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An ultrafine (6–7 nm) and well dispersed nano-SnO2/carbon nanotube hairball (SnO2/CNTH) composite material with a three-dimensional (3D) hierarchical structure is prepared by spray drying and solvo...

Inclusion of polymer additives is a known strategy to improve foam stability, but questions persist about the amount of polymer incorporated in the foam and the resulting structural changes that impact material performance. Here, we study these questions in sodium dodecyl sulfate (SDS)/hydroxypropyl methylcellulose (HPMC) foams using a combination of flow injection QTOF mass spectrometry and small-angle neutron scattering (SANS) measurements leveraging contrast matching. Mass spectrometry results demonstrate polymer incorporation and retention in the foam during drainage by measuring the HPMC-to-SDS ratio. The results confirm a ratio matching the parent solution and stability over the time of our measurements. The SANS measurements leverage precise contrast matching to reveal detailed descriptions of the micellar structure (size, shape, and aggregation number) along with the foam film thickness. The presence of HPMC leads to thicker films, correlating with increased foam stability over the first 15-20 min after foam production. Taken together, mass spectrometry and SANS present a structural and compositional picture of SDS/HPMC foams and an approach amenable to systematic study for foams, gathering mechanistic insights and providing formulation guidance for rational foam design.

Objective Patients with cardiac surgery–associated acute kidney injury are at risk of renal replacement therapy and in-hospital death. We aimed to develop and validate a novel predictive model for poor in-hospital outcomes among patients with cardiac surgery–associated acute kidney injury. Methods A total of 196 patients diagnosed with cardiac surgery–associated acute kidney injury were enrolled in this study as the training cohort, and 32 blood cytokines were measured. Least absolute shrinkage and selection operator regression and random forest quantile-classifier were performed to identify the key blood predictors for in-hospital composite outcomes (requiring renal replacement therapy or in-hospital death). The logistic regression model incorporating the selected predictors was validated internally using bootstrapping and externally in an independent cohort (n = 52). Results A change in serum creatinine (delta serum creatinine) and interleukin 16 and interleukin 8 were selected as key predictors for composite outcomes. The logistic regression model incorporating interleukin 16, interleukin 8, and delta serum creatinine yielded the optimal performance, with decent discrimination (area under the receiver operating characteristic curve: 0.947; area under the precision-recall curve: 0.809) and excellent calibration (Brier score: 0.056, Hosmer–Lemeshow test P = .651). Application of the model in the validation cohort yielded good discrimination. A nomogram was generated for clinical use, and decision curve analysis demonstrated that the new model adds more net benefit than delta serum creatinine. Conclusions We developed and validated a promising predictive model for in-hospital composite outcomes among patients with cardiac surgery–associated acute kidney injury and demonstrated interleukin-16 and interleukin-8 as useful predictors to improve risk stratification for poor in-hospital outcomes among those with cardiac surgery–associated acute kidney injury.

MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, have been implicated as critical regulatory molecules in ischemia-/reperfusion-induced cardiac injury. In the present study, we report on the role of miR-208a in myocardial I/R injury and the underlying cardio-protective mechanism. The gain-of-function and loss-of-function were used to explore the effects of miR-208a on cardiac injury induced by H2O2 in cardiomyocytes. As predicted, knockdown of endogenous miR-208a significantly decreased the level of cellular reactive oxygen species (ROS) and reduced cardiomyocyte apoptosis. In addition, miR-208a overexpression increased the ROS level and attenuated cell apoptosis in cardiomyocytes. Furthermore, protein tyrosine phosphatase receptor type G (PTPRG) and protein tyrosine phosphatase, non-receptor type 4 (PTPN4), which participate in regulating the level of cellular protein tyrosine phosphorylation balance, were predicted and verified as potential miR-208a targets using bioinformatics and luciferase assay. In summary, this study demonstrated that miR-208a plays a critical protective role in ROS-induced cardiac apoptosis.

In this work, switchable hydrophilicity solvent-based ultrasound-assisted liquid phase microextraction (SHS-UA-LPME) was combined with micro-volume UV-vis spectrophotometry for the determination of trace nickel in the medicinal plant Gentiana rigescens Franch. ex Hemsl. (G. rigescens) samples for the first time. Micro-volume spectrophotometry has the characteristics of microanalysis, economy and low operation cost, which is feasible to be combined with miniaturized enrichment method. Dipropylamine (DPA) as a switchable solvent completed the extraction during the phase conversion process. It was changed from hydrophobic to hydrophilic under the action of HCl, and then completed phase transition under the action of NaOH. The parameters influencing the extraction efficiency were investigated. Under the optimal conditions, the enhancement factor (EF), the limit of detection (LOD), the limit of quantitation (LOQ) and relative standard deviation (RSD, n = 7) were 28, 0.02 µg/L, 0.067 µg/L and 1.4%, respectively. The established method was applied to the determination of G. rigescens samples collected from the local field with satisfactory results.

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.

Two novel aggregation-induced emission (AIE) based fluorescent probes, TPANI-DNs and PCZNI-DNs, have been designed and synthesized for “turn-on” detection of H2S. Chromophore napthalimide fused triphenylamine (or phenylcarbazole) unit as fluorophore in combination with 2,4-dinitrobenzenesulfonyl as recognition moiety constructed probes. The design strategy of the twisted D-π-A structure can efficiently transform the aggregation-caused quenching (ACQ) system into the AIE system by strengthening the restriction of intramolecular motion and preventing the intermolecular π-π stacking. The consequences showed that both TPANI-DNs and PCZNI-DNs displayed large stokes shift (135 nm and 120 nm, respectively), high selective and sensitive detection. The response mechanisms and fluorescent properties were further investigated through the time-dependent density functional theory (TDDFT). Importantly, since the strong AIE properties, a H2S test board has been prepared and used to detect H2S onsite easily and sensitively, displaying potential practical applications.

Genipin is the major active component of Gardeniae fructus and has been shown to ameliorate diabetes and insulin resistance in rat models. In this study, we first investigated the effect of genipin on obesity and the related lipid metabolism mechanisms in diet-induced obese rats. Our results showed that genipin reduced body weight, food intake, and visceral fat mass; ameliorated dyslipidemia, glucose intolerance, insulin intolerance, adipocyte hypertrophy, and hepatic steatosis; and reduced serum tumor necrosis factor-α level in diet-induced obese rats. Quantitative real-time reverse-transcription polymerase chain reaction results further illustrated that genipin promoted lipolysis and β-oxidation of fatty acid by upregulating gene expressions of hormone-sensitive lipase and adipose triglyceride lipase in white adipose tissue (WAT) and peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase 1α in hepatic tissue. Moreover, genipin promoted browning of WAT by upregulating the mRNA and protein levels of uncoupling protein 1 and PRD1-BF1-RIZ1 homologous domain containing 16 in WAT. Additionally, genipin inhibited gene expressions of activin receptor-like kinase 7, tumor necrosis factor-α, and interlukin-6 in WAT. These results indicated that genipin had a potential therapeutic role in obesity, in which regulation of lipid mobilization and browning of WAT were involved.

The protective effects of dioscin, a natural steroidal saponin from some medicinal plants including Dioscorea nipponica Makino, against lipopolysaccharide (LPS)- induced acute liver and renal damages have been reported in our previous works. However, its effect on LPS-induced acute lung injury (ALI) remains unknown. In the present study, the effects and possible mechanisms of dioscin against LPS-induced ALI in vitro and in vivo were investigated. The results showed that dioscin effectively inhibited cell proliferation and markedly decreased reactive oxidative species (ROS) level in 16HBE cells treated by LPS. In addition, dioscin significantly attenuated LPS-induced histological alterations, suppressed the infiltration of inflammatory cells, as well as decreased the levels of MDA, SOD, NO and iNOS in mice and rats (p < 0.05). Mechanistically, dioscin significantly decreased the protein levels of TLR4, MyD88, TRAF6, TKB1, TRAF3, phosphorylation levels of PI3K, Akt, IκBα, NF-κB, and the mRNA levels of IL-1β, IL-6 and TNF-α against oxidative stress and inflammation (p < 0.05). TLR4 overexpression was also decreased by dioscin, leading to the markedly decreased the levels of MyD88, TRAF6, TKB1, TRAF3, p-PI3K, p-Akt, p-IκBα and p-NF-κB. These findings provide new insights that dioscin exhibited protective effect against LPS-induced ALI via adjusting TLR4/MyD88- mediated oxidative stress and inflammation, which should be developed as one potent candidate for the treatment of ALI in the future.

SynopsisObjective The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. Methods The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. Results In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. Conclusion This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS.

Introduction Ataluren functions by interacting with ribosomes to promote read-through of nonsense mutations in CF. Ataluren9s activity was shown to be inhibited by certain aminoglycosides such as tobramycin that also bind to the ribosome. Aim To investigate the effect of ataluren on lung function (LF) and pulmonary exacerbations. Methods A post-hoc analysis was performed on a recent randomized, double-blind, placebo-controlled, phase 3 study to determine the efficacy and safety of ataluren in patients with nonsense mutation cystic fibrosis (nmCF) (Kerem E, et al. Lancet Respir Med. 2014;2:539-47) on %-predicted FEV 1 (ppFEV 1 ) and exacerbations by use of chronic inhaled tobramycin at baseline. Results Patients not receiving chronic inhaled tobramycin (non-TOBI; n=146), showed a 5.7% difference in relative ppFEV 1 between ataluren and placebo (−0.7% vs −6.4%; p=0.0082) and 40% fewer exacerbations (1.42 vs 2.18; p=0.0061). Non-TOBI patients ≥6 to 1 between ataluren and placebo (4.9% vs −3.3%; p=0.026) and a 60% lower exacerbation rate favoring ataluren (p=0.030). In all intent-to-treat patients (N=232) at week 48, including tobramycin patients, neither relative change from baseline in ppFEV 1 (−2.5% vs −5.5%; p=0.12), nor number of exacerbations (1.42 vs 1.78; p=0.099) significantly differed between ataluren and placebo. Conclusions Ataluren significantly reduced exacerbations and improved LF in nmCF patients not receiving chronic inhaled tobramycin, with markedly improved treatment effect in children and adolescents. Ataluren thus shows promise as a disease-modifying therapy in nmCF.

Background Myocardial infarction (MI) remains the most common cause of heart failure (HF) worldwide. For almost 50 years, HF has been recognized as a determinant of adverse prognosis after MI, but efforts to promote myocardial repair have failed to be translated into clinical therapies. Aims In this study, we investigated the effects of BRD4 on cardiac function and the underlying mechanism. Material and methods The in vivo rat model of AMI and in vitro neonatal cardiomyocytes were established and cultured respectively, the BRD4 and NPPA/NPPB expression levels were detected by qPCR and Western blot, and interaction of BRD4 with acetylation RelA or NPPA/B promoters were examined by co-immunoprecipitation and chromatin immunoprecipitation assays, respectively. Results We found that BRD4 protein expression was significantly increased in cardiomyocytes of MI rat model and cardiomyocytes under hypoxia, accompanied by the expression of natriuretic peptide A (NPPA) and natriuretic peptide B (NPPB). Functionally, knockdown of BRD4 greatly downregulated the NPPA and NPPB in vivo and in vitro, improved the hemodynamic and biometric parameters in rat with heart failure, as well as decreased the apoptosis occurrence. In vitro studies further demonstrated that BRD4 bound with acetylated RelA to enhance the activation of NF-κb signaling, which resulted in activation of NPPA and NPPB transcriptions. Conclusions Taken together, our findings suggest that inhibition of BRD4 attenuated cardiomyocyte apoptosis via NF-κB pathway in myocardial infarction, and this study sheds light on developing new strategies to overcome myocardial damage.

This study aimed to investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). C57BL/6J mice were injected with MPTP for 8 days to establish a PD model. Nicotine was given for 10 days in the nicotine therapeutic group. Animals were examined behaviorally with the pole test and traction test. Tyrosine hydroxylase (TH) and γ-aminobutyric acid (GABA) were determined by using the immunocytochemistry (ICC) method. The ultrastructural changes of the caudate nucleus (CN) were observed under electron microscopy. The results showed that pretreatment with nicotine could improve the dyskinesia of PD mice markedly. Simultaneously, TH-positive (P

The competitive adsorption of the Na salt of phytic acid (PA) and 4-methyl-4H-1,2,4-triazole-3-thiol (4-MTTL) was investigated by using the surface-enhanced Raman scattering (SERS) technique and electrochemical measurements. On the basis of the recorded SERS spectra, it was found that PA molecules could competitively occupy the silver surface modified with 4-MTTL monolayers and that the competitive adsorption of 4-MTTL at the silver surface with the PA monolayers also happened, but the effect of coadsorption of PA on the structure of 4-MTTL monolayers was smaller than that of 4-MTTL on the PA monolayers. In addition, according to the electrochemical measurements, the only 4-MTTL monolayers or the only PA monolayers at the silver surface exhibited a reasonable stability. The coadsorption of 4-MTTL and PA formed a mixed film at the silver surface immersed in the solution with the coexistence of 4-MTTL and PA, and the film was unstable when a potential was applied.

SynopsisObjective Damage to hair from UV exposure has been well reported in the literature and is known to be a highly complex process involving initiation via absorption of UV light followed by formation and propagation of reactive oxygen species (ROS). The objective of this work was to understand these mechanisms, explain the role of copper in accelerating the formation of ROS and identify strategies to reduce the hair damage caused by these reactive species. Methods The location of copper in hair was measured by Transmission electron microscopy–(TEM) X-ray energy dispersive spectroscopy (XEDS) and levels measured by ICP-OES. Protein changes were measured as total protein loss via the Lowry assay, and MALDI ToF was used to identify the biomarker protein fragments. TBARS assay was used to measure lipid peroxide formation. Sensory methods and dry combing friction were used to measure hair damage due to copper and UV exposure and to demonstrate the efficacy of N,N' ethylenediamine disuccinic acid (EDDS) and histidine chelants to reduce this damage. Results In this work, a biomarker protein fragment formed during UV exposure is identified using mass spectrometry. This fragment originates from the calcium-binding protein S100A3. Also shown is the accelerated formation of this peptide fragment in hair containing low levels of copper absorbed from hair during washing with tap water containing copper ions. Transmission electron microscopy (TEM) X-ray energy dispersive spectroscopy (XEDS) studies indicate copper is located in the sulphur-poor endo-cuticle region, a region where the S100A3 protein is concentrated. A mechanism for formation of this peptide fragment is proposed in addition to the possible role of lipids in UV oxidation. A shampoo and conditioner containing chelants (EDDS in shampoo and histidine in conditioner) is shown to reduce copper uptake from tap water and reduce protein loss and formation of S100A3 protein fragment. In addition, the long-term consequences of UV oxidation and additional damage induced by copper are illustrated in a four-month wear study where hair was treated with a consumer relevant protocol of hair colouring treatments, UV exposure and regular shampoo and conditioning. Conclusions The role of copper in accelerating UV damage to hair has been demonstrated as well as the ability of chelants such as EDDS and histidine in shampoo and conditioner products to reduce this damage.

The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a FADD-like interferon converting enzyme or caspase 8 (FLICE) inhibitory protein (vFLIP) that prevents death receptor-mediated apoptosis by inhibiting the recruitment and activation of FLICE. Since vFLIP physically interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and TRAF2 mediates activation of the jun NH(2)-terminal kinase (JNK)/activation protein 1 (AP1) pathway, we hypothesized that vFLIP might also activate this pathway. To evaluate this hypothesis, we transiently and stably transfected a vFLIP expression construct and performed several complementary assays to document that vFLIP activates the JNK/AP1 pathway and does so in a TRAF-dependent fashion. As vFLIP also activates the nuclear factor kappaB (NF-kappaB) signaling pathway and the NF-kappaB and JNK/AP1 pathways both modulate cellular interleukin-6 (cIL-6) expression, we postulated that vFLIP induces expression of this cytokine. We show that vFLIP induces cIL-6 expression and activates the cIL-6 promoter, and maximal activation of the cIL-6 promoter by vFLIP requires NF-kappaB and AP1 activation. In addition, vFLIP and latency-associated nuclear antigen (LANA), another KSHV-encoded latent protein, potentiate each other's ability to activate the cIL-6 promoter. Gene silencing experiments by RNA interference demonstrate that vFLIP in BCBL-1 endogenously infected primary effusion lymphoma (PEL) cells mediates JNK/AP1 activation and cIL-6 expression. Thus, we conclude that vFLIP, in addition to its known effects on NF-kappaB activation, also modulates the JNK/AP1 pathway and induces gene expression from the cIL-6 promoter in a JNK/AP1-dependent fashion.

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/µES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright © 2000 John Wiley & Sons, Ltd.

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by β-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied β-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the β-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403. © 1998 John Wiley & Sons, Ltd.

Crystallins, the structural proteins of the lens, have been isolated from human lenses using a combination of gel filtration and reversed phase high performance liquid chromatography (HPLC). The molecular weights of the isolated crystallins have been determined by electrospray ionization mass spectrometry. The isolated crystallins have also been proteolytically digested into peptides, the peptides fractionated by HPLC, and the masses of the peptides determined by fast atom bombardment mass spectrometry. With these techniques, it is possible to confirm and/or correct known protein sequences and identify and locate post translational modifications. I NTRODU CTl ON Cataract, which can be defined as an opacity of the eye lens, is the leading cause of blindness worldwide. In countries where surgery to remove a cataractous lens and replace it with a synthetic lens is readily available, cataract may not be a serious impairment; however, in much of the world this surgery is not available, and the development of cataract leads to blindness. The two types of cataract, cortical and nuclear, both involve changes in the lens proteins, called the crystallins. For the lens to be transparent, the crystallins must be tightly and uniformly packed producing a lens with a uniform refractive index. In cortical cataract, the density of the crystallins is altered, causing a non-uniform density with variations in the lens refractive index and consequent light scattering (ref. 1). Nuclear cataract is associated with the formation of insoluble protein aggregates. In both cases, there are modifications to the lens crystallins that either prevent their normal close packing in the cortex or cause their aggregation in the nucleus. The focus of our research is to determine which proteins are modified, where the modifications are located, and the identity of the modifications. Cataract is most commonly associated with old age. Most people over age 80 have at least early indications of cataract. Cataract occurs earlier in people with diabetes, renal failure, chronic diarrhea or who have experienced prolonged exposure to W radiation. The mechanisms that have been proposed for the development of cataract vary, depending on the disease with which it is associated. Cataract associated with old age, diabetes, renal failure and chronic diarrhea are all proposed to occur because of modifications to the lysyl residues of the crystallins. In old age, the concentration of glutathione in the lens is lower. This permits an increase in the concentration of dehydroascorbic acid, which is normally reduced by glutathione in younger lenses. Dehydroascorbic acid can react with the amino groups of the lysyl residues, forming a Schiff base which can then rearrange or possibly form cross-links (ref. 2). In diabetes, the elevated concentrations of glucose may lead to formation of a similar Schiff base between the lysyl residues and glucose (ref. 3). Both renal failure and chronic diarrhea are associated with elevated urea, which forms an equilibrium with isocyanate. Isocyanate can also react with lysyl residues forming a carbamylated protein (refs. 4, 5). A different mechanism, oxidation of the tryptophan residues to form kynurenine, has been proposed

This paper described a facile method for preparation of gold nanoparticles with high efficient SERS activity within short reaction time in the presence of an environmentally benign and low-cost reagent named inositol hexaphosphate (IP(6)). IP(6) acted as a tunable cross-linker to obtain a suitable separation (about 2nm) between the neighboring particles for certain surface plasmons by adjusting the dosages of IP(6). When the molar ratio of Au to IP(6) was 10:1, gold particles presented in nano-pearl-necklace pattern, demonstrating the high enhancement factor (>10(7)) for 2-mercaptopyridine (2-Mpy) Raman scattering and high stability (>2months).

The main objective of this work was to use positive-ion fast atom bombardment mass spectrometry (FAB-MS) B/E linked scan spectra to investigate the possibility of differentiating positional isomers of various authentic glycine- and taurine-conjugated bile acids. Sodium salts of 14 conjugated bile acids were individually ionized by FAB-MS and characterized by scanning simultaneously the magnetic field B and the electric sector field E such that B/E remained constant throughout the scan. The dominant fragment ions could be related to cleavage of the aliphatic side chain with charge retention on the conjugated end of the bile acids. However, fragment ions arising from ring cleavages were also observed and could be used to distinguish the positions of substituent hydroxyl groups. For example, ring cleavage of conjugated dihydroxy bile acids at C-7/C-8 and C-9/C-10 permitted the differentiation of chenodeoxycholyltaurine (3 alpha,7 alpha-substitution pattern) from deoxycholyltaurine (3 alpha,12 alpha-substitution pattern) based on the presence of fragment ions at m/z 388 or m/z 404, which were indicative of hydroxyl group substitutions at either the 7- or 12-positions, respectively. It was concluded that B/E linked scans can be used to discriminate positional isomers of conjugated bile acids.

Publisher Summary Nano-electrospray (nES) is a new technique for characterizing biomolecules in small volumes (0.5–2 μl) at low picomole levels. In nES, signals from a single sample loading typically last more than 30 minutes, which permits optimization of instrument parameters and MS/MS sequencing with high sensitivity. Both nES/MS and nES/MS/MS data can be obtained from a single sample loading. These features make nES an attractive off-line technique for sequencing peptides collected from capillary electrophoresis (CE). In this chapter, an off-line approach that combines nES/MS analysis and Edman sequencing of peptide/protein fractions collected from CE is presented. Automatic peak collection is accomplished using a computer-controlled Beckman P/ACE 5000 instrument. An off-line approach that is simple and useful for peptide/protein sequencing using 5–10 picomoles of material has been demonstrated. Several different samples of material, including a peptide mixture (angiotensin-I, methionine enkephalin, and substance-P), a tryptic digest of cytochrome-C and proteins like myoglobin, insulin and lysozyme, are used to demonstrate this method. For this purpose, peptide and protein samples are first separated by capillary electrophoresis. Selected peaks are fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing.

alpha-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in the alpha-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination of in vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lens alpha-crystallins of renal failure patients.

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specifity with which peptide molecular weight is related to specific segments of the protein. These results demonstrate that the shift in peptide molecular weight, after the digest is treated with performic acid, leads to unique assignment of the six cysteinyl-containing peptides found in the digest.

Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology.