proteins and dry matter. In contrast, TI content in germinating Cicer arietinum seeds remained constant during the germination period; in this case, however, substantial changes in the total protein profile were not observed. TI isoform analyses indicated that compositional changes occurred during germination in both species. Copyright © 2004 Society of Chemical Industry
Yield and biosynthesis of nitrogenous compounds in fruits of green bean (Phaseolus vulgaris L cv Strike) in response to increasing N fertilisation
Authors: Sánchez Esteban , Rivero Rosa M , Ruiz Juan M , Romero Luis
Published: March 2004

The objective of the present work was to determine the effect of different dosages of N on the biosynthesis of organic N compounds in fruits and their influence on yield in green bean (Phaseolus vulgaris L cv Strike) plants. The nitrogen was applied to the nutrient solution as NH4NO3 at 1.5 (N1), 3 (N2), 6 (N3), 12 (N4), 18 (N5) and 24 mM (N6). Treatment N3 was considered optimal for efficient yield in green bean plants and also for the null presence of NO3−. Highest N dosages (18 and 24 mM) resulted in the accumulation of NO3− in pods and seeds. This accumulation encouraged nitrate reductase (NR) and nitrite reductase (NiR) activity in both tissues and treatments, and therefore NR activity might be considered as a good bioindicator of the presence of NO3− in edible fruits. The greater NH4+ assimilation by glutamine synthetase (GS) and glutamate synthase (GOGAT) occurred primarily in the pods of the N6 treatment, while the seeds acted as physiological sinks, these latter tissues presenting the highest concentrations of amino acids, proteins and organic N. The high accumulation of NO3− and NH4+ in both seeds and pods could be the direct cause of the reduction in fruit production, indicating that green bean plants are very sensitive to high N levels. Copyright © 2004 Society of Chemical Industry
Quantification of naphthalenes in jet fuel with GC×GC/Tri‐PLS and windowed rank minimization retention time alignment
Authors: Johnson Kevin J. , Prazen Bryan J. , Young Donald C. , Synovec Robert E.
Published: March 2004

Comprehensive, two‐dimensional gas chromatography (GC×GC) is used in conjunction with trilinear partial least squares (Tri‐PLS) to quantify the percent weight of naphthalenes (two‐ring aromatic compounds) in jet fuel samples. The increased peak capacity and selectivity of GC×GC makes the technique attractive for the rapid, and possibly less tedious analysis of jet fuel. The analysis of complex mixtures by GC×GC is further enhanced through the use of chemometric techniques, including those designed for use on 2‐D data such as Tri‐PLS. Unfortunately, retention time variation, unless corrected, can be an impediment to chemometric analysis. Previous work has demonstrated that the effects of retention time variation can be mitigated in sub‐regions of GC×GC chromatograms through the application of an objective retention time alignment algorithm based on rank minimization. Building upon this previous work, it is demonstrated here that the effects of retention time variation can be mitigated throughout an entire GC×GC chromatogram with an objective retention time alignment algorithm based on windowed rank minimization alignment. A significant decrease in calibration error is observed when the algorithm is applied to chromatograms prior to construction of Tri‐PLS models. Fourteen jet fuel samples with known weight percentages of naphthalenes (ASTM D1840) were obtained. Each sample was subjected to five replicate five‐minute GC×GC separations over a period of two days. A subset of nine samples spanning the range of weight percentages of naphthalenes was chosen as a calibration set and Tri‐PLS calibration models were subsequently developed in order to predict the naphthalene content of the samples from the GC×GC chromatograms of the remaining five samples. Calibration models constructed from GC×GC chromatograms that were retention time corrected are shown to exhibit a root mean square error of prediction of roughly half that of calibration models constructed from uncorrected chromatograms. The error of prediction is lowered further to a value that nearly matches the uncertainty in the standard percent weight values (ca. 1% of the median percent volume value) when the aligned chromatograms are truncated to include only regions of the chromatogram populated by naphthalenes and compounds of similar polarity and boiling point.
Assessment by Monte Carlo simulation of thermodynamic correlation of retention times in dual‐column temperature programmed comprehensive two‐dimensional gas chromatography
Authors: Davis Joe M.
Published: March 2004

The correlation of retention times in comprehensive two‐dimensional gas chromatography caused by correlation of enthalpy and entropy changes between two stationary phases, methylsilicone and poly(ethylene glycol), was examined using commercial GC software and in‐house Monte Carlo simulation. The enthalpy change, ΔH 0, and entropy change, ΔS 0, of 93 compounds were predicted from isothermal one‐dimensional gas chromatograms predicted by the software. These values then were mimicked by Monte Carlo simulation, which removed the strong correlation of ΔH 0 and modest correlation of ΔS 0 between the two phases. Retention times in a comprehensive two‐dimensional gas chromatogram (GC×GC) and in simulations of it were predicted for typical dual‐capillary temperature‐programmed conditions using the actual, correlated values of ΔH 0 and ΔS 0 and their uncorrelated Monte Carlo counterparts, respectively. The uncorrelated ΔH 0 and ΔS 0 values caused the retention‐time range of the simulations’ second dimension to expand substantially beyond that in the GC×GC. Other simulations were developed using a restricted range of uncorrelated ΔH 0 and ΔS 0 values to mimic more closely the retention‐time range of the GC×GC's second dimension. The intervals between nearest neighbor retention‐time coordinates were calculated in both the latter simulations and the GC×GC. The intervals were larger in the simulations than in the GC×GC because the former contained uncorrelated coordinates and the latter contained correlated ones, which clustered along or near the diagonal. The retention times in the first dimension of the GC×GC were Poisson distributed, as assessed by statistical‐overlap theory. In contrast, the two‐dimensional reduced retention‐time coordinates in the GC×GC were not Poisson distributed, because retention times were correlated. However, the reduced retention‐time coordinates in the simulations were Poisson distributed.
Speciation of nitrogen‐containing compounds in diesel fuel by comprehensive two‐dimensional gas chromatography
Authors: Wang Frank Cheng‐Yu , Robbins Winston K. , Greaney Mark A.
Published: March 2004

Nitrogen‐containing compounds in diesel fuel have been speciated by comprehensive two‐dimensional gas chromatography with nitrogen chemiluminescence detector (GC×GC‐NCD). The speciation of nitrogen‐containing compounds in diesel is difficult because of low concentration and complexity. The advantages of GC×GC are improved resolution and enhanced sensitivity. GC×GC‐NCD can achieve the type and class separation of nitrogen‐containing compounds with an appropriate separation column combination. Diesel contains both neutral (indoles and carbazoles) and basic (pyridines and quinolines) nitrogen‐containing compounds. Relative concentrations of each class as well as each carbon number family can be quantified by integrating their peak volumes. This study demonstrates the capability of GC×GC‐NCD for speciation of nitrogen‐containing compound classes.
Temperature requirements for thermal modulation in comprehensive two‐dimensional gas chromatography
Authors: Gaines Richard B. , Frysinger Glenn S.
Published: March 2004

Temperature requirements for trapping and release of compounds in a cryogenic gas loop‐type GC×GC modulator were determined. Maximum trapping temperatures on the uncoated, deactivated modulator capillary were determined for compounds from C4 (bp –0.5°C) to C40 (bp 522°C). The liquid‐nitrogen cooled gas flow rate was reduced from a high of 15.5 to 1.5 SLPM over the range to achieve the required trapping temperature. Excessive cold jet flow rates caused irreversible trapping and peak tailing for semi‐volatile compounds above C26. Alternate cold jet coolants were investigated. An ice water‐cooled jet was able to trap compounds with boiling points from C18 (bp 316°C) to C40 and a room temperature air‐cooled jet was able to trap compounds from C20 (bp 344°C) to C40. The hot jet produced launch temperatures approximately 40°C hotter than the elution temperature with heating time constants of 8 to 27 ms. Modulated compound peaks were symmetrical with half‐height peak widths of 43 to 56 ms for compounds with little second column retention, and 70 to 75 ms for compounds with more second column retention. The liquid nitrogen‐cooled loop modulator with gas flow programming was used to produce a GC×GC chromatogram for a crude oil that contained compounds from C7 to C47.
Application of comprehensive multidimensional gas chromatography combined with time‐of‐flight mass spectrometry (GC×GC‐TOFMS) for high resolution analysis of hop essential oil
Authors: Roberts Mark T. , Dufour Jean‐Pierre , Lewis Alastair C.
Published: March 2004

The selection and quality of hops is a major determinant in beer flavour. Brewers acknowledge that distinctive characteristics of different hop varieties can be traced to the composition of their essential oils. The difficulty in characterising complex mixtures such as hop oil using 1‐D chromatography is that many compounds co‐elute. With the introduction of comprehensive multidimensional capillary gas chromatography (GC×GC), there is a tremendous improvement in the separation power or peak capacity. Recent work using GC×GC with flame ionisation detection has suggested that there may be over 1,000 compounds in hop oil. This work describes the use of GC×GC combined with TOFMS detection (Leco Pegasus 4D instrument) to analyse Target hop oil. The TOFMS spectral acquisition rate of 60 Hz provided sufficient spectra per peak (2‐D peak base width of 0.1–0.2 s) for identification (119 components were identified with 45 previously unreported compounds). When analysing results, an advantage of GC×GC coupled to TOFMS is that 2‐D chromatograms can be viewed for individual masses that are characteristic of particular functional groups. This allows the analyst to view the various homologous series of compounds although in certain cases coelution may still be present as shown by the esters with mass 75.
Comprehensive two‐dimensional gas chromatography in the analysis of dietary fatty acids
Authors: Hyötyläinen Tuulia , Kallio Minna , Lehtonen Mari , Lintonen Stella , Peräjoki Päivi , Jussila Matti , Riekkola Marja‐Liisa
Published: March 2004

Comprehensive two‐dimensional gas chromatography utilising a semi‐rotating cryogenic modulator was applied to the analysis of dietary milk derived fatty acids. Four column combinations were tested: two polar‐nonpolar and two different nonpolar‐polar column combinations. Best results were obtained with a nonpolar‐polar column set, with narrow‐bore (50 μm ID) Carbowax column as the second column. The GC×GC method clearly improved the separation efficiency of the fatty acids in comparison with one‐dimensional GC analysis and proved to be suitable for the quantification of the analytes. The relative standard deviations (RSD) of the retention times in the first and second dimensions were on average 0.06% and 4.9%, respectively. The relative RSD for peak volumes was on average 2.9%.
Comonomer‐Unit Compositions, Physical Properties and Biodegradability of Bacterial Copolyhydroxyalkanoates
Authors: Feng Lidan , Yoshie Naoko , Asakawa Naoki , Inoue Yoshio
Published: March 2004

Summary: The comonomer‐unit compositions and their distribution of as‐produced bacterial copolyesters, including poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate), poly(3‐hydroxy‐butyrate‐co‐3‐hydroxypropionate), poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) and poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) are described in this paper. Each copolyester sample can be comonomer‐unit compositionally fractionated into several fractions, indicating that the original copolymers are mixtures of copolymers with different comonomer‐unit compositions. The effects of comonomer‐unit compositional distribution on thermal properties, crystallization, biodegradability and solid‐state phase behavior are investigated using comonomer compositionally fractionated copolymers.

The trend shown by the Tm values of the copolymers, P(3HB‐co‐3HV), P(3HB‐co‐3HP), P(3HB‐co‐3HH) and P(3HB‐co‐4HB), plotted against the content of the second monomer unit.

image

The trend shown by the Tm values of the copolymers, P(3HB‐co‐3HV), P(3HB‐co‐3HP), P(3HB‐co‐3HH) and P(3HB‐co‐4HB), plotted against the content of the second monomer unit.
Structure and Structural Changes of the Silk Fibroin from Samia cynthia ricini Using Nuclear Magnetic Resonance Spectroscopy
Authors: Asakura Tetsuo , Nakazawa Yasumoto
Published: March 2004

Summary: The structure of silk fibroin from a wild silkworm, S. c. ricini, the amino acid sequence of which consists of repeated poly‐Ala and Gly‐rich regions, was examined by using solution and solid‐state NMR methods. The structural transition of the silk fibroin in aqueous solution was monitored by using 13C solution NMR spectroscopy as a function of temperature. The fast exchange with respect to the chemical shift between the helix and coil conformations was observed in the poly‐Ala region and the slow conformational change from α‐helix to random coil was observed for the Gly residue adjacent to the N‐terminal Ala residue of the poly‐Ala region. The torsion angles of several Ala and Gly residues in the model peptide, GGAGGGYGGDGG(A)12GGA‐GDGYGAG, were determined by the conformation‐dependent 13C chemical shifts, rotational echo double resonance (REDOR) and 2D spin‐diffusion NMR methods. The solid‐state NMR analysis leads to the precise silk structure before spinning, where the poly‐Ala sequence takes a typical α‐helix pattern with a tightly winded helical structure at both terminal regions of the poly‐Ala sequence. This is expected to stabilize the α‐helical structure of the poly‐Ala region in S. c. ricini silk fibroin from the silkworm.

The proposed helical structure of poly(L‐alanine) region of S. c. ricini silk fibroin before spinning.

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The proposed helical structure of poly(L‐alanine) region of S. c. ricini silk fibroin before spinning.
Degradable Polyesters through Chain Linking for Packaging and Biomedical Applications
Authors: Seppälä Jukka V. , Helminen Antti O. , Korhonen Harri
Published: March 2004

Summary: The major route to convert lactic acid to high‐molecular‐weight polymers is ring‐opening polymerization of lactide. We have investigated alternative synthesis routes based on oligomerization and chain linking to produce high‐molecular‐weight thermoplastic degradable polymers cost‐effectively. Chain linking also offers new possibilities to prepare degradable polyesters for biomedical applications by extending the range of polymer properties achievable. In this paper, we briefly review different chain linking techniques used in our laboratory. Typically, lactic acid prepolymers with molecular weights of around 3 000–15 000 g · mol−1 have been prepared by direct polycondensation. Hydroxyl terminated oligomers have been chain linked by using diisocyanate coupling agents, preferably 1,4‐butane diisocyanate, forming poly(ester‐urethanes). Poly(ester‐amides) have been prepared by using 2,2′‐bis(2‐oxazoline) as coupling agent for carboxylic acid telechelic oligomers. Chain linking by end functionalization has been used in the preparation of poly(ester‐anhydrides). In addition, a variety of crosslinked degradable polymers and copolymers have been synthesized through different crosslinking routes, by using methacrylic, itaconic or maleic double bonds or triethoxysilane moieties. A biodegradation test and ecotoxicological evaluation of the degradation products were carried out in addition to hydrolysis tests. Lactic acid based chain linked polymers were biodegradable and the degradation products were harmless. In hydrolysis tests, enzymatic degradation was pronounced in the chain linked poly(ε‐caprolactone).

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Application of proteomics technologies in the investigation of the brain
Authors: Fountoulakis Michael
Published: March 2004

I.
Introduction
232

 II.
Proteomic Analysis
233

A.  Sample Preparation
233

B.  Two‐Dimensional (2‐D) Electrophoresis
234

C.  Protein Quantification
235

D.  Mass Spectrometry (MS)
236

    1.  Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectroscopy (MALDI‐TOF‐MS)
236

    2.  Tandem MS
237

  III.
2‐D Brain Protein Databases
238

A.  Database Construction
238

B.  Subcellular Location
240

C.  Frequency of Detection
240

D.  Protein Function
242

  IV.
Alterations in the Protein Levels
243

A.  Adult Brain
243

B.  Fetal Brain
245

  V.
Proteomic Studies on Rat Brain
245

A.  Differences Between Neonatal and Adult Brain
245

B.  Post‐Mortem Changes
248

C.  Toxicology Studies
248

  VI.
Limitations
248

A.  Brain Samples
248

B.  Protein Detection in 2‐D Gels
249

    1.  Low‐Abundance Proteins
249

    2.  Hydrophobic Proteins
250

    3.  Acidic and Basic Proteins
251

    4.  Low‐ and High‐Molecular Mass Proteins
251

    5.  Protein Heterogeneity
251

C.  Protein Indentification
252

 VII.
Perspectives
253

 VIII.
Conclusions
253

Acknowledgments
254

References
254

Approximately 30–50% of the genes in mammals are expressed in the nervous system. A differential expression of genes in distinct patterns is necessary for the generation of the large variety of neuronal phenotypes. Proteomic analysis of brain compartments may be useful to understand the complexity, to investigate disorders of the central nervous system, and to search for corresponding early markers. Up to now, proteomics has mainly studied the identity and levels of the abundant human, rat, and mouse brain proteins as well as changes of their levels and the modifications that result from various neurological disorders, like Alzheimer's disease and Down's syndrome in humans and in animal models of those diseases. The proteins, for which altered levels in these disorders have been observed, exert mainly neurotransmission, guidance, and signal‐transduction functions, or are involved in detoxification, metabolism, and conformational changes. Some of those proteins may be potential drug targets. Further improvement of proteomics technologies to increase sensitivity and efficiency of detection of certain protein classes is necessary for a more detailed analysis of the brain proteome. In this review, a description of the proteomics technologies applied in the investigation of the brain, the major findings that resulted from their application, and the potential and limitations of the current technologies are discussed. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 23:231–258, 2004
INK4a‐ARF mutations in skin carcinomas from UV irradiated hairless mice
Authors: Soufir N. , Queille S. , Mollier K. , Roux E. , Sarasin A. , de Gruijl F.R. , Fourtanier A.M. , Daya‐Grosjean L. , Basset‐Seguin N.
Published: March 2004

To characterize further the role of the INK4a‐ARF locus in the multistep process of skin carcinogenesis, we performed a mutational analysis of this locus in skin lesions from hairless mice either irradiated with UVB alone or with a solar simulator delivering UVA + B. INK4a‐ARF mutations were present in five of 57 squamous cell carcinomas (9%), but no mutation was detected in precancerous lesions. All mutations were C:G > T:A transitions located at dipyrimidic sites, the hallmark of UVB mutagenesis. Three mutations affected only the p19ARF reading frame, whereas two mutations affected only the p16INK4a transcript. This study demonstrates for the first time UV‐induced mutations of INK4a‐ARF that occur in a small percentage in late stages skin tumors. © 2004 Wiley‐Liss, Inc.
LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane
Authors: Ota Takahide , Maeda Masayo , Suto Shiho , Tatsuka Masaaki
Published: March 2004

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras‐transformed 1‐1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C‐terminal truncated form of LyGDI (ΔC‐LyGDI, 166–201 deletion), a member of the RhoGDIs. The stable expression of ΔC‐LyGDI induced pulmonary metastasis in 1‐1ras1000 cells, whereas expression of full‐length LyGDI did not induce metastasis. ΔC‐LyGDI was preferentially localized in the membrane, detected in a NP‐40‐insoluble fraction, and co‐purified with radixin, moesin, Rac1, Cdc42, and RhoA. In ΔC‐LyGDI transfectant, an activation state of Rac1 was elevated and ΔC‐LyGDI was associated with Rac1‐GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1‐GTP, ΔC‐LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis. © 2004 Wiley‐Liss, Inc.
Advances in molecular carcinogenesis: current and future use of mouse models to screen and validate molecularly targeted anticancer drugs
Authors: Frijhoff Anita F.W. , Conti Claudio J. , Senderowicz Adrian M.
Published: March 2004

Survival of patients with advanced solid tumors has not significantly improved over the past 30 years. Although molecularly targeted anticancer drugs offer promise, few drugs make it through the end of the Food and Drug Administration approval process. Animal models that more closely resemble human carcinogenesis may bridge the gap between preclinical success and benefits for patients. We discuss pros and cons of several mouse models, including genetically engineered mice that each represent different aspects of human cancer, and the screening of targeted drugs in these models. Published 2004 Wiley‐Liss, Inc.
Chediak‐Higashi syndrome with parkinsonism
Authors: Silveira‐Moriyama Laura , Moriyama Tais S. , Gabbi Tatiana V.B. , Ranvaud Ronald , Barbosa Egberto R.
Published: March 2004

Chediak‐Higashi syndrome (CHS), typically presents with partial albinism and severe hematological abnormalities. About 10% of the patients have a mild adult form associated with various neurological manifestations. We describe the case of a 24‐year‐old woman with parkinsonism that responded well to antiparkinsonian drugs. © 2003 Movement Disorder Society
Imaging of activated microglia with PET and [11C]PK 11195 in corticobasal degeneration
Authors: Henkel Karsten , Karitzky Jochen , Schmid Michaela , Mader Irina , Glatting Gerhard , Unger Jürgen W. , Neumaier Bernd , Ludolph Albert C. , Reske Sven N. , Landwehrmeyer G. Bernhard
Published: March 2004

Positron emission tomography (PET) using [11C]PK 11195, a ligand for peripheral benzodiazepine receptor binding sites, offers the opportunity to image activated microglia in vivo. This tool may therefore be used to display the occurrence of microglial activation in the course of neurodegeneration. A patient with the clinical diagnosis of corticobasal degeneration (CBD) and left‐sided symptoms was studied using fluorodeoxyglucose (FDG) and [11C]PK 11195 PET. We found a marked right hemispheric hypometabolism and asymmetric microglial activation in corresponding areas of the basal ganglia and right temporal and parietal cortex. [11C]PK 11195 PET suggests involvement of microglial activation in the pathogenesis of CBD. © 2004 Movement Disorder Society
Punding in Parkinson's disease: Its relation to the dopamine dysregulation syndrome
Authors: Evans Andrew H. , Katzenschlager Regina , Paviour Dominic , O'Sullivan John D. , Appel Silke , Lawrence Andrew D. , Lees Andrew J.
Published: March 2004

Punding is a term that was coined originally to describe complex prolonged, purposeless, and stereotyped behaviour in chronic amphetamine users. A structured interview of 50 patients with higher dopamine replacement therapy requirements (>800 levodopa equivalent units/day) from 123 unselected patients with Parkinson's disease (PD) from a PD clinic identified 17 (14%) patients with punding. Punding was acknowledged as disruptive and unproductive by the patients themselves, but forcible attempts by family to interrupt the behaviour led to irritability and dysphoria. Punding was associated with very high doses of dopamine replacement therapy often related to a pattern of chronic inappropriate overuse of dopaminergic medication. We believe that this is an underreported, socially disabling phenomenon that is commonly associated with the syndrome of dopamine dysregulation and is phenomenologically distinct from both obsessive‐compulsive disorder and mania. © 2004 Movement Disorder Society
Chorea‐acanthocytosis associated with tourettism
Authors: Saiki Shinji , Hirose Genjiro , Sakai Koichiro , Matsunari Ichiro , Higashi Kotaro , Saiki Misuzu , Kataoka Satoshi , Hori Ariyuki , Shimazaki Kohei
Published: March 2004

We report on a case of Chorea‐acanthocytosis (ChAc) in association with Tourettism that consisted of motor and vocal tics, attention deficit–hyperactivity disorder, and obsessive–compulsive disorder in addition to the typical symptoms of ChAc. The subject was compared with his elder sister who had the same disease but milder clinical profile and neuroradiological findings. The [18F]‐2‐fluoro‐2‐deoxyglucose positron emission tomography (FDG‐PET) findings did not explain the differences in symptomatology between the patient and his sister, although they may have correlated with severity. © 2004 Movement Disorder Society