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.

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.

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.

An investigation of the effects of TETRA RF fields on intracellular calcium in neurones and cardiac myocytes.

PURPOSE: This study aimed to determine whether Terrestrial Trunked Radio (TETRA) fields can affect intracellular calcium signalling in excitable cells. MATERIALS AND METHODS: Intracellular calcium concentration ([Ca(2 +) ](i)) was measured in cultured rat cerebellar granule cells and cardiac myocytes during exposure to TETRA fields (380.8875 MHz pulse modulated at 17.6 Hz, 25% duty cycle). [Ca(2 +) ](i) was measured as fura-PE3, fluo-3 or fluo-4 fluorescence by digital image analysis. RESULTS: Granule cells exposed at specific absorption rates (SARs) of 5, 10, 20, 50 or 400 mW x kg(-1) showed no significant changes in resting [Ca(2 +) ](i). Increases in [Ca(2 +) ](i) in response to potassium-induced depolarization were significantly different from sham controls in TETRA-exposed cells, but the majority of the difference was attributable to initial biological variation between cell cultures. No difference was found between fura-PE3 (UV excitation) and fluo-3 (visible light excitation) measurements in these cells. Exposure to TETRA (50 or 400 mW x kg(-1)) had no significant effect on either the rate or amplitude of spontaneous Ca(2 +) transients in cardiac myocytes. The cells showed normal responses to salbutamol (50 microM) and acetylcholine (10 microM). CONCLUSIONS: Overall, these results showed no evidence of any consistent or biologically relevant effect of TETRA fields on [Ca(2 + )](i) in granule cells and cardiac myocytes at any of the SAR tested.

Swiss ball enhances lumbar multifidus activity in chronic low back pain.

OBJECTIVES: This study examined the effects of sitting surfaces on the cross-sectional area of lumbar multifidus (LM) in patients with Chronic Low Back Pain (CLBP) and healthy controls (HC). DESIGN: Cross-Sectional Case Controlled Study. SETTING: Isle of Man Institute of Sport. PARTICIPANTS: 40 age and sex matched, sporting participants aged 18-45 years, recruited from private physiotherapy practice patients (n = 20 CLBP, 16 male, 4 female, and n = 20 healthy controls, 16 males and 4 females). MAIN OUTCOME MEASURES: Cross-sectional area of LM was measured using rehabilitative ultrasound imaging. RESULTS: Swiss Ball (SB) was more effective at stimulating LM than a Stable Surface (SS) in both groups: CLBP:SB:12.3 (cm(2)) (SD:3.6), SS:10.15 (SD:2.6), p < 0.0001; HC:SB:12.5 (SD:2.7), SS:11.3 (SD:2.9), p < 0.0001). No significant differences between groups were noted. No differences between left and right side cross-sectional areas between or within groups were noted. CONCLUSION: Cross-sectional area of LM increased as the lability of the surface increased, demonstrating that SB was more effective at stimulating LM activity than a non-labile surface. This confirms current clinical practice and supports the use of a labile surface in spinal rehabilitation. The lack of LM asymmetry within and between groups is discussed.

Markers of epidermal differentiation expressed by rat keratinocytes cultured by a modified feeder layer technique.

A broad range of analytical methods has been used to investigate the expression of key differentiation markers in keratinocytes cultured by a modified feeder layer technique. Cultures were stratified and showed many of the features characteristic of epidermal differentiation in vivo including tonofilaments, desmosomes, loss of organelles and thickening of the plasma membrane to form the cornified envelope. Profilaggrin synthesis was detected by 32P-incorporation and the presence of filaggrin suggested that it was broken down by the normal route. Staining with the lectin from Ulex europeus revealed the presence of a fucose-containing cell-surface glycoprotein. Keratin synthesis was shown by 3H-leucine incorporation and keratins were analysed by two-dimensional gel electrophoresis in comparison with those from different levels of the epidermis. Quantitative and qualitative differences were found between in vivo and in vitro epidermal differentiation. In particular, cornified envelope numbers were low, in keeping with the observation by electron microscopy of only one layer of cells with this structure. The absence of a true stratum corneum in vitro was also indicated by the virtual absence of histidase activity and stratum corneum keratins. The keratin species present in vitro most closely resembled those of the basal cells of the epidermis, although even in this case differences were observed. The evidence as a whole is consistent with the belief that epidermal cells do synthesise in vitro many of the important proteins involved in differentiation, but that they nevertheless do not develop a true keratinised stratum corneum.

Alterations in the metabolism of filaggrin in the skin after chemical- and ultraviolet-induced erythema.

We have investigated the effect on the normal synthesis and metabolism of filaggrin of treatment of guinea pig skin with a chemical irritant, hexadecane, or with erythemal doses of UV radiation. Examination of the skin by immunofluorescence with an antiserum against filaggrin demonstrates 3 phases of the response. The first phase is an apparent stabilization of the filaggrin present at the time of treatment. Thus, a zone of stratum corneum is produced which moves up toward the skin surface over the days following treatment, without the loss of immunoreactivity which normally results from the metabolism of filaggrin to free amino acids. The second phase of the reaction, which occurs during the first day after treatment, is a loss of immunoreactive material from the upper viable epidermis, which results over the next day in the formation of a zone of filaggrin-deficient stratum corneum. The third phase, 2-3 days after the treatment, is the reestablishment of immunoreactivity in the newly re-formed granular layer, followed by the formation of an immunoreactive zone at the bottom of the stratum corneum. This zone remains very thin despite the rapid passage of cells through it. This shows that the filaggrin being formed during this phase of the reaction is being broken down normally as the stratum corneum matures. Investigations of the kinetics of filaggrin synthesis and breakdown using a [3H]histidine pulse/chase method, confirm the impression gained from immunofluorescence studies that the time between formation and breakdown of the filaggrin is much reduced in the hyperplastic epidermis resulting from the irritation. Thus, although the hyperplasia is reflected in a thickening of malpighian and granular layers of the epidermis, it does not result in any thickening of the filaggrin-positive zone at the bottom of the stratum corneum. This suggests the action of a control mechanism designed to prevent the extension of this filaggrin-positive zone into the upper stratum corneum.

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.

Low doses of repetitive ultraviolet A induce morphologic changes in human skin.

Repetitive exposure of skin to sunlight is known to result in dermatoheliosis, characterized by photoaging and carcinogenesis. It has been demonstrated previously that relatively large amounts of ultraviolet (UV) A can produce photodamage and it is believed that UVB plays a major role in the induction of photodamage and photocarcinogenesis. The study reported here determines the cutaneous effects of minimal erythemal amounts of solar-simulated UV radiation as well as suberythemal and minimal erythemal doses of UVA. Previously non-sunexposed human skin was irradiated twice weekly for 24 weeks. Biopsies were obtained 12, 24 and 36 weeks after the initial irradiation and assessed for both epidermal and dermal alterations. Dermal elastic tissue content was measured via computerized image analysis. All UV treatment regimens produced observable epidermal and dermal changes. These alterations were observed after only 12 weeks of twice-weekly irradiation and were still evident 12 weeks after the final irradiation. Interestingly, UVA irradiation produced a decrease in elastic tissue content whereas solar-simulated UV produced a slight increase. Most notable were the changes produced by the suberythemal dose of UVA. Surprisingly, this relatively low UVA dose produced a reduction in elastic tissue content. The results of this investigation demonstrate that small amounts of UVA or solar-simulated UV are capable of producing cutaneous photodamage. These findings suggest that even suberythemal doses of repetitive UVA may lead to photoaging of the skin and that there is a need for daily broad spectrum UV protection.

Late paraplegia as a consequence of intraaortic balloon pump support.

There have been several reports of paraplegia after intraaortic balloon counterpulsation in the surgical literature. In each instance, the paraplegia occurred during the period of counterpulsation support. We describe a patient in whom late paraplegia occurred three days after the removal of an intraaortic balloon catheter.

Effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices.

Slices of rat hippocampus were exposed to 700 MHz continuous wave radiofrequency (RF) fields (25.2-71.0 V m(-1), 5-15 min exposure) in a stripline waveguide. At low field intensities, the predominant effect on the electrically evoked field potential in CA1 was a potentiation of the amplitude of the population spike by up to 20%, but higher intensity fields could produce either increases or decreases of up to 120 and 80%, respectively, in the amplitude of the population spike. To eliminate the possibility of RF-induced artefacts due to the metal stimulating electrode, the effect of RF exposure on spontaneous epileptiform activity induced in CA3 by 4-aminopyridine (50-100 microM) was investigated. Exposure to RF fields (50.0 V m(-1)) reduced or abolished epileptiform bursting in 36% of slices tested. The maximum field intensity used in these experiments, 71.0 V m(-1), was calculated to produce a specific absorption rate (SAR) of between 0.0016 and 0.0044 W kg(-1) in the slices. Measurements with a Luxtron fibreoptic probe confirmed that there was no detectable temperature change (+/- 0.1 degrees C) during a 15 min exposure to this field intensity. Furthermore, imposed temperature changes of up to 1 degrees C failed to mimic the effects of RF exposure. These results suggest that low-intensity RF fields can modulate the excitability of hippocampal tissue in vitro in the absence of gross thermal effects. The changes in excitability may be consistent with reported behavioural effects of RF fields.

Exchange arthroplasty for infected knee replacements. A new two-stage method.

We report a series of 17 exchange arthroplasties for infected knee prostheses, ten one-stage and seven two-stage procedures. The method proved successful in controlling infection and restoring function. In two-stage exchanges the interval between the stages was managed by using a prosthesis as a spacer, and acrylic cement beads containing the appropriate antibiotic to provide high local concentrations. Three one-stage procedures had recurrence of infection, but were successfully treated by further exchange operations. All patients had satisfactory function and there have been no serious complications. We recommend this modified two-stage technique for the management of infected knee arthroplasties.

Dry skin, moisturization and corneodesmolysis.

The process leading to the loss of corneocytes form the skin surface is termed desquamation. In healthy skin it is an orderly and essentially invisible process whereby individual or small groups of corneocytes detach from neighbouring cells to be lost to the environment and replaced by younger cells from the deeper layers. Desquamation is carefully controlled to ensure that corneum cohesion and integrity, and hence tissue thickness, is maintained. The most important components of the corneocytes contributing towards intercellular cohesion are the corneodesmosomes and lipids. Corneodesmosomes are proteinaceous complexes which effectively rivet corneocytes together. The intercellular lipids, primarily responsible for the water barrier, also provide part of the extracellular cement. In addition, the shape of the corneocyte itself plays a role in stratum corneum cohesion. Through interdigitation along their peripheral edges, adjacent corneocytes become physically locked together, a process which reinforces the integrity of the tissue. For effective desquamation to occur corneodesmosomes must be degraded: a process catalysed by serine proteases present within the intercellular space and facilitated by subtle changes in lipid composition and phase behaviour. Ultimately, it is the availability of free water which controls corneodesmolysis. In healthy skin this proteolytic process leaves relatively few corneodesmosomes intact in the most superficial layers. By contrast, in chronic and acute dry skin conditions, corneodesmosomal degradation and hence the final stages of desquamation are perturbed, leading to the characteristic formation of visible, powdery flakes on the skin surface. The inability to degrade these structures ultimately reflects a decreased hydrolytic activity of the desquamatory enzymes, either through reduced synthesis of the enzymes, inherent loss of activity, leaching from the surface layers of the corneum or changes in the surrounding lipid-rich microenvironment, which may indirectly reduce enzyme functionality. Increased understanding of the desquamation process is providing new insights into the mode of action of current moisturizing ingredients and is offering opportunities to develop novel therapies for preventing and correcting dry skin.

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.

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.

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.