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Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 163 records · Page 9Linked to original sources

Is nephrocalcin related to the urinary derivative (bikunin) of inter-alpha-trypsin inhibitor?

OBJECTIVE: To isolate, purify, sequence and characterize nephrocalcin (NC), a urinary protein that may be an important determinant of calcium oxalate (CaOx) kidney-stone disease. MATERIALS AND METHODS: Proteins were isolated from human urine using cellulose and resin columns and were sequenced using Edman degradation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of CaOx crystal growth by the isolated proteins was assessed by measuring the deposition of 14C-labelled CaOx. RESULTS: A protein assumed to be NC on the basis of SDS-PAGE, inhibitory and gel filtration properties was isolated from healthy human urine. Its molecular weight and the amino acid sequences of two of its peptides suggested it was identical to fragment HI-14 of the light chain (bikunin) of inter-alpha-trypsin inhibitor (ITI). CONCLUSIONS: NC represents a portion of the light chain of ITI, although this conclusion must remain tentative until confirmed using authentic NC.

Alpha-Globulins↗

Role of interleukin-6 in T-cell activation during primary and secondary infection with Listeria monocytogenes.

Injection of recombinant interleukin-6 (IL-6) into mice enhances recovery from infection with Listeria monocytogenes. In this study, the role of IL-6 during primary and secondary Listeria infection was further tested. Neutralization of IL-6 by polyclonal antibody exacerbated primary infection and significantly delayed gamma interferon production by cultured spleen cells. In contrast, administration of anti-IL-6 antibody at the time of secondary infection did not affect the recovery of mice from infection or gamma interferon production, showing that activated T cells are not dependent on IL-6.

Animals↗

Interaction of interleukin-6, tumour necrosis factor and interleukin-1 during Listeria infection.

Injected recombinant interleukin-6 (IL-6), tumour necrosis factor (TNF) and IL-1 all protect mice against experimental infection with Listeria monocytogenes. We have therefore investigated the interaction of these cytokines during infection. Treatment with recombinant (r)IL-6 enhanced TNF production by spleen cells during the first 2 days of infection. Anti-TNF antibody could totally abolish the protective effect of rIL-6, while the optimal protective function of TNF could not be achieved when IL-6 was neutralized by anti-IL-6 antibody. IL-1 induced a high level of IL-6 in the serum a short time after its administration, and neutralization of IL-6 totally abolished the protective function of rIL-1. The results thus provide further evidence for the complexity of cytokine interaction.

Animals↗

Computer-assisted trauma care prototype.

Each year, civilian accidental injury results in 150,000 deaths and 400,000 permanent disabilities in the United States alone. The timely creation of and access to dynamically updated trauma patient information at the point of injury is critical to improving the state of care. Such information is often non-existent, incomplete, or inaccurate, resulting in less than adequate treatment by medics and the loss of precious time by medical personnel at the hospital or battalion aid station as they attempt to reassess and treat the patient. The Trauma Care Information Management System (TCIMS) is a prototype system for facilitating information flow and patient processing decisions in the difficult circumstances of civilian and military trauma care activities. The program is jointly supported by the United States Advanced Research Projects Agency (ARPA) and a consortium of universities, medical centers, and private companies. The authors' focus has been the human-computer interface for the system. We are attempting to make TCIMS powerful in the functions it delivers to its users in the field while also making it easy to understand and operate. To develop such a usable system, an approach known as user-centered design is being followed. Medical personnel themselves are collaborating with the authors in its needs analysis, design, and evaluation. Specifically, the prototype being demonstrated was designed through observation of actual civilian trauma care episodes, military trauma care exercises onboard a hospital ship, interviews with civilian and military trauma care providers, repeated evaluation of evolving prototypes by potential users, and study of the literature on trauma care and human factors engineering. This presentation at MedInfo '95 is still another avenue for soliciting guidance from medical information system experts and users. The outcome of this process is a system that provides the functions trauma care personnel desire in a manner that can be easily and accurately used in urban, rural, and military field settings. his demonstration will focus on the user interfaces for the hand-held computer device included in TCIMS, the Field Medic Associate (FMA). The FMA prototype is a ruggedized, water-resistant personal computer, weighing approximately 5 lbs. It has an LCD graphical user interface display for patient record input and output, pen-based and audio input, audio output, and wireless communications capabilities. Automatic recording and dynamic, graphical display of time-stamped trends in patient vital signs will be simulated during the demonstration. Means for accessing existing patient record information (e.g., allergies to particular medications) and updating the record with the nature of the injury, its cause, and the treatments that were administered will be shown. These will include use of an electronic pen to mark up anatoglyphs (standard drawings of human body appearing on computer screen) to show where injuries occurred and where treatments were applied, and to input textual descriptions of the nature of the injury, its cause, what treatments were administered, etc. Computer recognition of handwritten inputs will be shown. Likewise, voice annotation and audio playback of patient record information by medics and hospital personnel will be illustrated. These latter technologies free the care providers' hands to treat the patient; they can therefore provide inputs to the patient record while information is fresh in their minds. The audio playback option allows hospital personnel to select more detailed voice annotations of specific portions of the patient record by simply touching the electronic pen to a particular place where an electronic pen marking was made by a medic in the field and then listening to the medic's corresponding audio commentary. Finally, the FMA's means for assisting the medic in simultaneously managing several injured patients will be shown. (abstract truncated)

Emergency Medical Services↗

Molecular cloning of a gene encoding an arabinogalactan-protein from pear (Pyrus communis) cell suspension culture.

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion of carbohydrate (typically > 90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala, Ser, and Thr. They are widely distributed in plants and may play a role in development. The structure of the carbohydrate of some AGPs is known in detail but information regarding the protein backbone is restricted to a few peptide sequences. Here we report isolation and partial amino acid sequencing of the protein backbone of an AGP. This AGP is a member of one of four major groups of AGPs isolated from the filtrate of pear cell suspension culture. A cDNA encoding this protein backbone (145 amino acids) was cloned; the deduced protein is rich in Hyp, Ala, Ser, and Thr, which together account for > 75% of total residues. It has three domains, an N-terminal secretion signal, a central hydrophilic domain containing all of the Pro residues, and a hydrophobic C-terminal domain that is predicted to be a transmembrane helix. Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.

Amino Acid Sequence↗

High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor, and gp-130.

The high affinity human interleukin-6 (IL-6) receptor complex consists of IL-6 and two membrane-associated receptor components, the IL-6 receptor (alpha-subunit) and the high affinity converter and signal transducing molecule, gp-130 (beta-subunit). Recombinant IL-6 and the extracellular ("soluble") components of the IL-6 receptor (sIL-6R) and gp-130 (sgp-130) have been prepared in order to investigate the stoichiometry and binding of these components in the low affinity (IL-6.sIL-6R) and high affinity (IL-6.sIL-6R.sgp-130) IL-6 receptor complexes. Using a combination of size-exclusion chromatography and analytical ultracentrifugation analysis, in the low affinity receptor complex, IL-6 was shown to bind sIL-6R in a stoichiometric ratio of 1:1, whereas the high affinity ternary complex is hexameric consisting of two molecules each of IL-6, sIL-6R, and sgp-130. This is the first direct demonstration of a higher order arrangement for receptor cytokine interactions that exhibit both high and low affinity complexes.

Amino Acid Sequence↗

Role of IL-6 in activation of T cells for acquired cellular resistance to Listeria monocytogenes.

We have previously shown that IL-6 is a major cytokine in the serum of mice infected with the intracellular bacterium Listeria monocytogenes, and that injection of rIL-6 before, but not after, infection promotes the recovery of mice from listeriosis. Here we demonstrate that IL-6 is required in the early stages of infection, in that injection of anti-IL-6 Ab 24 or 4 h before infection, but not 24 h after, led to a massive increase in bacterial numbers 4 or more days after infection. On the other hand, injection of rIL-6 before infection significantly suppressed bacterial numbers in the liver and spleen 3 to 4 days after infection and increased the production of IFN-gamma by in vitro cultured CD4+ and CD8+ T cells in response to specific Ag. rIL-6 did not protect SCID mice, which lack both T and B lymphocytes, against Listeria infection. The protective effect of rIL-6 was neutralized by the injection of Ab to IFN-gamma. We conclude that IL-6 plays an essential role in the activation of T cells to produce IFN-gamma, the cytokine that is central to acquired cellular resistance to intracellular bacteria.

Animals↗

Solution structure of synthetic peptides corresponding to the C-terminal helix of interleukin-6.

Two synthetic peptides corresponding to the C-terminal 19 residues of human and murine interleukin-6, respectively, have been synthesized and their structures in solution investigated using high-resolution 1H-NMR spectroscopy. Both peptides show a marked dependence of chemical-shift dispersion on pH, with a greater degree of structure apparent above pH 4.5, where their glutamate carboxyl groups are ionised. In purely aqueous solution, neither peptide adopts a well-defined structure, although the murine peptide has characteristics of a nascent helix. Titration of the murine peptide with trifluoroethanol produced a significant increase in structure, which was then investigated using two-dimensional NMR. In 50% (by vol.) trifluoroethanol the murine peptide consists of a well-defined central helix of 12 residues with unstructured N-terminal and C-terminal regions. These observations lend experimental support to the current model of the interleukin-6 structure, which proposes a four-helical bundle with the last helix encompassing the C-terminal 20-30 residues. Furthermore, the fact that synthetic peptides corresponding to part of the putative receptor-binding surface of interleukin-6 are able to adopt a similar conformation in solution to that proposed for the intact protein suggests that such peptide analogues should be useful starting points in the design of peptide agonists and antagonists of interleukin-6.

Animals↗

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

Amino Acid Sequence↗

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species.

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2 (PSA-2) fragment recognized three major polypeptides of approximate M(r) 96,000, 80,000 and 50,000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supernatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major showed they are similar but distinct, consistent with being members of a polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M(r) 35,000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Amino Acid Sequence↗

Isolation of the protein backbone of an arabinogalactan-protein from the styles of Nicotiana alata and characterization of a corresponding cDNA.

Arabinogalactan-proteins (AGPs) from the styles of Nicotiana alata were isolated by ion exchange and gel filtration chromatography. After deglycosylation by anhydrous hydrogen fluoride, the protein backbones were fractionated by reversed-phase HPLC. One of the protein backbones, containing mainly hydroxyproline, alanine, and serine residues (53% of total residues), was digested with proteases, and the peptides were isolated and sequenced. This sequence information allowed the cloning of a 712-bp cDNA, AGPNa1. AGPNa1 encodes a 132-amino acid protein with three domains: an N-terminal secretion signal sequence, which is cleaved from the mature protein; a central sequence, which contains most of the hydroxyproline/proline residues; and a C-terminal hydrophobic region. AGPNa1 is expressed in many tissues of N. alata and related species. The arrangement of domains and amino acid composition of the AGP encoded by AGPNa1 are similar to that of an AGP from pear cell suspension culture filtrate, although the only sequence identity is at the N termini of the mature proteins.

Amino Acid Sequence↗

Intestinal iron absorption studies in mouse models of iron-overload.

Three mouse strains have been evaluated as suitable models for investigations into the pathogenesis of iron-overload syndromes. Mice with hereditary heterozygous alpha-thalassaemia had moderately raised reticulocyte counts, but were not anaemic and showed little, if any, iron loading. In contrast, mice with homozygous beta-thalassaemia showed microcytic anaemia, reticulocytosis and splenomegaly. Iron-loading was marked, progressive with age and mainly confined to the spleen. Liver iron-loading increased until the age of 7-8 weeks, with no further increase over successive weeks. Although intestinal iron absorption was modestly increased due to enhanced mucosal uptake, the majority of the 'excess' liver and spleen iron could be accounted for by re-distribution of iron from the erythrocytic compartment. Homozygous hypotransferrinaemic mice, with approximately 1-2% of normal plasma transferrin levels, were markedly anaemic with hypochromic microcytic erythrocytes. Intestinal iron absorption increased 3-4-fold (predominantly due to changes in mucosal transfer), as compared to wild-type controls and heterozygotes, and was ascertained to be a major factor causing the marked hepatic iron overload. Heterozygous hypotransferrinamic mice, with over half normal plasma transferrin levels and a mild degree of hepatic iron loading, showed very similar characteristics to wild-type controls. Thus, of the three models, hpx/hpx mice showed the greatest enhancement in intestinal iron absorption and net iron-loading and provides a suitable animal model of spontaneous iron-overload. Comparison of iron absorption values between the models suggests that reticulocytes cannot account for the enhanced absorption seen in the hpx/hpx mice.

Animals↗

Isolation and characterization of group-I isoallergens from Bermuda grass pollen.

Two isoallergens of Cyn d I were isolated using preparative isoelectric focussing, and were designated Cyn d Ia and b. These isoallergens differ in their pI, molecular weight (Cyn d Ia, 32 kD and Cyn d Ib, 31 kD) and their NH2-terminal sequence. Four monoclonal antibodies (Mabs) were produced using Cyn d Ia as antigen. These Mabs recognized both Cyn d Ia and b. One of the Mabs recognized four different pI forms of Cyn d I on 2D gels. The Mabs also recognized cross-reactive epitopes on proteins from eight other grasses including rye grass, timothy grass, Kentucky bluegrass and Johnson grass.

Allergens↗

Consultation patterns in a community survey of men with benign prostatic hyperplasia.

BACKGROUND: The Stirling benign prostatic hyperplasia natural history group have previously reported a prevalence of this condition of 255 per 1000 in a community study of 1610 men aged 40-79 years. AIM: It was decided to examine the consultation patterns of men with benign prostatic hyperplasia in greater detail. METHOD: All participating men were invited to complete a previously validated lifestyle questionnaire including questions on consultations with their general practitioner during the previous year and previous history of prostatic problems. The men who had a urinary symptom score greater than 11, or who had a urinary flow rate of less than 15 ml per second were examined by transurethral ultrasonography for prostate size. RESULTS: Of 364 men with benign prostatic hyperplasia, 89% had not consulted their doctor about urinary symptoms in the year prior to the study. Men with moderate to severe urinary symptoms were six times more likely to have consulted their doctor than those with mild symptoms. Moderate to severe symptoms and greater interference with daily living activities were both associated with a greater likelihood of consultation, independent of age. Of all the men in the study referred to the specialist clinic for assessment of prostate size by transurethral ultrasonography, two thirds were referred because of low urinary flow rate and one third because of high urinary symptom scores. The reported consultation data showed a reverse ratio of one third of those consulting having a low urinary flow rate and approximately two thirds having urinary symptoms. CONCLUSION: While mass screening is unjustified, there is a need for patient education about benign prostatic hyperplasia in general and the recognition of declining strength of urinary flow as a symptom of benign prostatic hyperplasia and not of ageing alone. Furthermore, evaluation of primary care use of urinary flowmeters and the development of local protocols are suggested as elements of a case finding strategy for benign prostatic hyperplasia based on patient led consultation.

Adult↗