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Biomedical subjects

S T Jones

Publications and source records attributed to S T Jones.

At least 19 recordsLinked to original sources
PubMed2005

Storage stability of lutein during ripening of cheddar cheese.

Lutein (3,3'-dihydroxy-alpha-carotene) has been identified as a dietary factor that can delay the onset of age-related macular degeneration (AMD). However, available food sources of lutein contain only modest amounts of the carotenoid. Food fortification with lutein extract has been identified as a low-budget approach to prevent the onset or progression of AMD. The objectives of this study were to 1) incorporate various amounts of lutein into Cheddar cheese; 2) examine the color, pH, microbiological, and sensory characteristics of the Cheddar cheese during storage; and 3) analyze the stability of lutein during the cheese maturation process. Lutein extracted from corn was added to Cheddar cheese in quantities of 1, 3, and 6 mg per serving size. Measurements of the lutein stability were carried out by HPLC using a YMC C30 carotenoid column. Microbiological analyses of cheese samples included aerobic plate count, coliform, and yeast/mold counts. The color attributes a* and b* were significantly different between the treatment and control groups; however, no significant difference was observed in L* value and pH. Significant differences among 1, 3, and 6 mg lutein-enriched cheeses were observed in the aerobic plate count and yeast/mold compared with the control. Cheese samples contained no detectable levels of coliforms (< 10 cfu/g). The HPLC data showed quantitative recovery of lutein during the storage period, and no lutein degradation products were identified. These results indicate that lutein, a functional additive with purported ability to prevent or reduce the onset of AMD, can be incorporated into cheese adding value to this product.

Cheese↗
PubMed1997

Humanization of a mouse antibody against human alpha-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.

alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.

Amino Acid Sequence↗
PubMed1994

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Amino Acid Sequence↗
PubMed1993

Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering.

On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed in Escherichia coli, was purified and characterized. The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group. The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme. Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2. When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2. However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group. Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering.

Animals↗
PubMed1993

Molecular dynamics simulation of a phospholipase A2-substrate complex.

We have used knowledge of the three-dimensional structure of phospholipids and phospholipases A2 together with biochemical data, computer graphics modelling and a 48 ps molecular dynamics simulation to predict the structure of a phospholipase A2-substrate complex. There is remarkable similarity between this predicted structure of enzyme-substrate complex and the structure that can be deduced from the observed enzyme-inhibitor complex. Molecular dynamics simulation highlights the importance of the calcium-ion in substrate binding and the persistence of the His-48 to water-hydrogen bond is compatible with the proposed role of this water molecule as the nucleophile in catalysis.

Animals↗
PubMed1993

Possible clearance of effete polymorphonuclear leucocytes from synovial fluid by cytophagocytic mononuclear cells: implications for pathogenesis and chronicity in inflammatory arthritis.

A feature common to all forms of chronic inflammatory arthritis, irrespective of the possible underlying cause, is the persistent exudation of large numbers of polymorphonuclear leucocytes (PMNL) into synovial fluid. These cells possess potent degradative enzymes and proinflammatory mediators, and their removal is vital to normal inflammatory resolution. A major route of disposal of extravasated PMNL appears to be programmed cell death (apoptosis), followed by their rapid recognition, and intact phagocytosis, by mature tissue macrophages. Such macrophages, containing PMNL (cytophagocytic mononuclear cells (CPM)), long recognised in synovial fluid as Reiter cells, are commonly found in reactive arthritis, spondyloarthritis, and crystal arthritides, but only rarely in rheumatoid disease. In a retrospective analysis of 187 knee synovial fluid cytospins, the relation between the formation of CPM and the presence of apoptotic (pyknotic) PMNL was investigated. As long as the synovial fluid examined was fresh there was a high correlation between numbers of CPM (as a percentage of macrophages) and pyknotic numbers of PMNL in fluids containing CPM. This suggests that the formation of CPM occurs in vivo and is involved in the disposal of PMNL. Numbers of pyknotic PMNL increased rapidly in stored synovial fluid without a significant change in numbers of CPM, and were highest in synovial fluid which did not contain CPM. The presence or absence of CPM, or their disease associations, could not be explained simply by limiting numbers of macrophages, or apoptotic PMNL in synovial fluid. These findings are consistent with a regulatory role for CPM in synovial fluid, where they may be important in preventing autolysis of PMNL, and thus local tissue damage.

Apoptosis↗
PubMed1991

Serological evidence of infection with Helicobacter pylori may predict gastrointestinal intolerance to non-steroidal anti-inflammatory drug (NSAID) treatment in rheumatoid arthritis.

Specific circulating antibodies to the spiral gastric organism, Helicobacter pylori (HP) were detectable in 43% of 68 patients with rheumatoid arthritis by complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), a frequency comparable with that of a normal, age-matched population. Presence of these antibodies correlated strongly with a previous history of peptic ulcer disease (PUD) and to the severity of NSAID-related dyspeptic symptoms, the latter often leading to multiple drug intolerance. This contrasts with short term, prospective NSAID toxicity data, which show little relationship between ulceration and HP carriage. This result suggests, however, that HP may have a definite role in the pathogenesis of symptomatic PUD associated with more chronic NSAID usage, and may have important implications for ulcer prophylaxis in these patients.

Adult↗
PubMed1987

Hierarchy of the strength of Escherichia coli stringent control signals.

The quantitative effect of ppGpp, the effector of stringent control, on various Escherichia coli promoters was measured in an in vitro mixed transcription system. This allowed us to determine, among these promoters, the hierarchy of promoters according to their ppGpp susceptibility. The strength of the stringent control signal, however, was found to be altered when the test promoters were transcribed by ppGpp-insensitive RNA polymerases from relaxed mutants of E. coli, which carry substitutions in the beta subunit gene (rpoB). Thus, it was concluded that the activity of the stringent control signal depends on the nature of the RNA polymerase as well as that of the promoter.

DNA-Directed RNA Polymerases↗
PubMed1986

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VII. RNA polymerase is a target for ppGpp.

RNA polymerase was isolated from two suppressed rpoB amber strains exhibiting relaxed control over RNA synthesis in vivo. In vitro transcription analysis of these mutant holoenzymes carrying defined substitutions at known sites in the beta subunit clearly shows that single amino acid changes in beta render RNA polymerase resistant to ppGpp. This unambiguously demonstrates that RNA polymerase is indeed the target for ppGpp.

DNA Restriction Enzymes↗
PubMed1986

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VIII. Localisation of a region involved in promoter selectivity.

We have previously isolated an E. coli derivative carrying a small internal deletion (delta(rpoB)1570-1) of the beta structural gene. This RNA polymerase deletion mutant has no noticeable phenotype other than a slightly increased generation time in minimal medium. The deletion, which removes about 165 bp, has been localised to between codons 965 and 1,083, indicating it excises part of a tandem repeat structure present in the C-terminal region of beta. Analysis in vitro of purified RNA polymerase from the deletion mutant indicates that this enzyme has an altered promoter selectivity. These observations allow localisation of a site on the beta polypeptide of E. coli RNA polymerase involved in transcriptional initiation.

DNA-Directed RNA Polymerases↗
PubMed1985

Reducing children's psychological stress in the operating suite.

Children often experience extremely high levels of psychological stress in the operating suite because of anxiety associated with the separation from their parents, the unfamiliar and intimidating environment, the presence of strangers in unusual attire (masks, caps, and gowns), the act of being forcibly restrained, and the forceful pressing over the mouth and nose of an anesthetic mask that emits a foul-smelling gas and that seems to restrict breathing in a manner reminiscent of suffocation. The recovery room can be very frightening for children because of the separation from parents and the frequently overpowering scene of human injury and suffering. If the personnel in the operating suite are trained in child development, and if they are encouraged to practice elementary "preventive psychiatry" and to use common sense, then the psychological morbidity resulting from the experience in the operating suite can be minimized. Arrangements ought to be made for children undergoing relatively simple and short operations to go to a place different from the "intensive-care" type of recovery room.

Adolescent↗