The Clinical Utility of a Low Serum Ceruloplasmin Measurement in the Diagnosis of Wilson Disease.

The first step in screening for potential Wilson disease is serum ceruloplasmin testing, whereby a level of less than 0.2g/L is suggestive of the disease. We aimed to determine what proportion of an Irish population had a low ceruloplasmin level, whether low measurements were appropriately followed-up and what were the clinical outcomes. We conducted a retrospective review of all serum ceruloplasmin measurements between August 2003 and October 2009 in a large tertiary referral centre in Southern Ireland. Clinical data, serum ceruloplasmin, liver function tests, urinary copper and liver biopsy reports were all recorded where available. 1573 patients had a serum ceruloplasmin measurement during the 7-year study period. 96 patients (6.1%) had a ceruloplasmin level < 0.2g/L and of these only 3 patients had Wilson disease. There was only 1 new diagnosis. Only 27 patients (28.1%) had some form of confirmatory testing performed. In our centre's experience, the positive predictive value of a significantly low ceruloplasmin level is 11.1% (95% CI 2.91-30.3%). In practice a low serum ceruloplasmin measurement is often not followed by appropriate confirmatory testing. Measuring serum ceruloplasmin as a singular diagnostic test for Wilson disease or as part of the battery of unselected liver screening tests is inappropriate and low-yield.

Maternal serum cholesterol levels are elevated from the 1st trimester of pregnancy: a cross-sectional study.

Cholesterol is monitored in the non-pregnant adult population, where normal values are established. Although reported to be elevated in pregnancy, cholesterol is neither routinely measured nor treated. We aimed to investigate cholesterol levels throughout pregnancy and to establish reference values for cholesterol in healthy pregnant women. This was a cross-sectional analysis of serum cholesterol in healthy women with an uncomplicated singleton pregnancy. Pregnant women attending for antenatal care were recruited and cholesterol levels assayed at 12, 20, 28 and 36 weeks' gestation and on day 1-3 postpartum. A total of 222 women were recruited. The majority (95%) were white Irish, with a median age of 31 years (range 16-46). Median BMI was 25.9 kg/m2 (range 18-40) and 16% were smokers. Cholesterol levels were elevated in all trimesters of pregnancy, with median values from 1st trimester raised outside the non-pregnant adult range. High-density lipoprotein (HDL) levels ranged from 0.9 to 3.7 mmol/l and low-density lipoprotein (LDL) levels ranged from 1.3 to 6.1 mmol/l. Fasting, smoking and obesity did not have any significant effects on results. Total and LDL-cholesterol levels were raised throughout pregnancy. Levels were above non-pregnant adult ranges as early as the 1st trimester. The implications of this on fetus and mother are undetermined and deserve further investigation.

The Leu262Val polymorphism of presenilin associated rhomboid like protein (PARL) is associated with earlier onset of type 2 diabetes and increased urinary microalbumin creatinine ratio in an Irish case-control population.

AIMS: Environmental and genetic factors contribute to the evolution of type 2 diabetes (T2DM). Presenilin associated rhomboid like protein (PARL) is a mitochondrial protein that has been implicated in T2DM in both the rodent Psammomys obesus and in humans. The SNP variant (Leu262Val) in PARL has been shown to be associated with hyperinsulinaemia in an age-dependent manner in a US non-diabetic, cohort. However, this finding has not been replicated in UK cohorts. We studied Leu262Val associations in an Irish Caucasian T2DM case-control population. METHODS: An RFLP-PCR assay using BstN I was used to assess Leu262Val genotype in a total of 613 subjects, 421 with T2DM and 192 controls. RESULTS: In the control group genotype frequencies were as follows 27.37% (GG), 51.58% (CG) and 21.05% (CC), while in the group with T2DM 30.64% (GG), 47.74% (CG) and 21.62% (CC). We observed no association between Leu262Val variant and T2DM nor was there an association with plasma insulin concentrations or BMI. There was no interaction between age and fasting plasma insulin concentration. However, in the group with T2DM the C allele was associated with higher urinary albumin to creatinine ratio while the GG genotype was associated with an earlier age of onset of T2DM. CONCLUSION: The Leu262Val polymorphism of PARL is not associated with markers of insulin resistance. However, in subjects with T2DM, genetic variation at this locus may indicate earlier onset of T2DM and increased susceptibility to nephropathy and cardiovascular complications.

Photosensitizers for photodynamic therapy of cutaneous disease.

Photodynamic therapy is a rapidly developing treatment modality in dermatology. A sensitizer drug is activated by light in the presence of oxygen. This results in the release of reactive oxygen species that damage the target tissue. The ideal features of a photosensitizer are that it should be highly selective for lesional tissue, activated by light of a sufficiently long wavelength for tissue penetration, and have a high photodynamic yield (i.e. production of singlet oxygen). A short time interval between administration and its maximal accumulation in tumour tissue, followed by rapid tissue clearance, are also desirable. First-generation sensitizers were complex chemical mixtures, needing parenteral administration and causing troublesome and prolonged photosensitivity. A range of second-generation sensitizers of different chemical families show several advantages including purity, longer activation wavelength and less prolonged photosensitivity, but effective topical formulations have not been developed. Currently, the most convenient and widely practised form of PDT for cutaneous disorders is the topical application of the pro-drug delta-aminolevulinic acid or its methylated ester, which are activated by light following metabolism to the endogenous sensitizer protoporphyrin IX.

Polychlorinated biphenyl contamination and minisatellite DNA mutation rates of tree swallows.

The evidence that exposure to polychlorinated biphenyls (PCBs) leads to mutations is equivocal and controversial. Using multilocus DNA fingerprinting, we compared the mutation rate of tree swallows (Tachycineta bicolor) nesting at sites with high and low levels of contamination with PCBs. The upper Hudson River, USA, is highly contaminated with PCBs as a result of releases from two capacitor manufacturing plants in Hudson Falls and Fort Edward, New York, USA. Tree swallows nesting nearby have some of the highest known concentrations of PCBs in their tissues of any contemporary bird population (up to 114,000 ng PCB/g tissue). We found no difference in mutation rates between sites in New York with high PCB contamination and reference sites in Wisconsin, USA, and Ontario and Alberta, Canada, with known or presumably low levels of contamination. Thus, the mechanism behind altered reproductive behavior of tree swallows along the upper Hudson River is most likely physiological impairment, such as endocrine disruption, rather than mutation.

Topical tacrolimus in the management of peristomal pyoderma gangrenosum.

BACKGROUND: Peristomal pyoderma gangrenosum (PPG) is a potentially disabling disease in stoma patients. Topical tacrolimus has been shown to be effective in the management of pyoderma gangrenosum. Unfortunately, greasy topical treatments may be impractical for PPG because of impaired appliance adhesion. OBJECTIVE: The purpose of this open study was to evaluate the therapeutic effectiveness of topical tacrolimus 0.3% formulated in carmellose sodium paste compared with topical corticosteroid preparations in the management of PPG. RESULTS: A total of 11 patients with PPG received treatment with topical tacrolimus 0.3% in Orabase trade mark and 13 with topical clobetasol propionate 0.05% as monotherapy in each case. Seven of the tacrolimus-treated group healed completely (mean time to healing: 5.1 weeks) compared with five of the clobetasol propionate-treated group (mean time to healing: 6.5 weeks). Topical tacrolimus was significantly more effective than clobetasol propionate in managing larger PPG lesions (ulcer diameter > 2 cm). In six patients, who had failed to respond adequately to multiple systemic and topical treatments for PPG, the addition of topical tacrolimus was associated with healing of PPG within 6 weeks. CONCLUSION: These results suggest that topical tacrolimus 0.3% in Orabase trade mark is a more effective and expeditious treatment than clobetasol propionate 0.05% for PPG. It is significantly more effective than clobetasol propionate 0.05% in managing lesions larger than 2 cm in diameter. Topical tacrolimus may be highly effective when other systemic or topical treatments have been unsuccessful.

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development.

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.

A Drosophila complementary DNA resource.

Collections of nonredundant, full-length complementary DNA (cDNA) clones for each of the model organisms and humans will be important resources for studies of gene structure and function. We describe a general strategy for producing such collections and its implementation, which so far has generated a set of cDNAs corresponding to over 40% of the genes in the fruit fly Drosophila melanogaster.

The differentially conserved residues of carbamoyl-phosphate synthetase.

Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein. The larger of the two subunits (M(r) approximately 118,000) contains a pair of homologous domains of approximately 400 residues each that are approximately 40% identical in amino acid sequence. The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain approximately 79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other. The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled. True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other. Neither of the two possible chimeric proteins was structurally stable. These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent. This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues.

Topical sucralfate in the management of peristomal skin disease: an open study.

Dermatoses affecting the skin around stoma sites are common and difficult to treat. We have investigated the effectiveness of topical sucralfate in the management of peristomal dermatoses in adults using an open study design. Apart from forming a physical barrier to further irritation, sucralfate binds to basic fibroblast growth factor preventing its degradation and thereby promotes healing. In eight out of nine patients with faecal or urine erosions, daily, topical sucralfate treatment was associated with healing within 4 weeks. There was limited or no response to treatment in a further nine patients with traumatic ulcers, excoriated dermatitis or pyoderma gangrenosum. Topical sucralfate represents a safe, inexpensive and effective therapeutic intervention, particularly for those patients with high output or short stomas where repeated stoma leakage may be unavoidable.

Immobilized sample amplification for quantitative determination of retroviruses.

Immobilized sample amplification (ISA) is a novel method for amplification, detection, monitoring, and quantitative determination of nucleic acids from a minute amount of sample. We present here a novel quantitative ISA assay for retroviruses using a replication-defective recombinant retrovirus as a model retrovirus. Samples, as small as 5 to 10 microl or as large as 1 ml or more in volume, are readily immobilized on a nylon or polyester matrix. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. An ISA assay of a 10-microl viral sample generates results equal to or better than that of RT-PCR on equivalent amount RNA isolated from larger sample volumes. Recovery of RNA from small volumes, such as 10 microl, is almost impossible, whereas ISA assay detects retroviruses from as small as 1 to 5 microl of viral samples containing 10(4) cfu/ml determined by colony-forming assay. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. This method detects less than 10 infectious retroviral particles as determined by both colony-forming assay and electron microscope studies. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.

Influence of chloride ions on alpha1-adrenoceptor mediated contraction and Ca2+ influx in rat caudal artery.

The objective of the present investigation was to compare and contrast the effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8-Bromo-cyclic GMP), an analogue of guanosine 3':5'-cyclic monophosphate, felodipine, a dihydropyridine Ca2+ channel antagonist, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a putative chloride channel antagonist on alpha1-adrenoceptor mediated contraction and Ca2+ influx in rat caudal artery, in normal physiological salt solution and in chloride-free solution. Isometric contractions and 45Ca2+ influx were measured in isolated rat caudal arterial rings. Phenylephrine induced concentration-dependent contractions were inhibited by 8-Bromo-cyclic GMP (10 microM), felodipine (10 nM) and NPPB (3.0 microM). Removal of chloride ions also impaired phenylephrine-induced contractions. In chloride-free buffer, phenylephrine-induced contractions were partially inhibited by the presence of 8-Bromo-cGMP or felodipine, while NPPB had no effect. Phenylephrine induced 45Ca2+ influx was inhibited by the presence of 8-Bromo-cyclic GMP, felodipine and NPPB. Moreover, removal of chloride ions also inhibited phenylephrine-induced 45Ca2+ influx. The results of our study demonstrate that in the rat caudal artery the inhibitory effects of 8-Bromo-cyclic GMP, felodipine and NPPB, are mediated through a reduction of Ca2+ influx. In addition, chloride ions, in part, play a role in alpha1-adrenoceptor-mediated Ca2+ influx. However, the influence of removal of chloride ions on phenylephrine stimulated contraction is limited. Moreover, 8-Bromo-cyclic GMP and felodipine, but not NPBB, impair phenylephrine-induced contractions in the absence of chloride ions.

Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings.

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.

Cigarette smoking in Kentucky: smoking-attributable mortality and years of potential life lost.

To measure the adverse impact of smoking in Kentucky, a major tobacco-growing state with the nation's highest prevalence of smokers ages 18 and over, percentages of deaths and years of potential life lost attributable to smoking were estimated. Summaries of the analyses for Kentucky in 1996 are presented. These show that 23% of all deaths in Kentucky were attributable to smoking, compared to 19.5% for the nation. Because most smoking-related illnesses develop over a long time, Kentucky can expect to continue to have a substantial excess of smoking-attributable deaths in the future.

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes.

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10-50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis.

We used the differential hybridization technique for isolating developmentally regulated genes from the mouse metanephric kidney. In this screening, we identified the cDNA encoding high-mobility-group protein 17 (HMG-17), a chromosomal non-histone protein which modulates the conformation of transcriptionally active chromatin. Using Northern blot analysis, the HMG-17 mRNA was strongly expressed during embryogenesis and downregulated in various adult murine organs. At the histological level, the transcript localized to differentiating tissue regions and was apparently downregulated in mature structures indicating that HMG-17 expression is linked to cell differentiation. HMG-17 can thus be regarded as a general marker for tissues or cells undergoing differentiation during organogenesis.

Modulation of rigor and myosin ATPase activity in rat cardiomyocytes.

Ischaemic myocardium undergoes calcium-independent contracture at millimolar tissue ATP, though in actomyosin solutions ATP must be reduced to micromolar before rigor complexes form. This contracture is associated with myosin ATPase activity that may contribute to tissue de-energization. Here we used isolated rat cardiomyocytes permeabilized with digitonin to analyse in parallel how rigor and myosin ATPase activity are modulated by metabolic conditions that develop during ischaemia. At pH 7.1 and 37 degrees C rigor and myosin ATPase showed co-ordinated bell-shaped dependence on ATP concentration over 3-1000 microM. Rigor, but not myosin ATPase, was inhibited by acidosis (pH 6.2), indicating reduced efficiency of cross-bridge cycling, while both parameters were stimulated by ADP (< or = 1 mM) and unaffected by inorganic phosphate (Pi, 30 mM), AMP, Mg2+, lactate or inhibition of adenylate kinase with diadenosine pentaphosphate. Combined acidosis and high ADP inhibited rigor, while Pi attenuated the enhancement of rigor by ADP. Thus, rigor complex formation activates myosin ATPase in the intact myofilament array, modulated by ADP, Pi and acidosis in the ranges that occur in ischaemia. There was no evidence that adenylate kinase might attenuate falling ATP/ADP ratio at the myofilaments. In combination these effects are sufficient to resolve the apparent discrepancy between ATP concentrations triggering rigor in actomyosin and onset of contracture in ischaemic myocardium. Since rigor contracture activates myosin ATPase it is likely to exacerbate ATP depletion and thereby limit vital cell functions. This positive feedback is consistent with the abrupt depletion of ATP observed in individual cardiomyocytes undergoing deenergization contracture.

Lovastatin induces apoptosis by inhibiting mitotic and post-mitotic events in cultured mesangial cells.

Lovastatin, an inhibitor of protein prenylation, was reported to inhibit DNA synthesis and induce apoptosis in cultured cells. This report describes the morphological consequences of lovastatin treatment. Lovastatin (50 microM) induced mesangial cell rounding and disassembly of actin stress fibers within 24 to 48 h. After 48 to 72 h of lovastatin treatment, the cells detached from the substratum and underwent apoptotic cell death as evidenced by condensed nuclear chromatin, nuclear fragmentation, cell blebbing and decrease in cell size. Time lapse cinematography revealed that lovastatin caused cell rounding by either inhibiting cytokinesis or cell spreading following cytokinesis. Lovastatin-induced cell rounding, detachment, and apoptosis were dependent upon cell proliferation. These effects were prevented by serum deprivation to inhibit cell proliferation or by plating cells at densities which resulted in contact inhibition of cell growth. Lovastatin-induced mesangial cell rounding and apoptosis were also prevented by the inclusion of the isoprenoids all-trans-farnesol or all-trans-geranylgeraniol in the incubation medium. These results indicate that the effects of lovastatin were mediated by inhibition of protein isoprenylation because exogenous all-trans-geranylgeraniol can be used only in protein prenylation. The small GTP-binding protein RhoA, which may be important for cell spreading and cytokinesis, accumulated in the cytosol following treatment with lovastatin, suggestive of its inactivation. This effect was also prevented by the inclusion of either farnesol or geranylgeraniol in the incubation medium. Thus, lovastatin-induced apoptosis in mesangial cells occurs by interfering with prenylation dependent mitotic and post-mitotic events.