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Biomedical subjects

I R Scott

Publications and source records attributed to I R Scott.

29 records · Page 2Linked to original sources
PubMed1983

Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.

The urea-soluble protein profiles of guinea pig, rat, mouse and human epidermis have been compared by non-equilibrium pH gradient/sodium dodecyl sulphate two-dimensional gel electrophoresis. The histidine-rich proteins (filaggrins) were identified firstly by their characteristic specificity and kinetics of labelling with [3H]histidine and [32P]phosphate, and secondly by their ability in vitro to aggregate keratin filaments specifically into bundles. In all species the phosphorylated filaggrin precursor, profilaggrin, is resolved as a single or doublet band with an apparent molecular weight greater than 300,000 and a neutral or slightly acidic iso-electric point. In striking contrast, the strongly basic filaggrins produced from similar profilaggrins form molecular weight families that are clearly species specific. In rat and man there is a single, principal molecular weight form of filaggrin (Mr 45,000 and 38,000, respectively), while mouse and guinea pig have heterogeneous families, including high molecular weight variants (Mr greater than 200,000). Even filaggrins of a particular molecular weight are not homogeneous proteins, but consist of a number of iso-electric variants, some of which are considerably less basic than the bulk of the filaggrins. Incorporation studies using [3H]arginine and [32P]phosphate indicate that the iso-electric variance is not due to residual phosphate, following profilaggrin breakdown, but rather to a conversion of basic arginine residues into neutral citrulline residues. Filaggrins of all the molecular weights from all the species studied share the ability to aggregate keratin filaments into large, insoluble macrofibrils. However, the more acidic iso-electric variants have lower affinities for keratin, particularly in man and guinea pig where the most acidic filaggrins have completely lost the ability to aggregate keratins. We discuss the possibility that a loss of keratin binding ability, resulting in a loosening of the keratin fibre/filaggrin matrix is necessary before the normal complete proteolysis of the filaggrins can occur.

Amino Acids↗
PubMed1983

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels.

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.

Animals↗
PubMed1983

Pyrrolidone carboxylic acid synthesis in guinea pig epidermis.

To establish the in vivo mechanism of synthesis and accumulation of epidermal pyrrolidone carboxylic acid (PCA), enzymes potentially capable of PCA synthesis have been quantified and located within the guinea pig epidermis. Intermediates in the synthesis of [3H]PCA from a pulse of [3H]glutamine have been identified and quantified to determine which of the several possible metabolic routes occurs in vivo. PCA appears to be synthesized from substrate derived from the breakdown within the stratum corneum of protein synthesized several days earlier. The predominant route is probably via the nonenzymic cyclization of free glutamine liberated from this protein. In view of the high activity of gamma-glutamyl cyclotransferase in the stratum corneum, a minor contribution to PCA formation by the action of the enzyme on gamma-glutamyl peptides cannot be excluded.

Animals↗
PubMed1982

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination.

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.

Bacillus megaterium↗
PubMed1982

Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.

The pool of free amino acids, urocanic acid and pyrrolidone carboxylic acid in mammalian stratum corneum has been shown to be derived principally or totally from the histidine-rich protein of the keratohyalin granules. The time course of appearance of free amino acids and breakdown of the histidine-rich protein are similar, as are the analyses of the free amino acids and the histidine-rich protein. Quantitative studies show that between 70 and 100% of the total stratum corneum-free amino acids are derived from the histidine-rich protein.

Amino Acids↗
PubMed1981

Studies on the synthesis and degradation of a high molecular weight, histidine-rich phosphoprotein from mammalian epidermis.

The synthesis and subsequent fate of the histidine-rich proteins, which form a major component of keratohyalin granules in mammalian epidermis, have been studied in the guinea-pig and new-born rat. In both species the protein first synthesised is of very high molecular weight, approximately 340 000. It is short-lived and breaks down to lower molecular weight proteins 1-2 days after its synthesis. These smaller proteins differ in the two species. In the guinea-pig, the high molecular weight protein breaks down to proteins of molecular weight 250 000 and 200 000, which are themselves unstable and break down to low molecular weight species, probably amino acids. The initial breakdown of the high molecular weight protein coincides with the dispersion of the keratohyalin granules and the transition of the granular cell into the stratum corneum. This high molecular weight histidine-rich protein has been purified to homogeneity, despite its instability to several treatments during purification. The protein is highly phosphorylated, containing 6 mol% of phosphoserine, but is otherwise very basic. The possibility that dephosphorylation of the protein produces highly basic matrix proteins in the stratum corneum is discussed.

Amino Acids↗
PubMed1981

Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid.

The synthesis of urocanic acid by histidine ammonia-lyase in guinea-pig epidermis was investigated in various ways. 1. In epidermal homogenates the enzyme obeys Michaelis-Menten kinetics and shows marked dependence of its activity of pH, such that below pH 6 it is inactive. 2. Part-thickness skin samples cultured with radioactive histidine do not accumulate detectable radioactive urocanic acid during 3 days in culture. 3. Very little histidine ammonia-lyase activity can be detected in the living cells of the epidermis. The enzyme is almost completely restricted to the dead cells of the stratum corneum. 4. Radioactive histidine injected into living animals does not result immediately in the accumulation of radioactive urocanic acid. By 3 days after the injection, however, both radioactive urocanic acid and histidine appear, apparently at the expense of radioactive proteins, 5. In isolated stratum corneum, the residual histidine can be converted into urocanic acid by the histidine ammonia-lyase in the tissue only if the natural acidity of the tissue is neutralized. It is concluded from these observations that the biosynthesis of urocanic acid occurs naturally only in the stratum corneum, which contains only dead cells. The amount of urocanic acid accumulated is limited by the availability of free histidine produced by proteolysis of residual protein in these cells and by the penetration into the stratum corneum of the 'acid mantle' of the skin.

Ammonia-Lyases↗
PubMed1978

Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.

Adenine Nucleotides↗
PubMed1978

Metabolism and the triggering of germination of Bacillus megaterium. Use of L-[3H]alanine and tritiated water to detect metabolism.

L-[2,3-3H]Alanine was used to probe for metabolism of alanine during triggering of germination of spores of Bacillus megaterium KM. No detectable incorporation of label into any compound, including water, was found, indicating that any metabolism involving the alanine germinant must be at a very low rate and also that alanine racemase is absent from spores of this strain. Spores were germinated in 3H2O to find if any of the many metabolic reactions causing irreversible incorporation of 3H into reaction products took place during triggering of germination. No incorporation was detected until 2-3 min after addition of germinants. It is therefore concluded that a wide variety of metabolic routes, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and amino acid metabolism are either not involved in the reactions causing the triggering of germination or operate at an extremely low rate during this process.

Alanine↗
PubMed1978

Study of calcium dipicolinate release during bacterial spore germination by using a new, sensitive assay for dipicolinate.

The release of calcium and dipicolinic acid from spores of Bacillus megaterium KM during L-alanine-induced triggering of germination has been studied using a new, simple, and rapid assay for dipicolinic acid capable of detecting a concentration of 0.5 micron. The release of both calcium and dipicolinate started within seconds of exposure of the spores to L-alanine, thus preceding other measurable changes associated with germination. From the earliest times, the two substances were released in equimolar quantities, although later in germination calcium predominated.

Bacillus megaterium↗