Investigation of cutaneous photoadaptation to narrowband ultraviolet B.

BACKGROUND: Photoadaptation describes the skin's ability to withstand an increased dose of ultraviolet (UV) radiation with repeated exposure, and this is the reason for exposure doses being increased during a course of phototherapy. However, directly measured data on photoadaptation are available only for broadband (BB) and not narrowband (NB)-UVB. OBJECTIVES: To measure photoadaptation to narrowband UVB. METHODS: We measured the degree of photoadaptation in patients with psoriasis during a standard course of NB-UVB phototherapy. The minimal erythemal dose (MED) was measured before and towards the end of a course of phototherapy. An adaptation factor (AF) was calculated for each patient using the ratio of final MED to initial MED. Sigmoid dose-response curves were also constructed. RESULTS: MED results were available for 50 patients (mean age 44 years, 28 female). The mean AF was 2·7 (95% confidence interval 2·4-3·0). There was no significant correlation between AF and skin type or initial MED. Dose-response curves were right shifted and parallel after phototherapy, and there was no significant difference in the maximum slope (P = 0·73). CONCLUSIONS: The photoadaptation caused by NB-UVB is considerably less than that reported for BB-UVB. The variation in photoadaptation between patients was not explained by skin type or baseline MED. Physical factors (such as tanning and epidermal thickening) are probably sufficient to account for photoadaptation, rather than downregulation of the inflammatory response. These data should help in the design of phototherapy protocols for NB-UVB to achieve optimal clearance of psoriasis.

Clopidogrel: mechanisms of action and review of the evidence relating to use during skin surgery procedures.

Patients who have skin surgery may be taking medication that increases the likelihood of bleeding, such as clopidogrel, aspirin, warfarin, heparin and nonsteroidal anti-inflammatory drugs (NSAIDS). All of these may increase the risk of perioperative and postoperative bleeding. This article examines the mechanism of action of clopidogrel, current practice, and evidence for or against continuing its use during skin surgery. The mechanisms of action of aspirin, warfarin, heparin and NSAIDS will also be briefly discussed.

Aquagenic palmar wrinkling as a presenting feature of cystic fibrosis gene dysfunction.

Aquagenic palmar wrinkling (APW) is characterized by the rapid and transient oedematous wrinkling of the palms after brief immersion in water. APW has been associated with cystic fibrosis (CF). Since the discovery of the CF gene, the clinical spectrum of CF has broadened from classic severe CF to include milder 'atypical CF' and 'CF-related disorders'. We report an unusual case in which APW occurred in a patient with no lung disease, and in whom investigations showed evidence of CF gene dysfunction. APW may be a presenting feature of a CF-related disorder and should prompt investigation of CF gene dysfunction.

Assessment of the non-HLA-DR-DQ contribution to IDDM1 in British Caucasian families: analysis of LMP7 polymorphisms.

AIMS: Whilst HLA-DRB1 and HLA-DQ alleles contribute to IDDM1, the major determinant of genetic susceptibility to Type 1 diabetes mellitus, other major histocompatibility complex (MHC)-encoded genes may also be involved. The LMP7 (large multifunctional proteasome 7) gene is a potential candidate. The aim of this study was to assess whether LMP7 confers susceptibility to Type 1 diabetes independently of linkage disequilibrium with HLA-DRB1 and HLA-DQ. METHODS: The diallelic LMP7 polymorphism (LMP7*A or *B) was determined in 142 multiplex families from the British Diabetic Association Warren Repository. At least one parent was heterozygous for LMP7 in 112 families and these were informative for calculation of the statistic Tsp. This gives a valid chi2 test of the null hypothesis of no association or no linkage. RESULTS: An excess of transmissions of LMP7*A was observed from parents to affected offspring and the Tsp statistic was significant for association in the presence of linkage. LMP7*A was in positive, and LMP7*B in negative, linkage disequilibrium with the HLA-DRB1*03-DQ2, DRB1*04-DQ8 (group of all DRB1*04 subtypes), DRB1*0401-DQ8 and DRB1*0404-DQ8 haplotypes, although the linkage disequilibrium coefficient (delta) value was not statistically significant for DRB1*0404-DQ8. Analysis of HLA-DR-DQ-LMP7 haplotypes and Tsp analysis of HLA-matched-homozygous parents showed no association between LMP7 alleles and Type I diabetes independent of linkage disequilibrium with HLA-DR-DQ haplotypes associated with increased risk of disease. A contribution of LMP7 alleles to susceptibility to Type 1 diabetes in subjects with low-risk HLA-DR-DQ haplotypes could not be excluded. CONCLUSIONS: LMP7 alleles do not contribute to genetic susceptibility to Type 1 diabetes in subjects with high-risk-associated HLA-DR-DQ haplotypes.

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study.

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.

The role of magnesium in myocardial preservation.

The purpose of this review is to look at the role of magnesium in the formation of preservation and reperfusion solutions for the ischaemic heart. Preservation of the heart during cardiac surgery procedures, including cardiac transplantation, can be divided into distinct phases: arrest, cold storage in the case of transplantation, global ischaemia during implantation or cardiac surgery procedures, followed by reperfusion when the heart is rewarmed and restarted. Although the magnesium ion can play a significant role in myocardial protection, it is important to recognize the different types of protection required during these different phases of surgical procedures. The rationale for the inclusion of magnesium in cardioplegic solutions is threefold: (i) for its negative inotropic effect; (ii) to prevent ischaemia-induced magnesium loss; (iii) to influence cellular ionic movements. Preservation temperature as well as the concentration of other ionic constituents present in the preservation solution alter the effects of magnesium. Results obtained from animal models suggest that elevated magnesium (16 mM) is beneficial to the hypothermic preservation of hearts with extracellular type solutions, especially when calcium is elevated in the solution formulation. Research has shown that the amplitude of the inotropic effect of magnesium varies from one species to another so that the beneficial effect of magnesium is inferior in the less sensitive species. Using the human atrial trabecular preparation as a model for myocardial preservation, we have assessed the effects of elevated magnesium on the recovery of developed force, both for long-term preservation (24 h) during hypothermic arrest (4 degrees C) and for reperfusion during rewarming of the trabeculae. No clear pattern emerged when the ratio of calcium to magnesium was altered in St Thomas' I and II solutions used for the storage. However, when the atrial trabecular preparation was rewarmed in a Krebs Henseleit buffer containing an elevated level of magnesium (16 mM), a greater number of trabeculae reached a greater developed force and had higher levels of energetic metabolites than when the magnesium in the Krebs Henseleit buffer was 1.2 mM. Several studies have suggested that an elevated magnesium prevents calcium overload by competing with this ion at the membrane, and reduces the workload, while ATP reserves and ion homoeostasis are re-established. The role of the magnesium ion in hypothermic preservation of the human myocardium is still not clear after many clinical and experimental studies and requires further investigation.(ABSTRACT TRUNCATED AT 400 WORDS)

pH heterogeneity in aged hypertensive rat hearts distinguishes reperfused from persistently ischemic myocardium.

Myocardium reperfused following ischemia may contain regions that are adequately perfused but temporarily dysfunctional or metabolically abnormal, and regions that are persistently ischemic. By examining heterogeneity in the inorganic phosphate (Pi) resonance in 31P NMR spectra obtained for isolated perfused young v old hypertensive rat hearts made globally ischemic and then reperfused, areas of persistent intracellular acidosis (pHi about 6) were distinguished from recovering tissue (pHi about 7). The extent of persistent regional acidosis reported by Pi heterogeneity in the 31P NMR spectrum of the reperfused heart correlates well with (1) whole heart flow deficit measured as coronary flow, (2) the decrease in extracellular (primarily vascular) 23Na NMR resonance area measured using 23Na NMR and shift reagent, and (3) regional flow deficits identified using perfusate-borne dye markers. Persistent intracellular acidosis during reperfusion was observed only in the hearts of aged hypertensive rats, confirming that the vasculature of these animals is more susceptible to ischemic injury than both younger hypertensive rats and age-matched controls. The portion of the heart that is reperfused following only 16 min of global ischemia is metabolically abnormal, showing incomplete recovery of ATP and pH.

Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: A 31P, 23Na and 87Rb NMR study.

In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na+ concentration ([Na+]i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat hearts were treated with 2 mM DG in the presence of 10IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG + insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM), [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na+ content as assessed by shift reagent-aided 23Na NMR at the end of DG + insulin treatment (98 +/- 18%, 28-36 min of the protocol). In addition, there was no change in the Rb+ influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K+] = 3.76 mM, [Rb+] = 0.94 mM). During DG + insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate)3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ADP]) conditions induced by DG treatment do not inhibit Na+, K(+)-ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na+]i is not due to inhibition of the Na+ pump by reduced [ATP] or [ATP]/[ADP].

Simultaneous measurement of both lipid and lactate in isolated rat hearts by 1H NMR spectroscopy.

Myocardial lipid and lactate levels are sensitive indicators of biochemical status: lipid levels have been shown to increase in response to high fat diets, disease or metabolic stress and elevated lactate levels are indicative of reduced oxygen supply. Selective measurement of lactate or lipid levels by 1H NMR is not straightforward since both the lactate methyl group and lipid methylene groups resonate at 1.3 ppm. We have overcome this difficulty by employing spectral editing techniques to observe both lipid methylene and lactate methyl resonances, and have measured lipid and lactate levels in perfused rat hearts during control perfusion and in response to metabolic stress. Lactate increased during ischemia and decreased during reperfusion, and the ischemia-induced increase is inhibited by iodoacetate, as expected. In contrast, lipid levels increased during ischemia and remained elevated during reperfusion. Hearts from rats fed high fat diet show elevated lipid levels during control perfusion. Data obtained by 1H NMR are consistent with biochemical data, validating the technique.

Lipid accumulation in isolated perfused rat hearts has no apparent effect on mechanical function or energy metabolism as measured by 31P NMR.

Male Sprague-Dawley rats were fed diets that contained 20% by weight soybean oil or rapeseed oil (21% and 43% erucic acid) for 7 days. The rapeseed oil diets increased the cardiac triacylglycerol content 5-fold and 25-fold, respectively, above control values. Hearts were removed from the animals and perfused with modified Krebs-Henseleit buffer at 37 degrees C. The calculated rate-pressure product was used as a measure of contractile function. 31P NMR spectra were acquired throughout a protocol that consisted of 12 min control perfusion, followed by 12 min perfusion with 20 microM isoproterenol, 12 min washout, 12 min total global ischemia, and 28 min reperfusion. The steady state levels of creatine phosphate, ATP, intracellular pH, contractile function, and the free energy of ATP hydrolysis (delta GATP) were determined for all three groups of hearts. Isoproterenol more than doubled the rate-pressure product of the hearts on all diets and decreased the concentrations of creatine phosphate and ATP with a concomitant rise in Pi. After global ischemia, creatine phosphate levels recovered fully, ATP levels remained low, and most hearts developed ventricular fibrillation. Changes in intracellular pH were the same for all groups: pH was 7.1 throughout the equilibration and isoproterenol perfusion period, decreased to pH approximately 6.4 during ischemia, and returned to 7.0 during reperfusion. The results indicate that the fat accumulation that occurs in the hearts of rats fed diets rich in high erucic acid rapeseed oil does not interfere with the cardiac high energy phosphate metabolism or contractile function.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular volume and transsarcolemmal proton movement during ischemia and reperfusion: a 31P NMR spectroscopic study of the isovolumic rat heart.

We have measured, directly and simultaneously, changes in extracellular volume and intra- and extracellular pH during ischemia in the isolated rat heart using 31P NMR spectroscopy. Hearts were perfused with buffer containing 15 mM sodium phenylphosphonate at pH 7.4. Wash in and wash out experiments showed that phenylphosphonate entered only the extracellular (interstitial, vascular and chamber) space of the heart and had no adverse effects on myocardial energetics, contractile function or coronary flow rate. Hearts were subjected to 28 min of total, global ischemia, during which the phenylphosphonate resonance area in the 31P NMR spectra decreased by 83%, indicating that extracellular fluid had moved rapidly from the heart to the bath surrounding the heart, partly as a result of vascular collapse. A separate, morphological study confirmed that 95% of the vasculature had collapsed by 28 min ischemia. Intra- and extracellular pH were determined from the chemical shifts of the P(i) and the phenylphosphonate resonances, respectively. In the pre-ischemic rat heart, intracellular pH was 7.15 +/- 0.03 and extracellular pH was 7.39 +/- 0.03. By 4 min of ischemia, intra- and extracellular pH were the same and decreased concomitantly throughout the remainder of ischemia to final values of 6.09 +/- 0.19 and 6.16 +/- 0.23, respectively. On reperfusion, the extracellular volume and pH returned to pre-ischemic levels within 1 min, but restoration of intracellular pH took > 2.5 min. Thus, a large volume of extracellular fluid moves out of the rat heart to the surrounding bath and the intra- and extracellular pH become the same during total, global ischemia.

On the revised structure of the major phospholipid of Halobacterium salinarium.

Recent fast atom bombardment-mass spectrometry (FABMS) studies (Tsujimoto, K., Yorimitsu, S., Takahashi, T. and Ohashi, M. (1989) J. Chem. Commun. 668-670; Frederickson, H.L., De Leeuw, J.W., Tas, A.C., Van der Greef, J., LaVos, G.F. and Boon, J.J. (1989) Biomed. Environ. Mass. Spectrom. 18, 96-105; Kloppel, K.D. and Fredrickson, H.L. (1991) J. Chromatogr. 562, 369-376) have indicated that the structure of the major phospholipid of Halobacterium salinarium (formerly Halobacterium cutirubrum) is not 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'- phosphate (PGP), but the monomethylated derivative, 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'-methylphosphate (PGP-Me). We have now confirmed the structure of the major phospholipid of extremely halophilic archaebacteria as being this methylated structure (PGP-Me) by 1H- and 13C-NMR, FABMS and TLC of the native phospholipid and its product of mild acid hydrolysis PGP. The methylated structure (PGP-Me), rather than PGP itself, is also the major phospholipid in species of other genera of extreme halophiles examined so far, such as, Haloferax, Haloarcula, Halococcus, Natronobacterium and Natronococcus.

A role for unsaturated fatty acids in mitochondrial movement and inheritance.

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.

Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity.

The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.

Analysis of 23Na NMR spectra from isolated perfused hearts.

The 23Na NMR spectra obtained from isolated hearts perfused with buffer containing the paramagnetic shift reagent dysprosium triethylenetetraminehexaacetic acid, Dy(TTHA)3-, are complex and contain a number of overlapping peaks of different intensities. Spectra from rat, rabbit, guinea pig, and ferret hearts obtained during periods of control perfusion are similar and undergo similar changes when the hearts are subjected to periods of ischemia and reflow. The contributions from the intracellular, interstitial, vascular, and bath compartments to the multiple peaks in the spectra of rats hearts have been assigned. The significant contributions to these spectra of bulk magnetic susceptibility effects and incomplete mixing have been demonstrated through a series of modeling experiments. Since the spectra from hearts of different species are so similar, the peak assignments made for the rat are applicable to spectra from rabbit, guinea pig, and ferret hearts as well. This work provides a framework for quantitative analysis of the spectral changes which occur during conditions such as ischemia and reflow.

Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

Lateral proton conduction in monolayers of phospholipids from extreme halophiles.

Studies have been carried out on the lateral proton conductance properties of monolayers of the major and minor phospholipids of extremely halophilic archaebacteria, 2,3-diphytanyl-sn-glycero-1-phospho-3'-sn-glycerol 1'-phosphate (PGP) and 2,3-diphytanyl-sn-glycero-1-phospho-3'-sn-glycerol (PG), respectively, as well as on their respective deoxy analogues: 2,3-diphytanyl-sn-glycero-1-phospho-1'-propanediol 3'-phosphate (dPGP), 2,3-diphytanyl-sn-glycero-1-phospho-1'-1',3'-propanediol (dPG), and 2,3-diphytanyl-sn-glycero-1-phospho-1'-propanol (ddPG). Lateral proton conduction was found to occur with monolayers of all ether phospholipids examined at reduced surface pressure (pi greater than 25 mN/m) on subphases of low (1 mM) and high (4 M) ionic strength. Proton conduction was also detected in highly condensed monolayers (greater than 35 mN/m) of the naturally occurring phospholipids (PGP, PG) but was abruptly terminated in tightly packed monolayers (greater than 35 mN/m) of the corresponding deoxy compounds (dPGP, dPG, ddPG) on subphases with low ionic strength. conduction did occur, however, along monolayers of the deoxy compounds at high surface pressure when spread on a subphase of high ionic strength (4 M). The abrupt termination of conduction with monolayers of the deoxy compounds at low ionic strength cannot be attributed to a lipid phase transition or to changes in the lateral fluidity of the monolayers, nor was the pK of the fluorescent interfacial proton indicator affected at high surface pressures.(ABSTRACT TRUNCATED AT 250 WORDS)