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Erythropoietic protoporphyria (EPP) is a rare inherited photodermatosis that causes lifelong painful photosensitivity. Neither its full clinical spectrum nor its impact on quality of life (QOL) have been investigated in a large group of patients. We sought to document the epidemiology of EPP and its impact on QOL in a high proportion of all patients with EPP resident in the U.K. Subjects with EPP were identified from U.K. clinical databases and were assessed by the same clinical investigator over a 7-month period using a standardized proforma and Dermatology Life Quality Index questionnaires. Three hundred and eighty-nine living subjects with EPP were identified, giving a minimum U.K. prevalence of 1 in 136 000. Of these, 223 (114 females, 109 males; median age 34 years, range 5–87) were investigated. Mean total erythrocyte porphyrin (TEP) was higher in males (29·3 μmol L-1) than females (23·1 μmol L-1). The median age at onset (1·1 years) preceded that at diagnosis by 10·9 years. Median times to onset of symptoms after sun exposure, onset of signs (oedema, erythema) and resolution of signs were 20 min, 6 h and 3 days, respectively. Most subjects reported priming (85%), absence of protection by glass (92%), exacerbation by wind (68%), no family history of photosensitivity (58%), no symptoms during winter (56%) and had chronic skin lesions (79%). Symptoms changed little with age but improved during pregnancy in 47% of gravid women. Most subjects used protective clothing and a sunscreen; 28% were taking beta-carotene and a further 56% had taken it; 29% were not under regular medical care. Two subjects (0·9%) had liver failure and 8% had gallstone disease. QOL was markedly impaired with scores similar to those in severe dermatological disease. QOL scores correlated weakly with TEP ( r = 0·215) and time to onset of symptoms ( r = –0·267), but not with age at onset. In conclusion, we have reported the epidemiology of EPP. It is a persistent, socially disabling disease with a marked impact on QOL. Its diagnosis is often overlooked. Neither TEP, age at onset nor time to onset of symptoms are useful predictors of impaired QOL in individual patients. [ABSTRACT FROM AUTHOR]

Acute neurovisceral attacks of porphyria can be life threatening. They are rare and notoriously difficult to diagnose clinically, but should be considered, particularly in female patients with unexplained abdominal pain, and associated neurological or psychiatric features or hyponatraemia. The diagnosis might be suggested by altered urine colour and can be confirmed by finding an elevated porphobilinogen concentration in fresh urine protected from light. Severe attacks require treatment with intravenous haem arginate and supportive management with safe drugs, including adequate analgesia. Intravenous glucose in water solutions are contraindicated as they aggravate hyponatraemia, which can prove fatal.

Erythropoietic protoporphyria (EPP) is a syndrome in which accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver leads to acute photosensitivity and, in about 2% of patients, liver disease. More than 95% of unrelated patients have ferrochelatase (FECH) deficiency (MIM 177000) while about 2% have X-linked dominant protoporphyria (XLDPP) (MIM 300752) caused by gain-of-function mutations in the ALAS2 gene. Most FECH-deficient patients are compound heterozygotes for a hypomorphic allele (FECH IVS3-48C) and a deleterious FECH mutation that together lower FECH activity to around 30% of normal. The frequency of the IVS3-48C allele varies between populations, ranging from less than 1% to 45%. About 4% of unrelated FECH-deficient patients are compound heterozygotes or homozygotes for rare FECH mutations and have lower enzyme activities. Acquired somatic mutation of FECH secondary to myeloid disease may rarely cause EPP. The risk of liver disease is increased in XLDPP and in FECH-deficient patients who are hetero- or homoallelic for rare FECH mutations. Inherited FECH-deficient EPP is an autosomal recessive disorder with some families showing pseudodominant inheritance; the proportion of such families being determined by the population frequency of the IVS3-48C allele.

The porphyrias comprise a group of disorders of the haem biosynthesis pathway that can present with acute neurovisceral symptoms, skin lesions or both. Acute porphyrias present with severe abdominal pain, confusion and seizures which may be life-threatening. Specific treatment with haem preparations should be instituted as soon as possible following confirmation of increased excretion of porphobilinogen in the urine. Supportive treatment includes opiate analgesia, monitoring for and treating complications such as hypertension and hyponatraemia. Follow-up should include counselling on lifestyle modification involving avoidance of alcohol, smoking and known porphyrogenic drugs and diet. Identification and counselling of at risk relatives is essential. The cutaneous porphyrias result from porphyrin-induced photosensitivity and can present with either acute photosensitivity or skin fragility and blisters. All cutaneous porphyrias can be alleviated by avoidance of sunlight. Treatment of erythropoietic protoporphyria involves administering large doses of beta-carotene, which may improve tolerance to sunlight. Porphyria cutanea tarda can be effectively treated by phlebotomy or low dose chloroquine. Congenital erythropoietic porphyria is a rare, early onset, severe, photomutilating condition for which bone marrow transplantation has been shown to be successful.

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.

We review and present current evidence supporting independent regulation of nuclear Ca2+ ([Ca2+]n). The nucleus and nuclear envelope contain proteins to both regulate and respond to changes in [Ca2+]n. However, this does not prove that [Ca2+]n is independently regulated from cytosolic Ca2+ ([Ca2+]c). Studies using fluorescent dyes suggested that changes in [Ca2+]n differed in magnitude from changes in [Ca2+]c. These studies have been criticised as the nuclear environment alters the fluorescent characteristics of these dyes. We have evaluated this question with aequorin targeted to the nucleus and cytoplasm and shown that the characteristics of the indicators are not altered in their respective environments. We have demonstrated that different stimuli induce changes in [Ca2+]n and [Ca2+]c that vary both temporally and in magnitude. The nucleus appeared to be shielded from increases in [Ca2+]c, either through a mechanism involving the nuclear envelope or by cytosolic buffering of localised increases in Ca2+. In addition, agonist stimulation resulted in an increase in [Ca2+]n, consistent with release from the perinuclear Ca2+ store. There was a stimulus dependence of the relation between [Ca2+]n and [Ca2+]c suggesting differential regulation of [Ca2+]n. These results have important implications for the role of Ca2+ as a specific regulator of nuclear events through Ca2+ binding proteins. In addition, they highlight the advantages of using targeted aequorin in intact cells to monitor changes in organelle [Ca2+].

We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1-100 microM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 microM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.

Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We have engineered the Ca2+ sensitive photoprotein aequorin to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum. DNA encoding the engineered aequorins have been inserted into a replication deficient adenovirus (Ad) type 5 E1-vector, under control of the cytomegalovirus (CMV) major immediate early promoter. The Ad vector provides a simple and efficient method to express the photoproteins in a wide variety of mammalian cell types. Efficient targeting of the photoproteins to the appropriate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical agents which affect the activity of the CMV promoter. In HeLa cells expressing nuclear targeted aequorin or cytosolic aequorin, ATP or histamine induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. In addition to epithelial type adherent cell lines (COS7 and HeLa), aequorin expression was also readily detected in non-adherent cells of myeloid lineage (K562 and HL60) and non-adherent primary cells polymorphonuclear leucocytes (neutrophils). The Ad vectors can, therefore, be used to express targeted aequorin in a range of different cell types and represents a novel method to monitor changes in free [Ca2+] in cellular organelles.

Recombinant acquorin is widely used as an intracellular Ca2+ indicator within live cells. Our data shows that recombinant apoacquorin was unstable within the cytosol, with a half life of approximately 20 minutes. Targeting of the protein to subcellular organelles resulted in an increase in stability which may be due to either stabilisation of the photoprotein structure or the absence of the relevant proteases within the organelles. When the apoprotein was reconstituted with the prosthetic group coelenterazine, there was a substantial increase in it's half life in the cytosol. We propose that this variable stability makes acquorin an ideal reporter of gene expression.

The Ca(2+)-activated photoprotein aequorin has been targeted to the nucleus of live cells by engineering nucleoplasmin, a nuclear structural protein from Xenopus laevis, onto the amino terminus. Successful targeting of the apoprotein was demonstrated by immunolocalization and selective permeabilization of the plasma membrane. Functional protein was reconstituted in live cells by incubation with coelenterazine. The effect of elevating cytosolic Ca2+ on nuclear Ca2+ was investigated in populations of live COS7 cells expressing either cytosolic aequorin or nuclear aequorin. Incubation of cells with ionomycin, in the absence of external Ca2+, released Ca2+ from internal stores causing an increase in chemiluminescent light emission from cytosolic aequorin but not nuclear aequorin. Nonagonist-dependent movement of Ca2+ across the plasma membrane, induced by the membrane attack complex of complement, causes a large increase in cytosolic Ca2+ which triggered 95% of cytosolic aequorin but only 50-60% of nuclear aequorin. These results provide clear evidence for a nucleocytoplasmic barrier to Ca2+.

The Ca(2+)-activated photoprotein aequorin has been engineered with the endoplasmic reticulum (ER)-targeting sequence from calreticulin at the N-terminus and the KDEL sequence at the C-terminus so that it locates in the ER of living cells. Targeting of apoaequorin to the ER of COS7 cells was demonstrated by immunolocalization. Selective permeabilization of cells expressing the modified protein suggested that targeting was highly efficient. Functional photoprotein was reconstituted in live cells by incubating them with coelenterazine. Light emission from cells expressing ER aequorin showed that the estimated free Ca2+ within the ER of live cells at 37 degrees C was 0.3-1.0 microM, some 10 times that in the cytosol. An increase in the rate constant for aequorin light emission was demonstrated when the cells were warmed from 4 degrees C. This increase could be in part, but not wholly, explained by an increase in rate constants for aequorin at higher temperatures and a change in kinetics as a result of the ER targeting of aequorin. The increase in rate constants in the cells was inhibited by thapsigargin and occurred in the presence or absence of extracellular Ca2+. These results highlight the importance of converting aequorin light emission to rate constants and of calibrating any variants if qualitative and quantitative conclusions are to be drawn about free Ca2+ in intracellular compartments.

Fluorimetric titrations of mammalian [Arg8] LH-RH, chicken [Gln8] LH-RH and an analogue [Lys8] LH-RH revealed pK values of 5.80, 6.22 and 6.01 for His2, and 9.65, 9.88 and 9.88 for Tyr5. The titration ranges for His2 were 1.72, 2.03 and 1.71 while the range for Tyr5 was rather similar (approximately 1.7) for all three peptides. Biological activity and receptor binding in the mammalian system for chicken LH-RH was 1% relative to mammalian LH-RH while [Lys8] LH-RH had a relative activity of approximately 10%. In contrast, mammalian and chicken LH-RH were equipotent in stimulating LH release from chicken pituitary cells. The results indicate differences in the receptors related to the conformations of LH-RH and position 8-substituted analogues.