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Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 91 records · Page 5Linked to original sources
PubMed1998

Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

3T3 Cells↗
PubMed1998

Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in the rat.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal damage by a non-prostaglandin (PG) dependent "topical" action and by inhibiting cyclooxygenase. AIMS: To discriminate between these two effects by studying some key pathophysiological steps in NSAID enteropathy following administration of (R)- and (S)-flurbiprofen, the racemic mixture, and an uncoupler, dinitrophenol. METHODS: The effects of dinitrophenol, racemic, (R)-, and (S)-flurbiprofen on mitochondria were assessed in vitro and on key pathophysiological features of small intestinal damage in vivo (ultrastructure by electron microscopy, mucosal prostanoid concentrations, intestinal permeability, inflammation, and ulcer count) in rats. RESULTS: All the drugs uncoupled mitochondrial oxidative phosphorylation in vitro, caused mitochondrial damage in vivo, and increased intestinal permeability. Dinitrophenol and (R)-flurbiprofen caused no significant decreases in mucosal prostanoid concentrations (apart from a decrease in thromboxane (TX) B2 concentrations following (R)-flurbiprofen) while racemic and (S)- flurbiprofen reduced mucosal prostanoids significantly (PGE, TXB2, and 6-keto-PGF1alpha concentrations by 73-95%). Intestinal inflammation was significantly greater following administration of (S)-flurbiprofen and racemate than with dinitrophenol and (R)-flurbiprofen. No small intestinal ulcers were found following dinitrophenol or (R)-flurbiprofen while both racemic and (S)-flurbiprofen caused numerous ulcers. CONCLUSIONS: Dinitrophenol and (R)-flurbiprofen show similarities in their actions to uncouple mitochondrial oxidative phosphorylation in vitro, alter mitochondrial morphology in vivo, increase intestinal permeability, and cause mild inflammation without ulcers. Concurrent severe decreases in mucosal prostanoids seem to be the driving force for the development of severe inflammation and ulcers.

Animals↗
PubMed1998

The natural history of untreated lower urinary tract symptoms in middle-aged and elderly men over a period of five years.

OBJECTIVES: To describe changes in untreated lower urinary tract symptoms (LUTS), levels of bothersomeness and interference in selected living activities arising from LUTS in a cohort of men followed up for a period of 5 years. METHODS: 1,994 men aged 40-79 years registered in four health centres in Scotland completed a urinary symptom questionnaire in the community. 1,177 (71% of original eligible participants) were followed up at 5 years. RESULTS: The mean level of LUTS and the level of bothersomeness they caused increased universally between baseline and 5 years. The largest increases in mean symptom levels occurred for straining, hesitancy, incomplete emptying, weak stream and nocturia, and for mean levels of bothersomeness, i.e. dribbling, incomplete emptying, weak stream, frequency, nocturia and intermittency. The increase in mean interference level in selected living activities arising from LUTS was less marked. The progression of LUTS over time in individual men was very variable; however, the overall trend was one of continuing deterioration. CONCLUSION: If left untreated, overall levels of middle-aged and elderly mens' LUTS, bothersomeness and interference in selected activities caused by these symptoms will increase over time.

Adult↗
PubMed1997

Performance assessment model for guideline-recommended pharmacotherapy in the secondary prevention of coronary artery disease and treatment of left ventricular dysfunction.

The Agency for Health Care Policy and Research, the National Heart Lung and Blood Institute of the National Institutes of Health, the American Heart Association, and the American College of Cardiology have all developed guidelines for improving the care of patients with cardiovascular disease. The guidelines include recommendations for intensive lipid-lowering therapy in patients with coronary artery disease (CAD) and angiotensin-converting enzyme (ACE) inhibitors in those patients with symptomatic heart failure and asymptomatic left ventricular dysfunction. Despite clinical trial evidence and consensus that these therapies improve survival in high-risk patients, data suggest that there is wide variation in the delivery of guideline-based care. To investigate whether evidence-based assessment of provider practice patterns can impact the delivery of quality cost-effective care, Merck and Company, in conjunction with leading cardiology group practices, the University of North Carolina at Chapel Hill, and Medical Review of North Carolina developed an ambulatory medical record abstraction study. This quality assurance initiative was conducted at practices beginning in the spring of 1996 and continues. Medical records and administrative claims of patients with ischemic heart disease or heart failure were abstracted by a healthcare consulting organization to maintain patient and physician confidentiality. As of mid-July 1997, 626 group practices had completed the medical record abstraction process, with > 1,136 practices participating at some stage of the project; >6,000 physicians participated in the project and >270,000 patients charts were abstracted. Analysis of these data will provide insight and benchmark patterns of care in the pharmacologic management of heart failure and CAD. This project represents a unique collaboration between a pharmaceutical company, an academic institution, a Peer Review Organization, and practicing physicians, to support evidence-based best medical practices.

Aged↗
PubMed1997

Improving care for unstable angina patients in a multiple hospital project sponsored by a federally designated quality improvement organization.

In 1992, the Health Care Financing Administration (HCFA) implemented a major change in the methodology of the quality of care oversight activities conducted by Medicare Peer Review Organizations. The Health Care Quality Improvement Program (HCQIP) represented a shift in oversight activity direction from identifying and dealing with individual clinical errors to helping providers improve mainstream care. The change in the oversight activities of Peer Review Organizations has been so substantial that the organizations are now commonly referred to as Quality Improvement Organizations (QIOs). Since its introduction, the HCQIP has developed multiple cooperative projects between QIOs and participating hospitals to examine specific processes of care and to ultimately improve the quality of care provided to Medicare patients. This report describes one project in North Carolina focusing on inpatient treatment of patients with a principal diagnosis of unstable angina, one of the most frequent causes of hospital admissions for Medicare patients. Based on the guidelines for treating unstable angina issued by the Agency for Health Care Policy and Research, 5 measures of good medical care for these patients were selected as quality of care indicators. A total of 16 hospitals in North Carolina each provided medical records of approximately 50 Medicare patients discharged with a principal diagnosis of unstable angina. Our findings indicated that guidelines-recommended standard of care were met in only a minority of patients. These indicators of care--including ordering an electrocardiogram within the first hour of admission and admitting high-risk patients to the intensive care unit--all occurred in <50% of the patients. Moreover, use of drugs that improve outcomes in patients with unstable angina was lower than expected. Only 17% of eligible patients with unstable angina were discharged on a lipid-lowering medication. Although there was variation in compliance with the guidelines between types of hospitals, all hospitals had an opportunity to improve in at least one quality of care indicator. The data demonstrate that significant variances exist between published guidelines and actual practices. Given the high rates of readmission for patients with coronary disease, there is opportunity to improve compliance with recommended guidelines of good care. The new oversight activity direction taken by Medicare should ultimately improve care for more patients than could ever be achieved through individual case review.

Aged↗
PubMed1997

Characterization of posttranslational modifications of human A33 antigen, a novel palmitoylated surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33 recognizes a differentiation antigen (A33) expressed in normal human gastrointestinal epithelium and in 95% of human colon cancers. Murine mAb A33 shows specific targeting of colon cancer in humans and a humanized A33 antibody is currently being evaluated in the clinic. The cDNA for the human A33 antigen has recently been cloned, and sequence comparison indicated that the A33 antigen is a novel human cell surface molecule of the immunoglobulin superfamily. Because mAb A33 recognizes a conformational epitope, only a partial characterization of the A33 antigen has been carried out to date. In this report we show that the A33 antigen is (I) N-glycosylated, containing approximately 8 K of N-linked carbohydrate and there is no evidence for O-glycosylation, sialylation or glycophosphatidylinositol, and (ii) S-acylated in vitro, incorporating [3H] palmitic acid linked through a hydroxylamine-sensitive thioester bond. The S-palmitoylation may be involved in regulating the internalization process initiated by binding of mAb A33 to cell surface A33 antigen.

Acylation↗
PubMed1997

Roles of histidine 31 and tryptophan 34 in the structure, self-association, and folding of murine interleukin-6.

Interleukin-6 (IL-6) is a multifunctional cytokine which is involved in a broad spectrum of activities such as immune defense, hematopoiesis, and the acute phase response, as well as in the pathogenesis of multiple myeloma. A series of murine IL-6 (mIL-6) mutants, H31A, W34A, and H31A/W34A, were constructed to investigate the roles of His31 and Trp34 in the structure, conformational stability, time-dependent aggregation, folding, and spectral properties of mIL-6. The characteristic pH-dependent quenching of fluorescence of mIL-6 at low pH was shown to be caused by an interaction between Trp34 and protonated His31 at low pH and not associated with Trp157. Denaturant-induced equilibrium unfolding experiments monitored by fluorescence and far-UV CD showed that the increased quantum yield and blue shift of the wavelength of the emission maximum observed for mIL-6 at moderate denaturant concentrations were also associated with Trp34, rather than Trp157. The tendency to form aggregation-prone unfolding intermediates, as judged by poor fits to a two-state unfolding mechanism, low m values (slopes of the unfolding curve in the transition region), and the range of denaturant concentrations over which these intermediates formed, was shown to be higher for H31A than mIL-6 but significantly lower for W34A and H31A/W34A. These differences were most pronounced at pH 7.4 and correlated with the tendencies of the proteins to aggregate at high protein concentrations in the absence of denaturant. As judged by the 1H NMR chemical shifts of the aromatic residues, the global conformations of H31A and W34A were not significantly different from that of mIL-6. Nuclear Overhauser effects (NOE) between the side chains of His31 and Trp34 were consistent with the indole side chain of Trp34 being oriented toward the face of the imidazolium side chain of His31, an arrangement consistent with our estimates of a low interaction energy (0.4-0.6 kcal/mol) between these side chains. A shift in the pKa of the His31 side chain in W34A (+0.3 unit) suggested that, in the absence of Trp34, His31 could interact with other residues. Further mutations in this region should yield forms of mIL-6, even less prone to aggregation, which would be more suitable for NMR studies. Mutation of His31 and Trp34 to alanine did not significantly alter the mitogenic activity of the mutants on mouse hybridoma 7TD1 cells, even though the corresponding region of human IL-6 has been shown to be important for biological activity.

Amino Acid Sequence↗
PubMed1997

Mechanisms involved in increased iron uptake across rat duodenal brush-border membrane during hypoxia.

1. Chronic hypoxia enhances intestinal iron transport but the cellular processes involved are poorly understood. In order to assess the effects of 3 days of hypoxia on iron uptake across the duodenal brush-border membrane, we have measured the membrane potential difference (Vm) of villus-attached enterocytes by direct microelectrode impalement and have used semi-quantitative autoradiography to study changes in expression of iron uptake during enterocyte maturation. 2. Hypoxia increased duodenal Vm (-57.7 vs. -49.3 mV, P < 0.001). Ion substitution experiments revealed that hyperpolarization was due, at least in part, to a reduction in brush-border Na+ permeability. 3. Autoradiography revealed that hypoxia increased by 6-fold the rate of iron accumulation during enterocyte transit along the lower villus and enhanced by 3-fold the maximal accumulation of iron. Depolarization of the brush border, using a high-K(+)-containing buffer, caused a proportionally greater reduction in iron uptake in control compared with hypoxic tissue suggesting that the raised iron uptake is only partly driven by brush-border hyperpolarization. 4. We conclude that hypoxia increases the expression of iron transport in duodenal brush-border membrane and an enhanced electrical driving force may be involved in this response.

Animals↗
PubMed1997

Identification, purification, and characterization of a soluble interleukin (IL)-13-binding protein. Evidence that it is distinct from the cloned Il-13 receptor and Il-4 receptor alpha-chains.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are structurally and functionally related cytokines which play an important role in the regulation of the immune response to infection. The functional similarity of IL-4 and IL-13 can be explained, at least in part, by the common components that form their cell surface receptors, namely the IL-4 receptor alpha-chain (IL-4Ralpha) and the IL-13 receptor alpha-chain (IL-13Ralpha). Soluble forms of the IL-4Ralpha have also been described and implicated in modulating the effect of IL-4. In this paper we describe the presence of a 45,000-50,000 Mr IL-13-binding protein (IL-13BP) in the serum and urine of mice. This protein binds IL-13 with a 100-300-fold higher affinity (KD = 20-90 pM) than does the cloned IL-13Ralpha (KD = 3-10 nM). In addition to this functional difference, the IL-13BP appears to be structurally and antigenically distinct from the IL-13Ralpha. Finally, unlike the cloned receptor, the IL-13BP acts as a potent inhibitor of IL-13 binding to its cell surface receptor, raising the possibility that it may be used to modulate the effects of IL-13 in vivo.

Animals↗
PubMed1997

Disruption of the disulfide bonds of recombinant murine interleukin-6 induces formation of a partially unfolded state.

A chemical modification approach was used to investigate the role of the two disulfide bonds of recombinant murine interleukin-6 (mIL-6) in terms of biological activity and conformational stability. Disruption of the disulfide bonds of mIL-6 by treatment with iodoacetic acid (IAA-IL-6) or iodoacetamide (IAM-IL-6) reduced the biological activity, in the murine hybridoma growth factor assay, by 500- and 200-fold, respectively. Both alkylated derivatives as well as the fully reduced (but not modified) molecule (DTT-IL-6) retained a high degree of alpha-helical structure as measured by far-UV CD (37-51%) when compared to the mIL-6 (59%). However, the intensity of the near-UV CD signal of the S-alkylated derivatives was very low relative to that of mIL-6, suggesting a reduction in fixed tertiary interactions. Both IAA-IL-6 and IAM-IL-6 exhibit native-like unfolding properties at pH 4.0, characteristic of a two-state unfolding mechanism, and are destabilized relative to mIL-6, by 0.3 +/- 1.6 and 2.4 +/- 1.2 kcal/mol, respectively. At pH 7.4, however, both modified proteins display stable unfolding intermediates. These intermediates are stable over a wide range of GdnHCl concentrations (0.5-2 M) and are characterized by increased fluorescence quantum yield and a blue shift of lambda(max) from 345 nm, for wild-type recombinant mIL-6, to 335 nm. These properties were identical to those observed for DTT-IL-6 in the absence of denaturant. DTT-IL-6 appears to form a partially unfolded and highly aggregated conformation under all conditions studied, as showed by a high propensity to self-associate (demonstrated using a biosensor employing surface plasmon resonance), and an increased ability to bind the hydrophobic probe 8-anilino-1-naphthalenesulfonic acid. The observed protein concentration dependence of the fluorescence characteristics of these mIL-6 derivatives is consistent with the aggregation of partially folded forms of DTT-IL-6, IAM-IL-6, and IAA-IL-6 during denaturant-induced unfolding. For all forms of the protein studied here, the aggregated intermediates unfold at similar denaturant concentrations (2.1-2.9 M GdnHCl), suggesting that the alpha-helical structure and nonspecific hydrophobic interprotein interactions are of similar strength in all cases.

Alkylation↗
PubMed1997

Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Amino Acid Sequence↗
PubMed1997

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1997

Interleukin-6: structure-function relationships.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor, and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affinity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.

Amino Acid Sequence↗
PubMed1997

Molecular evidence for the involvement of alpha crystallin in the colouration/crosslinking of crystallins in age-related nuclear cataract.

The proteins of the lens which become insoluble, crosslinked and coloured as a result of the onset of human nuclear cataract have been studied using a combination of enzymatic digestion and HPLC/mass spectrometry (MS). The objective was to determine if such an approach could provide information on the identities of the polypeptide components involved in the colouration and crosslinking and to discover whether any crystallins predominate in this characteristic post-translational modification process. Initially, coloured high molecular weight peptides were isolated from a tryptic/chymotryptic digest of the 6 M guanidine hydrochloride-insoluble lens protein fraction. These tryptic/chymotryptic peptides were then incubated with pronase and the small peptides released, purified by gel filtration. All but one of the peptides analysed by HPLC/MS/MS were found to contain proline. Peptides derived from alpha-crystallin were found to comprise the great majority of the peptides characterised. No gamma-crystallin peptides were observed. Both alpha A-crystallin and alpha B-crystallin were represented. Further, all but one of these peptides were derived from the N-terminal region of the alpha-crystallin subunits: a region recently implicated in the chaperone activity of alpha-crystallin. This finding suggests that the putative N-terminal domain of alpha-crystallin may be involved at the molecular level in the process of crosslinking and colouration which is known to be characteristic of age-related nuclear cataract. It is, therefore, conceivable that an early stage of these cataractous modifications may involve alpha-crystallin acting as a molecular chaperone.

Aging↗
PubMed1997

The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum.

We have previously shown that a radioiodinated photoreactive analogue of chloroquine, [125I]N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ([125I]ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L. J Biol Chem 1994:269:6955-6961). We now report the identification of the 33 kDa protein. The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with [125I]ASA-Q to monitor the enrichment process. N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P. falciparum lactate dehydrogenase (PfLDH). The plasmodial enzyme was cloned and expressed in E. coli and the recombinant protein used to produce a rabbit antiserum. Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein. The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ. PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.

Affinity Labels↗
PubMed1997

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice.

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of 26Al and 67Ga, has been compared with that of gallium, a proposed tracer for aluminium. 26Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7 ml 100 g-1; renal cortex 102.9 ml 100 g-1; liver 13.0 ml 100 g-1; spleen 8.4 ml 100 g-1 and brain 0.8 ml 100 g-1. 67Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for 26Al, respectively. In the hypotransferrinaemic mice, uptake of 67Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. 26Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced 67Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on 26AI uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.

Aluminum↗
PubMed1997

Fabrication of stable packed capillary reversed-phase columns for protein structural analysis.

Capillary column (< or = 320-micron ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-micron ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300-400 bar) enabled their use for rapid chromatography (> 3400 cm/hr; i.e., approximately 40 microliters/min for 200-micron ID columns) and the loading of large sample volumes (up to 500 microliters). The accurate low flow rates (0.4-4.0 microliters/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.

Amino Acid Sequence↗