Medical biochemistry and metabolomics

In our previous studies, the opioid receptors located on pinealocytes have been identified and characterized, and these receptors have been found to play a stimulatory role in melatonin synthesis by activating the rate limiting enzyme, N-acetyltransferase (NAT). In the present study, by using reverse transcriptase polymerase chain reaction (RT-PCR) followed by nested-PCR, segments of delta and mu opioid receptors have been amplified from mRNA of rat pineal gland and cerebral cortex. In addition, segments of delta and mu opioid receptors have also been amplified from mRNA of human pineal gland. Furthermore, G ␣i/o -and G ␤ -protein-coupled receptor mRNAs have been amplified and identified from rat pineal gland. The regulatory effects of morphine on G ␣i/o and G ␤ mRNA levels have been semiquantitatively analyzed. Acute morphine administration caused significant increase in G ␣i/o , and G ␤ , mRNA levels in rat pineal gland, but not in other brain regions. Further studies are needed in order to elaborate the mechanisms of these opioid receptors in regulating G-protein expression in pineal gland.

The Maastricht Utrecht Adherence in Hypertension (MUAH) questionnaire provides clinicians with information about the causes of a patient's poor adherence to antihypertensive drugs. In this study, the authors aimed to develop and validate a short version of the MUAH questionnaire. After an exploratory factor analysis, the number of MUAH items was reduced. The original MUAH questionnaire (model 1) was compared with the 16-item MUAH short version (model 2). Next, this short version of MUAH (MUAH-16) with all factors correlated (model 2a) was compared with the short version of MUAH with four subscales that contribute to a global factor of adherence (model 2b). Model 1 had a poor fit to the data (χ(2) 269 = 663.41, P < .001, comparative fit index = 0.695, root mean square error of approximation = 0.06), and model 2 had a very good fit to the data (χ(2) 100 = 171.07, P < .001, comparative fit index = 0.92, root mean square error of approximation = 0.04). When comparing model 2a ...

Conclusion: We have shown that two equations selected from the literature give incorrect RMR values. The case of Harris Benedict equation, that overestimates the RMR of our patients by 1.3 10%, has particular clinical relevance since this equation is the most widely used in clinical practice. The equation of Mifflin-ST Jeor, that is not commonly used in clinical practice, gives the most reliable results in all patients.

Large but not small cholangiocytes: (i) secrete bicarbonate by interaction with secretin receptors (SR) through activation of cystic fibrosis transmembrane regulator (CFTR), chloride bicarbonate anion exchanger 2 (Cl − /HCO 3 − AE2) and adenylyl cyclase 8 (AC8) (proteins regulating large biliary functions); and (ii) proliferate in response to bile duct ligation (BDL) by activation of cAMP signaling. Small, mitotically dormant cholangiocytes are activated during damage of large cholangiocytes by activation of IP 3 /Ca 2+-CaMK I. GABA affects cell functions by modulation of Ca 2+-dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca 2+ /CaMK I-dependent AC 8. Methods-In vivo, BDL mice were treated with GABA in the absence/presence of BAPTA/AM or W7 before evaluating apoptosis and ductal mass (IBDM) of small and large cholangiocytes. In vitro, control-or CaMK I-silenced small cholangiocytes were treated with GABA for 3 days

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (M⌽). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of M⌽. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose threedimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2-DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high-abundance protein depletion, (ii) non-specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non-specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the ''deepest'' depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow-through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2-DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.

three strains of mutant rats with congenital conjugated The workshop covered three major areas: Unconjuhyperbilirubinemia. (2) The roles of the classical and gated bilirubin (UCB) chemistry and physical chemistry; newer molecular biological approaches to identification UCB transport and intracellular trafficking; and evaluaof these transporters were contrasted, and their limitation and therapy of neonatal and congenital hyperbilitions were discussed. (3) The relative roles of the multirubinemias. Findings of studies in the chemistry and ple carriers in UCB transport under different conditions physical chemistry area were as follows. (1) Nuclear and substrate concentrations were discussed. (4) Cytomagnetic resonance (NMR) studies of highly enriched solic UCB-binding proteins (e.g., ligandin) were shown 13 COOH mesobilirubin in water-dimethyl sulfoxide systo promote transcellular movement of UCB by solubiliztems indicated that the pK a values of the carboxyl ing and transporting the pigment in the aqueous phase groups are 4.2 and 4.9, respectively. This finding differs while limiting binding of UCB to the relatively immobile from some reports that suggest that the two pK a values membranes of cell organelles. (5) Mechanisms were prein aqueous systems are near or above pH 7.0. (2) Consented for translocation of UDP-glucuronic acid (UDtrasting views of the hydrophobic interactions of UCB PGA) into the lumenal location of UDPGA transferase with bile salts were presented: one suggested that multiin the endoplasmic reticulum, as well as the enhanceple bile salt monomers bind to one UCB molecule; the ment of this process by N-acetyl-glucosamine. Studies other suggested that UCB binds to the nonpolar surface in the neonatal and congenital jaundice area were as of helical bile salt micelles. (3) Structures were proposed follows. (1) Criteria were reviewed for initiating treatfor the varied calcium and copper bilirubinate salts ment of neonatal jaundice, emphasizing the primacy of formed at various pH values and cation/UCB ratios. (4) serum bilirubin levels, gestational age, and hemolysis Studies of binding of UCB to human serum albumin as risk factors for kernicterus. (2) New methods were (HSA) showed marked diminution of UCB-binding affinpresented for frequent, automated monitoring of serum ity as albumin and chloride concentrations increased. bilirubin levels and breath CO levels as an index of rates (5) A unique UCB derivative, bilirubin-C10-sulfonic acid, of formation of UCB from heme. (3) The current status was identified as the major bile pigment in bullfrog bile. and limitations of new approaches to treatment of se-(6) New methods were presented for removal of impurivere unconjugated hyperbilirubinemia were discussed: ties from preparations of bile salts and UCB. Findings hepatocyte transplantation and gene therapy, still in the of studies in the transport area were as follows. (1) Four stage of development in animal models, have provided putative basolateral and two putative canalicular hepaonly partial and temporary relief of hyperbilirubinemia; tocytic transporters of UCB and related organic anions extracorporeal liver assist devices have had some sucwere described. Special emphasis was given to the adencess in initial human studies; and inhibition of heme osine triphosphate (ATP)-dependent canalicular multioxygenase (HO) with metalloporphyrins, especially tin specific organic anion transporter that is defective in mesoporphyrin, which markedly decreases bilirubin production for prolonged periods, is a new alternative Abbreviations: UCB, unconjugated bilirubin; NMR, nuclear magnetic resonance; NOE, to phototherapy. (4) The ontogeny of the two HO isonuclear Overhauser enhancement; DMSO, dimethyl sulfoxide; TC, taurocholate; CMC, criti-zymes was contrasted in the liver, spleen, kidney, and cal micellar concentration; CD, circular dichroism; Bf, concentration of unbound UCB; Kf,

Advance care planning (ACP) and goals of care designation (GCD) are being integrated into modern health care. In cirrhosis, uptake and adoption of these practices have been limited with physicians citing many perceived barriers and limitations. Recognizing the many tangible benefits of ACP and GCD processes in patients with life-limiting chronic diseases, the onus is on health practitioners to initiate and direct these conversations with their patients and surrogates. Drawing upon the literature and our experiences in palliative care and cirrhosis, we provide an actionable framework that can be readily implemented into a busy clinical setting by a practitioner. Conversation starters, visual aids, educational resources (for patients and practitioners), and videos of mock physician-patient scenarios are presented and discussed. Importantly, we have customized each of these tools to meet the unique health care needs of patients with cirrhosis. The inherent flexibility of our approach to ACP discussions and GCD can be further modified to accommodate practitioner preferences. Conclusion: In our clinics, this assemblage of "best practice tools" has been well received by patients and surrogates enabling us to increase the number of outpatients with cirrhosis who have actively contributed to their GCD before acute health events and are supported by well-informed surrogates.

Endothelial-derived microparticles (EMPs) are a novel biological marker of endothelium injury and vasomotion disorders that are involved in pathogenesis of cardiovascular, metabolic, and inflammatory diseases. Circulating levels of EMPs are thought to reflect a balance between cell stimulation, proliferation, apoptosis, and cell death. Increased EMPs may be defined in several cardiovascular diseases, such as stable and unstable coronary artery disease, acute and chronic heart failure, hypertension, arrhythmias, thromboembolism, asymptomatic atherosclerosis as well as renal failure, metabolic disorders (including type two diabetes mellitus, abdominal obesity, metabolic syndrome, insulin resistance) and dyslipidemia. This review highlights the controversial opinions regarding impact of circulating EMPs in major cardiovascular and metabolic diseases and summarizes the perspective implementation of the EMPs in risk stratification models.

We measured apolipoproteins (apo) A-I and B by rate immunonephelometry (rate INA) during Phase 1 of the National Health and Nutrition Examination Survey (NHANES) III. We also made the measurements by radial immunodiffusion (RID) in a 20% subset of the samples. Aliquots of this subset were also analyzed in the Northwest Lipid Research Laboratories by fixed-time INA calibrated to the World Health Organization (WHO)-International Federation of Clinical Chemistry (IFCC) First International Reference Materials for Apolipoproteins A-I and B. The CVs for the rate INA and RID measurements were: apoA-I, 4.5-7.7% and 2.5-7.6%, respectively; apoB, 2.3-5.3% and 2.3-6.4%, respectively. In NHANES III, rate INA values (x) can be transformed to WHO-IFCC Reference Material-based values (y) as follows: for apoA-I, y = 0.87x + 251.8 mg/L (r = 0.93, SEslope = 0.13, SEintercept = 17, n = 708); for apoB (mg/L), y = 1.068x + 112.8 mg/L (r = 0.98, SEslope = 0.08, SEintercept = 7, n = 646).

HeLa cells were studied with the use of light microscopical immunocytochemistry to obtain more information on the nucleolar localization of nucleophosmin (B23 protein) and RH-II/Gu protein. In control cells these proteins are colocalized. After structural rearrangement of nucleoli induced by actinomycin D, nucleophosmin was present in the nucleolar periphery and nucleolar peripheral caps. In contrast, RH-II/Gu protein was observed mainly in central regions of nucleoli as a compact body. However, in some nucleoli the proteins colocalized in a small portion of the peripheral nucleolar cap. The proteins were also colocalized in micronucleoli but occasionally RH-II/Gu protein was absent or both proteins were not present.

Telomeric DNA consists of G-and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G-G base pairs. The term "G-DNA" was coined for this class of structures . On the basis of gel electrophoresis, imino proton N M R , and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 m M phosphate buffers) specifically induces conformational transitions in the G-rich telomeric D N A from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of > 2 5 O , as monitored by loss of the imino proton N M R signals. N M R semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 m M potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 m M sodium phosphate (pH 7) buffer (Nap) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I),], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG,)4, results in less effective K+-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na+-stabilized structure dissociates quickly to form a W C double helix. This demonstrates that under some circumstances the K+-stabilized G-DNA structure can be kinetically preferred over W C DNA.

Supplemental Figure 1. Ribose-614 cleavage is unaffected by the addition of 10% complementary strand. Addition of equimolar amounts of the complement greatly lowers cleavage rate and plateau. (A) Cleavage profile for ribose-614 (100 nM)(triangles), or ribose-614 (115 nM) plus complementary strand (11.5 nM)(squares). (B) Cleavage profile for ribose-614 (222 nM)(triangles), or ribose-614 (200 nM) plus equimolar complementary strand (circles). Unless otherwise stated, all kinetics were performed under standard reaction conditions, namely 1M NaCl, 1 mM MgCl 2 , 50 mM HEPES, 25°C, and errors in percent cleaved are < ±2 percentage points. Supplemental Figure 2. Cleavage products do not affect ribose-614 cleavage, and only minimally alter ribose-library cleavage. Ribose-614 or ribose-library were incubated either alone (400 nM), or with the addition of the 28-nt product of 614 cleavage (400 nM), the 78-nt product of 614 cleavage (400 nM), or both (400 nM each). Data are fit to a single exponential curve. Errors in percent cleaved are < ±2 percentage points. Supplemental Figure 3. Gel-purification of ribose-614 at cleavage plateau results in additional cleaveage, indicating a fraction of 614 is folded into an inactive conformation. Ribose-614 (222 nM) was allowed to self-cleave for 140 hours, nearing cleavage plateau

Introduction: The emergency laboratory in Hacettepe University Hospitals receives specimens from emergency departments (EDs), inpatient services and intensive care units (ICUs). The samples are accepted according to the rejection criteria of the laboratory. In this study, we aimed to evaluate the sample rejection ratios according to the types of pre-preanalytical errors and collection areas. Materials and methods: The samples sent to the emergency laboratory were recorded during 12 months between January to December, 2013 in which 453,171 samples were received and 27,067 specimens were rejected. Results: Rejection ratios was 2.5% for biochemistry tests, 3.2% for complete blood count (CBC), 9.8% for blood gases, 9.2% for urine analysis, 13.3% for coagulation tests, 12.8% for therapeutic drug monitoring, 3.5% for cardiac markers and 12% for hormone tests. The most frequent rejection reasons were fibrin clots (28%) and inadequate volume (9%) for biochemical tests. Clotted samples (35%) and inadequate volume (13%) were the major causes for coagulation tests, blood gas analyses and CBC. The ratio of rejected specimens was higher in the EDs (40%) compared to ICUs (30%) and inpatient services (28%). The highest rejection ratio was observed in neurology ICU (14%) among the ICUs and internal medicine inpatient service (10%) within inpatient clinics. Conclusions: We detected an overall specimen rejection rate of 6% in emergency laboratory. By documentation of rejected samples and periodic training of healthcare personnel, we expect to decrease sample rejection ratios below 2%, improve total quality management of the emergency laboratory and promote patient safety.

BACKGROUND: The aim of this study was to compare the effects of 10 weeks of resistance or treadmill exercises on glycemic indices levels prior to and immediately following exercise in adults with type 2 diabetes. RESEARCH DESIGN AND METHOD: Twenty inactive subjects (mean age 53.5 years) with type 2 diabetes enrolled in the study. Baseline HbA1c, blood glucose levels, heart

Orotic acid (ORA) is an important biochemical marker for uridine monophosphate synthase deficiency, an autoso-mal recessive disease characterized by macrocytic hypo-chromic megaloblastic anemia, growth retardation, orotic aciduria, and crystalluria (1). In addition, patients with urea cycle diseases excrete increased amounts of urinary ORA (2). Thus, ORA aciduria is observed in patients with ornithine carbamoylasetransferase deficiency (OCTD), an X-linked disorder, and could reveal heterozygosity after a protein load, and in citrullinemia, argininosuccinic acid-uria, and argininemia (2, 3). Currently there are two widely accepted approaches to the determination of urinary ORA, stable-isotope-dilution gas chromatography–mass spectrometry (GC/MS), and ion-exchange HPLC methods with ultraviolet detection,

Objectives: Development and validation of a direct enzymatic HbA1c assay that utilizes a single channel on chemistry auto-analyzers without the need to run separate glycated hemoglobin and total hemoglobin assays. Design and methods: An enzyme based single channel assay was developed to measure %HbA1c in human whole blood samples. The performance characteristics of the Diazyme Direct Enzymatic HbA1c Assay were evaluated on the Hitachi 917 auto-analyzer using whole blood samples, appropriate controls and a reference lot of manufactured reagents. Accuracy studies were completed by comparing the Direct Enzymatic Assay to existing HPLC and immunoassay methods. Interference testing was performed to determine the effect of total hemoglobin, glycated serum proteins, chemical substances and hemoglobin variants in patient samples. Results: The Direct Enzymatic HbA1c Assay showed within run precision and total precision results of ≤ 2% CV for both normal and abnormal level samples. Method comparison studies showed that there was a good correlation between the Direct Enzymatic HbA1c and the HPLC (R 2 = 0.98) or the immunoassay (R 2 = 0.97) methods. The assay measured within the range of 4-16% HbA1c and showed excellent performance with variant hemoglobin in samples. Conclusions: Diazyme Direct Enzymatic HbA1c Assay is accurate and precise when compared to currently marketed medical devices. The assay is designed to report %HbA1c values directly without need for a separate measurement of total hemoglobin and is not adversely affected by interferences from common hemoglobin variants in samples. It is a cost effective, user-friendly method and is adaptable to most general chemistry analyzers.

Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and eff...

Lipoxygenases are a group of closely related enzymes responsible for the dioxygenation of various polyenoic fatty acids containing all-cis-methylene-interrupted double bonds. The nomenclature for lipoxygenases is based on the site of insertion of molecular oxygen on the fatty acid molecule, in this

Introduction: The association of the mutations in HBs Ag gene has not been studied adequately in patients with occult HBV infection (OBI). The current study was aimed to investigate the HBs Ag mutation, genotype of HBV and co-infection with HCV in OBI in central part of Iran. Materials and Methods: In this experimental study, 3700 plasma samples were examined for the presence of HBs Ag, anti-HBc and HBV-DNA. The HBs Ag-/ anti-HBc+ / HBV-DNA + samples were regarded as OBI. Genotype of HBV was determined using Gap-PCR and RT-PCR was used for HCV detection. The direct sequencing was performed to analysis mutations of S gene of HBV. All the statistical analyses carried out using SPSS version 14. Chi-square and fisher test used for determination of significance of association. The p ≤ 0.05 was considered significant. Results: Overall, 352 (9.5%) cases were HBs Ag-/anti-HBc+ of which HBV-DNA was detected in 57 (16.1%). All of the patients carrying D genotype and direct sequencing indicated two mutations out of determinant range. Moreover, none of the OBI patients had HCV infection. Conclusion: The absence of mutations in a-determinant of HBs Ag confirmed the fact that this antigen could be detected by commercial ELISA kits; thus, it can be concluded that suitable amount of HBs Ag to be detected by ELISA was not expressed by HBV in the OBI patients. Low level expression of HBs Ag might be related to D genotype of the virus and it seems that this is not related to HCV infection.

Significance: It is of common sense that the world population is aging and life expectancy is increasing. However, as the population ages, there is also an exponential risk to live into the ages where the brain-related frailties and neurodegenerative diseases develop. Hand in hand with those events, the world is witnessing a major upsurge in diabetes diagnostics. Remarkably, all of this seems to be narrowly related, and clinical and research communities highlight for the upcoming threat that it will represent for the present and future generations. Recent Advances: It is of utmost importance to clarify the influence of diabetes-related metabolic features on brain health and the mechanisms underlying the increased likelihood of developing neurodegenerative diseases, in particular Alzheimer's disease. Thereupon, a wealth of evidence suggests that mitochondria and associated oxidative stress are at the root of the link between diabetes and co-occurring disorders in the brain. Critical Issues: The scientific community has been challenged with constant failures of clinical trials raising major issues in the advance of the therapeutic field to fight chronic diseases epidemics. Thus, a change of paradigms is urgently needed. Future Directions: It has become urgent to identify new and solid candidates able to clinically reproduce the positive outcomes obtained in preclinical studies. On this basis, strategies settled to counteract diabetes-induced neurodegeneration encompassing mitochondrial dysfunction, redox status imbalance, and/or insulin dysregulation seem worth to follow. Hopefully, ongoing innovative research based on reliable experimental tools will soon bring the desired answers allowing pharmaceutical industry to apply such knowledge to human medicine. Antioxid. Redox Signal. 00, 000-000.

Aggressive periodontitis (AgP), a severe and early onset type of periodontitis, is thought to be subject to significant genetic background effects. Formyl peptide receptor 1 (FPR1) is a gene strongly implicated in AgP. To determine whether variations in this gene are associated with AgP, we performed an association study with 49 AgP patients and 373 controls using 30 variations identified by sequencing the 21.1-kb gene region. Five polymorphisms (À12915C > T, À10056T > C, À8430A > G, 301G > C, and 546C > A) showed significant association with AgP. Polymorphonuclear neutrophils from subjects carrying the À12915T allele expressed significantly lower levels of FPR1 transcripts than those homozygous for the À12915C allele. Furthermore, the À12915T allele decreased activity of transcriptional regulation in a luciferase assay. Haplotype association analysis with three SNPs (À12915C > T, 301G > C, and 546C > A) revealed that one haplotype (À12915T-301G-546C) was significantly represented in AgP patients (p = 0.000020). Thus, altered FPR1 function might confer increased risk to AgP.

The effects of a delayed and/or disturbed sleep-wake pattern on mental functioning, frequently found in university students, were confirmed by several studies. The sleep-wake pattern of university students is known to be delayed as their grade progresses. However, some literature argues that the environmental changes at the beginning of university life are crucial to students mental hygiene. These two factors had been examined separately. We investigated the effects of the sleep-wake pattern and environmental changes, especially residential status, on mental functioning, and explored the change of these effects with grade progress in university students. University freshmen participated in this survey in the first and the second semester. The second or third year students participated in the first semester. A total of 196 students' data were used for analysis. The data were analyzed cross-sectionally. The results showed that the second or third year students reported the most delayed bedtime (0 : 52) and rise time (7 : 51) on weekdays. Students living with their families reported earlier bedtimes and rise times on weekdays than students living alone (bedtime: 0 : 07 vs. 0 : 38; rise time: 7 : 02 vs. 7 : 50). Multiple regression analyses revealed that the freshmen living with their families showed a better psychological state only immediately after entering university. In contrast, the sleep-wake pattern had no relationship with the psychological state of freshmen, but second or third year students who reported earlier bedtimes showed better psychological states. The impact of residential status on psychological distress might decrease while that of sleep-wake pattern might increase with grade progress.

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 R N A polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent R N A polymerase. Replication involves the synthesis of both R N A and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of R N A transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce R N A transcripts. Furthermore, each R N A transcript encodes the R N A polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7,

incubation times. Otherwise UV-Vis results showed the reduced absorbance in soret region to be a constant value in less time of incubation. Conclusion: These two different observations demonstrate that amount of fluorescence heme degradation products' accumulation is independent from the soret band depletion. Therefore, heme with important physiological functions is so sensitive to the fructation. So that some changes would be appear in oxygen transportation (the most Hb vital index) in diabetic patients.

Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/ alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/ alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1 H (plasmalogen-type lipid) or 9 H in a ratio < 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1 H -enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.

Nightshift is a common work schedule in health environments, and is associated with decreased alertness and increased adverse events at work. This decrease in alertness can be predicted from biological models of sleep homeostasis and circadian influences. Naps can provide a short-term alleviation of sleep need, and the benefits of naps have been demonstrated in laboratory-based studies, and in specific controlled work environments. The efficacy of brief naps has not been demonstrated in health workers in their usual work environment. The current study examined the effects of a 30min nap break during the nightshift in a cohort of nursing staff and scientists in their usual work environment. Measures of both subjective sleepiness and objective alertness were taken at hourly intervals throughout the nightshift, on nights where a scheduled nap was taken and nights where there was no nap. Following a nap, psychomotor performance metrics (response speed and fastest 10% reactions times) improved and self-reported sleepiness was reduced compared to nights without a nap. These improvements persisted to the end of the nightshift. Effect size estimates suggested that 20-50% of variance in these measures was explained by the interaction of time on shift and the nap intervention. Differences were found in the timing of increases in subjective and objective alertness after the nap. These findings support the effectiveness of a scheduled nap break during a nightshift to maintain alertness in health workers.

Hexachlorobenzene-induced porphyria is iron dependent and characterized by the decreased activity of uroporphyrinogen decarbowlase and the accumulation of porphyrins in the liver. To examine the relationship between iron and porphyrins in liver tissue, we performed a biochemical and morphological (histological, ultrastructural and morphometrical) study in the livers of C57BL/10 mice. Mice were treated with hexachlorobenzene, iron dextran or the combination of hexachlorobenzene and iron dextran. An accumulation of porphyrins and an increased total iron content were found not only in the livers of mice treated with hexachlorobenzene and iron dextran but also in mice treated with iron dextran alone. In contrast, the amount of porphyrins was only slightly increased in the livers of mice treated with hexachlorobenzene alone. Needle-like structures, representing uroporphyrin crystals, were observed, histologically and ultrastructurdy, in hepatocytes of mice treated with hexachlorobenzene and iron dextran and with iron dextran alone. Uroporphyrin crystals and ferritin

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, atherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH 2 , CWSMMGFQCGGGS-NH 2 , CWEWGGWHCGGSS-OH, CNW TLGGYKCGGGS-NH 2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study.

This paper was my first as a graduate student of Professor Peter Perlmann at Stockholm University. At the time, RIAs were in full bloom, but they were too sophisticated for many areas of research and diagnosis because they required expensive equipment and used antigens and antibodies labeled with radioactive isotopes with short half-lives. We wanted something simpler with the same sensitivity. Techniques for labeling antibodies with enzymes had been described for immunohistochemistry (1), and we thought they could be useful for serologic assays as well. First, we gave the prospective enzyme immunoassay a name: enzymelinked immunosorbent assay, or ELISA (an important decision). Second, we chose to use an enzyme for which there was, at the time, a soluble and sensitive substrateproduct system, namely alkaline phosphatase. Third, we used the procedure of a standard RIA, i.e., a com

BACKGROUND High-sensitivity cardiac troponin T (hs-cTnT) is a biomarker used in diagnosing myocardial injury. The clinical utility and the variation of this biomarker over time remain unclear in hemodialysis (HD) and peritoneal dialysis (PD) patients. We sought to determine whether hs-cTnT concentrations were predictive of myocardial infarction (MI) and death and to examine hs-cTnT variability over a 1-year period. METHODS A total of 393 nonacute HD and PD patients (70% HD and 30% PD) were followed in a prospective observational study for new MI and death. RESULTS Median hs-cTnT was 57 ng/L (interquartile range, 36–101 ng/L) with no observed difference between HD and PD patients (P = 0.11). Incremental increases in mortality (P = 0.024) and MI (P = 0.001) were observed with increasing hs-cTnT quartiles. MI incidence increased significantly across quartiles in both HD and PD patients (P = 0.012 and P = 0.025, respectively), whereas mortality increased only in HD patients (P = 0.015)....

The influence of membrane environment on human cannabinoid 1 (hCB 1) receptor transmembrane helix (TMH) conformational dynamics was investigated by solid-state NMR and site-directed spin labeling/EPR with a synthetic peptide, hCB 1 (T377-E416), corresponding to the receptor's Cterminal component, i.e., TMH7 and its intracellular α-helical extension (H8) (TMH7/H8). Solidstate NMR experiments with mechanically aligned hCB 1 (T377-E416) specifically 2 H-or 15 Nlabeled at Ala380 and reconstituted in membrane-mimetic dimyristoylphosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) bilayers demonstrate that the conformation of the TMH7/H8 peptide is more heterogeneous in the thinner DMPC bilayer than in the thicker POPC bilayer. As revealed by EPR studies on hCB 1 (T377-E416) spin-labeled at Cys382 and reconstituted into the phospholipid bilayers, the spin label partitions actively between hydrophobic and hydrophilic environments. In the DMPC bilayer, the hydrophobic component dominates, regardless of temperature. Mobility parameters (ΔH 0-1) are 0.3 and 0.73 gauss for the peptide in the DMPC or POPC bilayer environment, respectively. Interspin distances of doubly-labeled hCB 1 (T377-E416) peptide reconstituted into a TFE/H 2 0 mixture or a POPC or DMPC bilayer were estimated to be 10.6 ± 0.5, 16.8 ± 1, and 11.6 ± 0.8 Å, respectively. The extent of coupling (≥ 50%) between spin labels located at i and i+4 in a TFE/H 2 0 mixture or a POPC bilayer is indicative of an α-helical TMH conformation, whereas the much lower coupling (14%) when the peptide is in a DMPC bilayer suggests a high degree of peptide conformational heterogeneity. These data demonstrate that hCB 1 (T377-E416) backbone dynamics as well as spin-label rotameric freedom are sensitive to and altered by the peptide's phospholipid bilayer environment, which exerts a dynamic influence on the conformation of a TMH critical to signal transmission by the hCB 1 receptor. Signal-transducing G protein-coupled receptors (GPCRs) 1 are proteins that share as a common defining structural signature an intracellular carboxyl terminus, seven hydrophobic transmembrane helices (TMHs) connected by intra-and extracellular loops, and an extracellular amino terminus (1). Agonist binding is believed to induce ligand-specific GPCR structural changes that allow the receptor to assume, likely through a continuum of intermediate conformations, a functionally active state (2-4). GPCR conformation, activity, and intracellular trafficking may also be modulated by local membrane properties (e.g., tension, fluidity, lateral † This study was supported by National Institutes of Health/National Institute on Drug Abuse grants DA009158-10S2 (EKT) and DA3801 (AM) and a National Science Foundation instrumentation grant CHE-0466616 (DEB).

It is well recognized that variation in the geographical distribution of prevalence of multiple sclerosis (MS) exists: increasing the latitude its prevalence increases as well, but the underlying causes of such dissimilarity still remained elusive as of today. Currently, the most accredited hypothesis is that the closer to the equator the more pronounced is the amount of sunlight which, in turn, increases the production of vitamin D. Cholecalciferol is indeed deficient in MS patients, but this factor does not explain by itself the etiopathogenesis of the disease. In the present study, to search for a pattern and provide a model of the disease's etiology consistent with this regional factor, as well with its changing ethnic, sex-ratio, lifestyle variations and the other unexplained aspects of MS, an extensive analysis of peer-reviewed literature and data was conducted. The arisen hypothesis was that, increasing the latitude, the factor that varies and can have the stronger effect on the human organism, is the continuous and ever-increasing diversity of the natural light-dark cycle. The consequent effort of the suprachiasmatic nucleus to entrain the organism's circadian rhythm affects the hypothalamic-pituitary-adrenal axis resulting in desynchronizing the central and peripheral circadian clocks and pathologizing the immunitary system. To verify such hypothesis, a theoretical framework of the etiopathogenesis, coherent with the gathered literature, was conceived and a demonstration to corroborate it was eventually devised and performed. The results underscored that people living in countries subjected to a further circadian disruptive factor, as daylight saving time, have a 6.35 times higher prevalence of MS than States placed on their same latitude that do not observe it, thus strongly supporting the hypothesis. As further reinforcement of the conclusions, it is worth mentioning that the levels of polyamines rise abruptly in autoimmune diseases. Moreover, among their numerous roles, these polycations participate to the regulation of the circadian clock so their sudden variation might disrupt it. Following these interesting findings, new perspectives in therapies are, therefore, proposed.

In order to realize the goal of stratified and/or personalized medicine in the clinic, significant advances in the field of biomarker discovery are necessary. Adding to the abundance of nucleic acid biomarkers being characterized, additional protein biomarkers will be needed to satisfy diverse clinical needs. An appropriate source for finding these biomarkers is within blood, as it contains tissue leakage factors as well as additional proteins that reside in blood that can be linked to the presence of disease. Unfortunately, high abundant proteins and complexity of the blood proteome present significant challenges for the discovery of protein biomarkers from blood. Animal models often enable the discovery of biomarkers that can later be translated to humans. Therefore, determining appropriate sample preparation of proteomic samples in rodent models is an important research goal. Here, we examined both mouse and rat blood samples (including both serum and plasma), for appropriate high abundant protein removal techniques for subsequent gel-based proteomic experiments. We assessed four methods of albumin removal: antibody-based affinity chromatography (MARS), Cibacron Ò Blue-based affinity depletion (SwellGel Ò Blue Albumin Removal Kit), protein-based affinity depletion (ProteaPrep Albumin Depletion Kit) and TCA/acetone precipitation. Albumin removal was quantified for each method and SDS-PAGE and 2-DE gels were used to quantify the number of protein spots obtained following albumin removal. Our results suggest that while all four approaches can effectively remove high abundant proteins, antibody-based affinity chromatography is superior to the other three methods.

Introduction: Normetanephrine and metanephrine are intermediate metabolites of noradrenaline and adrenaline metabolism. To assess whether normetanephrine and metanephrine analysis may aid in the diagnosis of Neuroblastoma, a reference interval for these metabolites must first be established. Aim: The overall aim of this study was to establish a paediatric age-related reference interval for the measurement of total fractionated metanephrines. Methods: A total of 267 urine samples were analysed following acid hydrolysis. This releases the metanephrines from their sulphate-bound metabolites. The samples were analysed using reverse phase high-performance liquid chromatography with electro-chemical detection on a Gilson automated sequential trace enrichment of dialysate sample system. Results: Data were analysed using Minitab Release version 14. Outliers were removed using the Dixon/Reed one-third rule. Partitioning of the age groups was achieved using Harris and Boyd's standard normal deviate test. Non-parametric analysis of the data was performed, followed by the establishment of the 2.5th and the 97.5th reference limits. Conclusions: The established reference intervals are described in Table 2.

Vimentin is a type III Intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and poor prognosis. We have analysed migration and adhesion to collagenous matrix of a panel of carcinoma cell lines. In vitro invasiveness was highest in vimentin-positive SW480 colon cancer and MDA-MB-231 breast cancer cells and the role of vimentin in these cell lines was investigated by RNA interference. Down-regulation of vimentin expression resulted in impaired migration in both scratchwound experiments and in invasion assays through cell culture inserts coated with collagen gel. Compromised migration was observed in both cell lines, whereas cell attachment assays revealed impaired adhesion to fibrillar collagen in MDA-MB-231 cells while the adhesion of vimentin-ablated SW480 cells, that express both vimentin and keratin intermediate filaments was not affected. In conclusion, ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines.

Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and proteinbound forms. In the present review, we compare and evaluate several techniques forthe determination of total homocystelne. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound homocysteine through disulfide interchange does not affect the results, and total homocysteine can be measured in stored samples. Total homocysteine in whole blood increases at room temperature because of a continuous production and release of homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mol/L in fasting subjects are considered normal. Total homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases only when they are treated with the deficient vitamin. Total homocysteine is therefore of value for the diagnosis and follow-up of these deficiency states and may compensate for weaknesses of the traditional laboratory tests. In addition, total homocysteine is an independent risk factor for premature cardiovascular diseases. These disorders justify introduction of the total homocysteine assay in the routine clinical chemistry laboratory.

A novel toxic cyclopeptide from Amanita suballiacea (Murr.) mushrooms that possesses structural features similar to viroidin is described. This peptide, alloviroidin, is identical with viroidin in mass, affinity for actin, and all amino acids except for one. The single discernible difference between the two peptides exists in the configuration at carbon 4 of the 4,5-dihydroxyleucine residues, as shown by a combination of chemical modification and magnetic resonance experiments. The configuration of this residue in viroidin is similar to that of phalloidin and is 2S,4R, while that in alloviroidin is established to be 2S,4S. This peptide is thus unique in its hydroxylation pattern among both the virotoxins and phallotoxins and may be an intermediate for more highly hydroxylated virotoxins, such as viroisin.