bluuebunny/crossref_metadata_2025_split huggingface dataset:
Origin of the conformation dependence of protein charge‐state distributions in electrospray ionization mass spectrometry
Authors: Grandori Rita 
Published: January 2003

AbstractThe influence of tertiary structure on the electrospray ionization mass spectra of proteins is a well known and broadly exploited phenomenon. However, the underlying mechanism is not well understood. This paper discusses the bases and the implications of the two current hypotheses (solvent accessibility and Coulombic repulsions), pointing out the remaining open questions. Evidence reported here supports a third hypothesis, i.e. that intramolecular interactions in folded proteins play a key role in determining the observed charge‐state distributions. It is proposed that native protein structures stabilize to a large extent pre‐existing charges of the opposite polarity to the net charge of the ion, preventing their neutralization during the electrospray process. Thus, the higher charge states of unfolded conformations, relative to the folded structure, would not derive from a more extensive ionization of the former, but rather from a higher content of neutralizing charges in the latter. This interpretation allows several other problematic observations to be explained, including the different shapes of the spectra of folded and unfolded proteins, the discrepancies between observed and predicted gas‐phase reactivity of protein ions and the apparent inconsistency of positive‐ and negative‐ion mode results. Copyright © 2003 John Wiley & Sons, Ltd.
Feasibility of a liquid‐phase microextraction sample clean‐up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples
Authors: Kuuranne Tiia , Kotiaho Tapio , Pedersen‐Bjergaard Stig , Einar Rasmussen Knut , Leinonen Antti , Westwood Steven , Kostiainen Risto 
Published: January 2003

AbstractAnabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid‐phase microextraction (LPME) sample clean‐up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid–liquid extraction (LLE) and solid‐phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium‐labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean‐up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2–20 ng ml−1 for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses. Copyright © 2003 John Wiley & Sons, Ltd.
Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao)
Authors: Sánchez‐Rabaneda Ferran , Jáuregui Olga , Casals Isidre , Andrés‐Lacueva Cristina , Izquierdo‐Pulido Maria , Lamuela‐Raventós Rosa M. 
Published: January 2003

AbstractLiquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection was used for the identification of a variety of phenolic compounds in a cocoa sample. Gradient elution with water and acetonitrile, both containing 0.1% HCOOH, was used. Standard solutions of 31 phenolic compounds, including benzoic and cinnamic acids and flavonoid compounds, were studied in the negative ion mode using MS/MS product ion scans. At low collisional activation, the deprotonated molecule [M − H]− was observed for all the compounds studied. For cinnamic and benzoic acids, losses of CO2 or formation of [M − CH3]−· in the case of methoxylated compounds were observed. However, for flavonol and flavone glycosides, the spectra present both the deprotonated molecule [M − H]− of the glycoside and the ion corresponding to the deprotonated aglycone [A − H]−. The latter ion is formed by loss of the rhamnose, glucose, galactose or arabinose residue from the glycosides. Different fragmentation patterns were observed in MS/MS experiments for flavone‐C‐glycosides which showed fragmentation in the sugar part. Fragmentation of aglycones provided characteristic ions for each family of flavonoids. The optimum LC/MS/MS conditions were applied to the characterization of a cocoa sample that had been subjected to an extraction/clean‐up procedure which involved chromatography on Sephadex LH20 and thin‐layer chromatographic monitoring. In addition to compounds described in the literature, such as epicatechin and catechin, quercetin, isoquercitrin (quercetin‐3‐O‐glucoside) and quercetin‐3‐O‐arabinose, other compounds were identified for the first time in cocoa samples, such as hyperoside (quercetin‐3‐O‐galactoside), naringenin, luteolin, apigenin and some O‐glucosides and C‐glucosides of these compounds. Copyright © 2003 John Wiley & Sons, Ltd.
Clustering of nucleosides in the presence of alkali metals: Biologically relevant quartets of guanosine, deoxyguanosine and uridine observed by ESI‐MS/MS
Authors: Aggerholm Tenna , Nanita Sergio C. , Koch Kim J. , Cooks R. Graham 
Published: January 2003

AbstractElectrospray ionization (ESI) mass spectra of nucleosides, recorded in the presence of alkali metals, display alkali metal ion‐bound quartets and other clusters that may have implications for understanding non‐covalent interactions in DNA and RNA. The tetramers of guanosine and deoxyguanosine and also their metaclusters (clusters of clusters), cationized by alkali metals, were observed as unusually abundant magic number clusters. The observation of these species in the gas phase parallels previous condensed‐phase studies, which show that guanine derivatives can form quartets and metaclusters of quartets in solution in the presence of metal cations. This parallel behavior and also internal evidence suggest that bonding in the guanosine tetramers involves the bases rather than the sugar units. The nucleobases thymine and uracil are known to form magic number pentameric adducts with K+, Cs+ and NH4+ in the gas phase. In sharp contrast, we now show that the nucleosides uridine and deoxythymidine do not form the pentameric clusters characteristic of the corresponding bases. More subtle effects of the sugars are evident in the fact that adenosine and cytidine form numerous higher order clusters with alkali metals, whereas deoxyadenosine and deoxycytidine show no clustering. It is suggested that hydrogen bonding between the bases in the tetramers of dG and rG are the dominant interactions in the clusters, hence changing the ribose group to deoxyribose (and vice versa) generally has little effect. However, the additional hydroxyl group of RNA nucleosides enhances the non‐selective formation of higher‐order aggregates for adenosine and cytidine and results in the lack of highly stable magic number clusters. Some clusters are the result of aggregation in the course of ionization (ESI) whereas others appear to be intrinsic to the solution being examined. Copyright © 2003 John Wiley & Sons, Ltd.
Liquid chromatographic/tandem mass spectrometric method for the determination of the tobacco‐specific nitrosamine metabolite NNAL in smokers' urine
Authors: Byrd Gary D. , Ogden Michael W. 
Published: January 2003

AbstractA specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for NNAL, a metabolite of the tobacco‐specific nitrosamine metabolite NNK. The metabolite was detected in smokers' urine with a limit of quantitation (LOQ) of 20 pg ml−1 and a linear range up to 1000 pg ml−1. The method features a single solid‐phase extraction step and MS/MS monitoring following electrospray ionization. Fragmentation pathways for the protonated molecular ion are proposed. The sample preparation is simpler than that for gas chromatographic methods reported in the literature and maintains sensitivity adequate for determining NNAL in smokers' urine. By using enzyme hydrolysis to determine total NNAL in urine, the amount of NNAL‐glucuronide was calculated. A standard pooled smokers' urine sample used for development gave values of 176 ± 8 pg ml−1 free NNAL and 675 ± 26 pg ml−1 total NNAL following enzyme hydrolysis. The method was applied to a group of seven smokers; the free NNAL level for the group was 101–256 pg ml−1 with NNAL‐glucuronides at 247–566 pg ml−1. The ratio of conjugated to free NNAL was in the range 0.98–2.95. The variability in total daily amount of NNAL excreted (ng per 24 h) had RSDs of 6–21% for free NNAL, 7–22% for conjugated NNAL and 6–20% for total NNAL excreted. When normalized to the number of cigarettes smoked, the amounts of NNAL excreted per cigarette smoked were in the range of amounts of NNK yields reported for cigarettes in the literature. Copyright © 2003 John Wiley & Sons, Ltd.
Human and simian immunodeficiency virus‐infected chimpanzees do not have increased intracellular levels of β‐chemokines in contrast to infected humans
Authors: Ondoa Pascale , Vereecken Chris , Fransen Katrien , Colebunders Robert , van der Groen Guido , Heeney Jonathan L. , Kestens Luc 
Published: January 2003

AbstractThis study was undertaken to explain why chimpanzees infected with HIV‐1 (human immunodeficiency virus type 1) or SIVcpz (simian immunodeficiency virus of chimpanzee) are relatively resistant to AIDS (acquired immunodeficiency syndrome). The numbers of β‐chemokine‐positive cells were compared between uninfected and infected humans and chimpanzees using three‐color cytofluorometry. In humans, the percentage of β‐chemokine‐positive cells was significantly higher in CD8+ T and natural killer (NK) cells than in CD4+ T cells in both uninfected and HIV‐1‐infected individuals. In the presence of HIV‐1 infection, however, both CD8+ and CD4+ T cell subsets contained significantly more β‐chemokine‐positive cells than in the absence of infection. Interestingly, in chimpanzees two important differences were noted. First, their percentage of β‐chemokine‐positive CD8+ T and NK cells was significantly higher than in uninfected humans. Second, in contrast to humans, infection with either HIV‐1 or with SIVcpz was not associated with increased numbers of β‐chemokine‐positive cells. These results indicate that: constitutive high levels of intracellular β‐chemokines in chimpanzees' CD8 lymphocytes and NK cells do not necessarily correspond to lower levels of virus replication during the chronic phase of infection; and increased percentages of β‐chemokine‐positive cells in HIV‐infection are not a correlate of disease resistance. The data suggest that neither pre‐nor post‐exposure levels of intracellular β‐chemokines are correlated with the subsequent control of disease progression. J. Med. Virol. 69:297–305, 2003. © 2003 Wiley‐Liss, Inc.
Hepatitis B virus genotypes and serotypes in western India: Lack of clinical significance
Authors: Gandhe Swati S. , Chadha Mandeep S. , Arankalle Vidya A. 
Published: January 2003

AbstractTo determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection. J. Med. Virol. 69:324–330, 2003. © 2003 Wiley‐Liss, Inc.
Changes in hypervariable region 1 of the envelope 2 glycoprotein of hepatitis C virus in children and adults with humoral immune defects
Authors: Gaud Umesh , Langer Barbara , Petropoulou Theoni , Thomas Howard C. , Karayiannis Peter 
Published: January 2003

AbstractThe N‐terminal end of the hepatitis C virus (HCV) envelope glycoprotein E2 contains a stretch of 27 amino acids that exhibit increased variability. This hypervariable region 1 (HVR‐1), as it is normally referred to, is thought to contain epitopes that come under humoral immune attack. In the present study, 10 patients (5 children and 5 adults) with humoral immune defects and chronic HCV infection were investigated, to see how HVR‐1 sequences behave over time in these patients who are unable to produce antibodies. Amplicons of this region showed little or no variation at all over time, indicating that quasispecies variation in this region is driven by the host's humoral immune response. J. Med. Virol. 69:350–356, 2003. © 2003 Wiley‐Liss, Inc.
Processing of hepatitis C virus core protein is regulated by its C‐terminal sequence
Authors: Kato Takanobu , Miyamoto Michiko , Furusaka Akihiro , Date Tomoko , Yasui Kotaro , Kato Junko , Matsushima Shozo , Komatsu Tatsuji , Wakita Takaji 
Published: January 2003

AbstractPolyprotein processing of plus‐strand RNA viruses is important in the regulation of gene production and replication. The core protein of hepatitis C virus (HCV), constructing the viral particle, is processed from its precursor polyprotein and observed as two forms, p23 and p21. Production of p21 by cleavage at the C‐terminus of p23 is considered crucial to viral assembly and replication. In this study, this processing step was compared between clones isolated from two patients with fulminant hepatitis and from five patients with chronic hepatitis by an in vitro translation assay and cell transfection assay. The p21 core protein was predominant from the clone isolated from one of the fulminant hepatitis patient (p21 core protein production was 65.98%), while p23 was abundant with clones from five chronic hepatitis patients (p21 core protein production was 7.11±1.62%) and clone from another fulminant hepatitis patient (p21 core protein production was 13.36%). Investigations with chimeric and mutation‐introduced constructs revealed that four amino acid residues in the C‐terminus of the core region are responsible for this difference. The data suggest that core protein processing is regulated by C‐terminus mutations. J. Med. Virol. 69:357–366, 2003. © 2003 Wiley‐Liss, Inc.
Quantitation of cytomegalovirus (CMV) DNA by real‐time PCR for occurrence of CMV disease in HIV‐infected patients receiving highly active antiretroviral therapy
Authors: Gourlain Karine , Salmon Dominique , Gault Elyanne , Leport Catherine , Katlama Christine , Matheron Sophie , Costagliola Dominique , Mazeron Marie‐Christine , Fillet Anne‐Marie 
Published: January 2003

AbstractIn HIV‐infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan® PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan® PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow‐up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan® PCR >10/150,000 cells (RR: 27.6, IC95: 7.1–107.2), the CD4 cell count <75 cells/mm3 and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan® PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml. J. Med. Virol. 69:401–407, 2003. © 2003 Wiley‐Liss, Inc.
Induction of cellular immunity to varicella‐zoster virus glycoproteins tested with pernasal coadministration of escherichia coli enterotoxin in mice
Authors: Tsuji Takao , Shiraki Kimiyasu , Sato Hitoshi , Sasaki Keiko , Arita Michiko , Kato Michio , Takahashi Tsuyoshi , Ochi Sadayuki , Ichinose Yoshio , Yokochi Takashi , Asano Yoshizo 
Published: January 2003

AbstractA mutant of Escherichia coli enterotoxin promotes the induction of cellular immunity to a live varicella vaccine (the Oka strain) as a mucosal adjuvant in mice. An investigation was carried out to determine which of the purified glycoproteins of the virus among three induced cellular immunity with a single nasal administration. Spleen cells from mice immunized nasally with the vaccine and toxin produced interleukin‐2 (IL‐2) at the same level on restimulation in vitro with glycoprotein H: glycoprotein L (gH:gL), gB, and gE:gI, but not IL‐4. The spleen cells from mice immunized with gH:gL, gB, or gE:gI and toxin produced IL‐2 on restimulation with gH:gL, gB, or gE:gI, respectively, and the vaccine, but not IL‐4. Immunization with gH:gL and the toxin showed increased thymidine uptake and production of IL‐2 and interferon‐γ (IFN‐γ) of the spleen cells, but not IL‐4, depending on the dose of gH:gL used for immunization and restimulation in vitro. Purified gE:gI and gB have been reported to be the strongest stimulators of cellular immunity to varicella upon subcutaneous injection and are useful as a subunit vaccine. All the glycoproteins tested are excellent stimulators of cellular immunity to the virus and itself on nasal co‐immunization with the toxin. J. Med. Virol. 69:451–458, 2003. © 2003 Wiley‐Liss, Inc.
Apolipoprotein E expression after spinal cord injury in the mouse
Authors: Seitz Alexander , Kragol Maja , Aglow Elsa , Showe Louise , Heber‐Katz Ellen 
Published: January 2003

AbstractApolipoprotein E (apo‐E), a protein involved in lipid metabolism and cholesterol transport, has been found to be up‐regulated in CNS injury and is associated with Alzheimer's disease in humans. In this study, we show that apo‐E is also up‐regulated after complete spinal cord transection in the C57BL/6 mouse. In the uninjured cord, the cellular localization of apo‐E protein is in astrocytes, in individual neurons throughout the laminae except for the dorsal horn, and in endothelial cells of capillaries in the immediate vicinity of those neurons. After injury, RNA levels are elevated as early as 4 days and reach a maximal level between 1 and 2 weeks. Protein levels follow closely but remain up‐regulated beyond 3 weeks. Early on, the protein can be found in neutrophils and macrophages at the injury site and only at later times in astrocytes during the remodeling of white matter tracts, most prominently in degenerating parts of the fasciculus gracilis. © 2002 Wiley‐Liss, Inc.
Caspase activation pathways induced by staurosporine and low potassium: Role of caspase‐2
Authors: Caballero‐Benítez Andrea , Morán Julio 
Published: January 2003

AbstractApoptotic death is a physiological process with regulatory mechanisms that are under the control of different molecules such as caspases. These are classified as initiators, such as caspases‐8 and ‐9, and effectors, such as caspases‐3 and ‐7. The participation of caspase‐2 in the effector phase of apoptosis has been commonly observed in many cell types; however, it is able to act as an initiator caspase, depending on the apoptotic stimulus. Cerebellar granule cells (CGCs) undergo apoptosis when they are transferred from high potassium (K25) to low potassium (K5); this process seems to be mediated by caspase‐3 activation. Staurosporine (STS), a full strength inhibitor of kinase proteins, also induces apoptosis in these cells. To characterize the caspase cascade induced by two stimuli in the same cell type we studied the activation of different caspases in CGCs treated with STS or K5. We found that both K5 and STS induce the activation of caspase‐3. This result was confirmed by the proteolytic cleavage of poly (ADP‐ribose) polymerase (PARP), an endogenous caspase‐3 substrate. Caspase‐2 was activated preferentially by STS, which showed a temporal course suggesting that this caspase was induced before caspase‐3. The initiator caspase‐9 was also activated by both K5 and STS, as well as cytochrome‐c release. The results obtained in this study suggest that STS and K5 induced different activation caspase pathways for apoptotic cell death of CGCs. © 2002 Wiley‐Liss, Inc.
Nitrergic dendrites in the superficial layers of the rat superior colliculus: Retinal afferents and alternatively spliced isoforms in normal and deafferented animals
Authors: Batista Claudia M. C. , Carneiro Kátia , Ernesto de Bittencourt‐Navarrete Ruben , Soares‐Mota Marcia , Cavalcante Leny A. , Mendez‐Otero Rosalia 
Published: January 2003

AbstractThe superficial layers of the rat superior colliculus (sSC) receive innervation from the retina and include nitrergic neurons. We have shown previously that in sSC, eye enucleation reduces NADPH diaphorase staining considerably in all but the most proximal dendrites of nitrergic neurons. We have used immunocytochemistry for neuronal nitric oxide synthase (nNOS) at light and electron microscopic levels and bilateral eye enucleation with varied survival times to determine the regulatory changes imposed by the direct and indirect loss of retinal input on apparent nNOS amount and subcellular distribution. In addition, we have used SDS‐PAGE and immunoblotting to test alternatively spliced isoforms in normal and deafferented animals. Our results show that unambiguously identified retinal terminals contact nitrergic neurons. In normal dendrites, nNOS immunoreactivity was distributed almost completely within the cytoplasm of the dendrite and along the postsynaptic membrane at synaptic junctions, in association with endoplasmic reticulum, ribosomes and external mitochondrial membranes. In contrast, nNOS labeling was greatly reduced in sSC deprived of retinal projections, and could only be observed in association with mitochondrial membranes and postsynaptic densities. Immunoblots of the soluble fraction from sSC revealed a surprisingly high proportion of the β isoform with respect to the α counterpart in normal colliculi, suggesting an increase in isoform proportion after enucleation, or at least maintenance of the same proportion. It is suggested that ultrastructural alterations observed in sSC cells of enucleated animals may be consequent to plastic reactions of the sSC cells in response to the removal of retinal afferents. © 2002 Wiley‐Liss, Inc.