Abnormal Enzyme Activity Research Articles

Recent progress in two-photon small molecule fluorescent probes for enzymes

Enzymes are macromolecular biological catalysts which can accelerate chemical reactions in living organisms. Almost all the physiological metabolism activities in the cell need enzymes to sustain life via rapid catalysis. Currently, medical research has proved that abnormal enzyme activity is associated with numerous diseases, such as Parkinson’s disease (PD), Alzheimer's disease (AD) and cancers. On the other hand, early diagnosis of those diseases is of great significance to improve the survival rate and cure rate. In the current diagnostic tools, two-photon fluorescent probes (TPFPs) are developing rapidly due to their unique advantages, such as higher spatial resolution, deeper imaging depth, and lower biotoxicity. Therefore, the design and synthesis of two-photon (TP) small molecule enzymatic probes have broad prospects for early diagnosis and treatment of diseases. As of now, scientists have developed many TP small molecule enzymatic probes. This review aims to summarize the TP small molecule enzymatic probes and expound the reaction mechanism.

Frontiers in Probing Alzheimer's Disease Biomarkers with Fluorescent Small Molecules.

Alzheimer’s disease (AD) is the most common form of dementia. The pathogenesis of the disease is associated with aggregated amyloid-β, hyperphosphorylated tau, a high level of metal ions, abnormal enzyme activities, and reactive astrocytes. This outlook gives an overview of fluorescent small molecules targeting AD biomarkers for ex vivo and in vivo imaging. These chemical imaging probes are categorized based on the potential biomarkers, and their pros and cons are discussed. Guidelines for designing new sensing strategies as well as the desirable properties to be pursued for AD fluorescence imaging are also provided.

A Chronology for the Identification and Disclosure of Adverse Effects of Succinylcholine

BackgroundNew therapies are created to address specific problems and enjoy popularity as they enter widespread clinical use. Broader use can reveal unknown adverse effects and impact the life cycle significantly. Succinylcholine, a depolarizing neuromuscular blocker, was the product of decades of research surrounding the ancient compound, curare. It was introduced into practice in the 1950s by Burroughs Wellcome and Company (BW Co) and was welcomed due to its rapidly acting muscle relaxation effects. Global clinical use revealed adverse effects, both minor and major, in particular, hyperkalemia and malignant hyperthermia. We investigated when practitioners and the manufacturer became aware of these adverse effects, how information about these side effects was disseminated, and whether the manufacturer met the regulatory requirements of the time, specifically regarding the timely reporting of adverse effects. SourcesPrimary literature search using online and archived documents was conducted at the Wood Library-Museum of Anesthesiology, Schaumburg, IL. We consulted documents submitted by BW Co to federal authorities, through the Freedom of Information Act (FOIA), Food and Drug Administration (FDA) reports, promotional advertisements, package inserts, published articles, and textbooks. ResultsInitial clinical testing in humans in 1952 found no adverse effects on cardiovascular or respiratory systems. Fasciculations and myalgia were early side effects described in case reports in 1952. Large-scale clinical trials in 1953 found abnormally long recovery times among some patients; the discovery of abnormal pseudocholinesterase enzyme activity was not fully demonstrated until the early 1960s. Bradycardia was first reported in 1957 in children, and in 1959 in adults. In 1960, animal studies reported a transient increase in plasma potassium; further experiments in 1969 clearly demonstrated succinylcholine-induced hyperkalemia in burn patients. Malignant hyperthermia was first described in 1966. Similar cases of elevated temperatures and muscle rigidity were described globally but the underlying mechanism was not elucidated until the 1990s. Standard anesthesia textbooks did not report major side effects of succinylcholine until 1960 and included newly documented side effects with each edition. BW Co's packaging contained warnings as early as the 1950s but were later updated in 1962 and beyond to reflect the newly discovered hyperkalemia and malignant hyperthermia. ConclusionParticularly given the regulatory environment of the time, BW Co appropriately reported the adverse effects of succinylcholine after market entry; it updated promotional and packaging material in a timely manner to reflect newly discovered adverse effects. The toxicity, though alarming and put clinicians on alert, did not seem to heavily impact succinylcholine's use, given its various desirable properties. It is still a choice muscle relaxant used today, although there are efforts to develop superior agents to replace succinylcholine.

Mutation of glucose-6-phosphate dehydrogenase deficiency in Chinese Han children in eastern Fujian.

We aim to investigate the mutation types of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese Han children in eastern Fujian Province.A total of 904 Chinese Han neonates (male: 733 with positive G6PD deficiency and 28 with weakly positive deficiency; female: 73 with positive G6PD deficiency and 70 with weakly positive deficiency) received G6PD screening in our center from January 2014 to December 2016 were included in this study. Additionally, 904 age-matched normal Chinese Han individuals (male: 761; female: 143) were selected as control. Neonatal G6PD deficiency screening was performed through blood sample collection from the heels, using the commercial kits. Multicolor melting curve analysis (MMCA) method was used to determine the G6PD mutation type in the 904 cases. If it failed to detect mutations in the cases with abnormal enzyme activity, the polymerase chain reaction (PCR) and gene sequencing were used to determine the mutation sites. PCR and gene sequencing were used to determine the mutation sites in the 904 individuals with normal enzyme activity. Three most common mutation types in Chinese population were compared between Fujian and other provinces.Among the 904 neonates with abnormal G6PD enzyme activity, 17 mutation types were detected including 15 single point mutations and 7 complex mutations. Three most common mutation types were c.1376G > T, c.1388G > A, and c.95A > G accounted for 72.6% of the total mutations in eastern Fujian.The proportion of mutational types in G6PD and the degree of enzyme activity change in various mutational types were found in the neonates of Fujian Province. Our study may enrich the molecular diagnosis of G6PD deficiency meaning Fujian Province.

Analysis of Soil Degradation Causes in Phyllostachys edulis Forests with Different Mulching Years

Moso bamboo (Phyllostachys edulis (Carrière) J.Houz.) is famous for its fast growth and biomass accumulation, as well as high annual output for timber and bamboo shoots. Organic mulches are widely used to improve shoots’ production in moso bamboo forests. However, continuous mulching management may cause bamboo forest degradation and affect sustainable development. The objective of this study was to identify the degradation mechanism and to provide a theoretical basis for recovery. A complete randomized block design with four treatments was conducted, including mulching for one year (M1), two years (M2), three years (M3) and no-mulching management (NM). Soil nutrient contents, enzyme activities and microbial biomass were determined. With the increase of mulching years, the soil pH value gradually reduced, causing soil acidification, but the content of soil organic matter was inclined to ascend. Soil total nitrogen (TN), total phosphorus (TP) and total potassium (TK) contents showed an increasing trend, and they were significantly higher in mulching stands than those in NM (p < 0.05). Contents of soil available nutrients (AN, AP and AK) increased, then decreased with the increase of mulching years and peaked in M1. With the increase of mulching years, the soil stoichiometry ratio (C/N, C/P and N/P) gradually increased. Soil invertase, urease and acid phosphatase activities presented a single-peak curve and reached the maximum within one year after mulching. Total microbial biomass and that of individual groups changed greatly after mulching. Soil microbial biomass increased first and then decreased, and it was the largest in M1. The fungi:bacteria ratio decreased in the first year and then began to rise, while the aerobic:anaerobic ratio showed the opposite trend. According to the overall results, M3 leads to soil acidification, imbalance of the nutrients’ proportion, abnormal enzyme activity and change of soil microbial flora, and rotated mulching management (mulching one year and then recuperating one year) should be recommended in practice.

Safety and efficacy of nivolumab in Japanese patients with malignant melanoma: An interim analysis of a postmarketing surveillance.

A postmarketing surveillance study is ongoing to evaluate nivolumab treatment for Japanese patients with malignant melanoma and accumulate data on all adverse events (AE) and efficacy. In this interim analysis, we evaluated data from approximately 100 Japanese medical institutions obtained from the nivolumab approval date in Japan (4 July 2014) through 3 July 2016. Patients were monitored during the first 12 months of treatment. Nivolumab was administrated by i.v. infusion (2 mg/kg every 3 weeks). A total of 680 and 610 patients were evaluated for safety and efficacy, respectively. The incidences of adverse drug reactions (ADR) and grade 3 or higher ADR were 53.53% and 12.35%, respectively. Predominant ADR included hypothyroidism (11.32%) and abnormal enzyme activity, such as increase of aspartate aminotransferase (7.79%), alanine aminotransferase (6.76%), alkaline phosphatase (6.18%) and γ-glutamyltransferase (5.44%). Grade 3 or higher ADR of special interest with an incidence of 1% or higher were hepatic function disorder (2.50%), colitis/diarrhea (2.06%) and infusion reaction (1.32%). No cases of encephalitis or venous thromboembolism, other AE of special interest, were observed. The estimated median overall survival was 379 days (95% confidence interval [CI], 290-not reached [NR]) in the overall population, NR (95% CI, 305-NR) for cutaneous melanoma and 340 days (95% CI, 275-NR) for mucosal melanoma. The improvement rate based on the antitumor response at the last evaluation was 22.2% (131/590 patients). No new safety concerns were raised, and serious ADR of special interest were infrequent. Nivolumab showed equivalent efficacy in patients with mucosal melanoma and those with cutaneous melanoma.

A high-throughput UPC2-MS/MS method for the separation and quantification of C19 and C21 steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately.Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites –one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.

Where should the safe limits of alcohol consumption stand in light of liver enzyme abnormalities in alcohol consumers?

ObjectivesTo estimate the prevalence and risk factors for abnormal liver enzymes in a large age- and gender stratified population-based sample of apparently healthy individuals with or without alcohol consumption and other health-related risk factors (adiposity, physical inactivity, smoking).MethodsData on alcohol use, smoking, diet and physical activity were recorded using structured questionnaires from 13,976 subjects (6513 men, 7463 women, aged 25–74 years) in the national FINRISK studies. Alcohol data was used to categorize the participants into abstainers, light drinkers, moderate drinkers and heavy drinkers. Serum gamma-glutamyltransferase (GGT) and alanine aminotransferase (ALT) activities were measured using standard kinetic methods.ResultsMale light drinkers, moderate drinkers and heavy drinkers showed significantly higher relative risks of abnormal GGT than abstainers: 1.37 (95% confidence interval 1.11 to 1.71, p < 0.01), 2.72 (2.08 to 3.56, p < 0.0005), and 6.10 (4.55 to 7.17, p < 0.0005), respectively. Corresponding values for women were 1.22 (0.99 to 1.51, p = 0.065), 1.90 (1.44 to 2.51, p < 0.0005), and 5.91 (3.80 to 9.17, p < 0.0005). Estimated threshold doses for a significant GGT elevation was 14 standard weekly alcohol doses for men and 7 for women. Excess body weight and age over 40 years modulated the thresholds towards smaller quantities of alcohol. The risk of abnormal GGT was also significantly influenced by physical inactivity and smoking. The relative risks of abnormal ALT activities were increased in male heavy drinkers, especially in those presenting with adiposity and sedentary lifestyle.ConclusionsAlcohol use markedly increases the risk for abnormal liver enzyme activities in those presenting with age over 40 years, obesity, smoking or sedentary lifestyle. The data should be considered in public health recommendations and in the definitions of safe limits of alcohol use.

Identification of novel missense HEXB gene mutation in Iranian-child with juvenile Sandhoff disease

Sandhoff disease is an autosomal recessive lysosomal storage disease caused by mutation in the HEXB gene. Accumulation of GM2 gangliosides resulting in advanced motor neuron manifestation and death from respiratory failure and infections in infantiles. Pathogenic mutations in the HEXB gene were detected that leads to enzyme activity reduction and therefore interruption of normal metabolic cycles of GM2 ganglioside in Sandhoff patients. We included one Iranian 5-years-old female with typical biochemical and clinical symptoms of the juvenile Sandhoff disease. After DNA extraction and amplification, proband and her parents, were assessed by direct sequencing of the amplicons. In addition, homology modeling of the novel mutant protein has been conducted to reveal the structural defects caused by the mutation. New unreported pathogenic missense mutation c.821T>A (p.Val274Glu; Homozygous) in exon 7 of the HEXB gene was identified. The structural analysis also showed that the novel mutation is the cause of a serious structural deficiency and leads to abnormal enzyme activity. We concluded that missense mutation p.Val274Glu is pathogenic because of amino acid alteration and its location on the conserved region of the protein. Also, biochemical and structural analysis demonstrated defects in the normal function of beta-hexosaminidase protein.

α-Tocopherol mediated amelioration of camptothecin-induced free radical damage to avert cardiotoxicities.

Reactive oxygen species (ROS) such as O2(-), hydrogen peroxide, and OH(-) are highly toxic to cells. Cellular antioxidant enzymes and free radical scavengers normally protect a cell from toxic effects of ROS. However, when generation of ROS overtakes the antioxidant defense of the cells, it leads to various pathological conditions. The present study investigated the protective efficacy of α-tocopherol on the peroxidative damage and abnormal antioxidant levels in the myocardial tissue of camptothecin (CPT), administered at the dosage of 6 mg/kg/day in male Wistar rats. CPT-administered rats showed significant increase (p < 0.001) in lipid peroxidation and abnormal changes in the activities/levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione S-transferase) and nonenzymic antioxidants (reduced glutathione and vitamin E). Alterations in the levels of tissue alkaline phosphatase, lactate dehydrogenase (p < 0.01), alanine transaminase (p < 0.001), and aspartate transaminase (p < 0.001) were also observed in CPT-treated rats. In contrast, rats pretreated with α-tocopherol showed significant revision of elevated levels of lipid peroxides and abnormal antioxidant enzyme activity suggesting the ameliorative property of vitamin E. Histopathological alterations in the heart tissue observed after CPT administration were also protected in animals that were pretreated with vitamin E. Based on our results, we conclude that supplementation of vitamin E may improve the efficacy of standard and experimental cancer therapies by subsiding the toxic effect of the antineoplastic agent.

Steatohepatitis Secondary to Long-Term Glucocorticoid Treatment for Congenital Adrenal Hyperplasia: A Potential Diagnostic Pitfall

A 24-year-old woman with congenital adrenal hyperplasia (CAH) was referred for evaluation of elevated liver enzyme activities over the preceding 6 months. The patient was diagnosed with CAH at the age 12 when she presented with irregular menses and hirsutism. Since then, she had been on dexamethasone to maintain a normal menstrual cycle and prevent hirsutism and acne. She had no history of chronic liver disease and drank alcohol socially. An extensive workup for other treatable causes of liver disease was unrevealing. Therefore, a liver biopsy was performed, which revealed extensive ballooned degenerative hepatocytes containing Mallory-Denk hyalines. The ballooned hepatocytes were located predominantly in centrilobular areas and without any accompanying steatosis. Even though the histopathologic features are most compatible with alcoholic and/or nonalcoholic steatohepatitis, it was not supported by the patient's medical history and clinical presentation. The patient had a normal body mass index and only occasional alcohol use. Based on the biopsy finding and clinical presentation, we postulated that the abnormal liver enzyme and pathological features seen on the liver biopsy were secondary to CAH and long-term use of glucocorticoid. A few studies have shown that patients with CAH often develop metabolic abnormalities and insulin resistance, particularly women treated with glucocorticoid for several years. To our knowledge, this is the first report describing steatohepatitis secondary to CAH and prolonged glucocorticoid treatment. It is important to be aware that steatohepatitis can develop in these patients due to long-term glucocorticoid use and potentially lead to progressive liver damage. Furthermore, in patients with CAH who develop abnormal liver enzyme activities a liver biopsy is warranted to assess for steatohepatitis and any associated fibrosis. If indeed fibrosis is already present, a consultation with the endocrinologist should be undertaken in an effort to lower the dose of the glucocorticoids as much as possible while still controlling the symptoms of the disease.

Selected Pharmaceutical Excipient Prevent Isoniazid and Rifampicin Induced Hepatotoxicity

The incidence of isoniazid (INH)- and rifampicin (RIF)-induced abnormal liver enzyme activity is 27% but only 19% with INH alone. Cytochrome P450 2E1 (CYP2E1) is thought to contribute to the synergistic effects of RIF and INH. Pharmaceutical excipients are inactive ingredients that are added to a pharmaceutical compound. The purpose of this study was to screen excipients for CYP2E1 inhibition and identify whether the screened excipients prevented INH/RIF-induced hepatotoxicity. Fifty-five known pharmaceutical excipients were screened for CYP2E1 inhibition. The hepatotoxic doses of INH and RIF were 50 and 100 mg/kg/day, respectively. Hepatotoxicity was assessed by the galactose single point (GSP) method (a US Food and Drug Administration (FDA) recommended quantitative liver function test), liver histopathology, malondialdehyde (MDA) assay, and measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. We chose the CYP2E1-specific substrate chlorzoxazone to assess CYP2E1 activity in animal and human. Mannitol inhibited CYP2E1 activity by 54% in mice with INH/RIF-induced hepatotoxicity (p < 0.005). Serum AST, ALT and GSP levels were significantly increased 3.8- to 7.8-fold in these mice (p < 0.005), and these levels could be lowered by mannitol. Mannitol significantly alleviated the depletion of hepatic glutathione (GSH) and partially reversed the increase in MDA formation in mice treated with INH/RIF (p < 0.005). Mannitol also decreased CYP2E1 activity by 58% in humans (p < 0.005). Furthermore, an antituberculosis (TB) efficacy assay revealed that mannitol did not affect the anti-TB effects of INH/RIF. Mannitol, an FDA-approved excipient, was found to be a CYP2E1 inhibitor. Mannitol may be a useful adjuvant for drugs that induce hepatotoxicity through CYP2E1, such as INH and RIF.

A Case of Quadruple Viral Infections and Elevated Aminotransferase Activities

A 40-year-old man with human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infection was referred for evaluation of abnormal liver enzyme activities. The patient was maintained on antiretroviral therapy for HIV as well as medication to suppress HBV and had previously undergone treatment for HCV with durable sustained virologic response. The patient was clinically well without any symptoms or evidence of liver decompensation. Laboratory findings were notable for aminotransferase activities in the 200 to 225 U/L range that had been persistent for several months. An extensive workup for the etiology of the aminotransferase elevation ensued. Imaging studies showed no evidence of biliary obstruction. Serology revealed negative autoantibodies, negative serum HCV-RNA, and low level HBV-DNA by polymerase chain reaction. Further testing revealed positive hepatitis delta virus (HDV) antibody and positive HDV RNA in the serum. A percutaneous liver biopsy was performed to further elucidate the cause of the elevated aminotransferase activities. Based on histology, serology, and clinical presentation, a diagnosis of chronic HDV infection was made. HDV infection should be considered in patients with known chronic viral hepatitis B with low viral load, who present with worsening liver function or elevation in aminotransferase activities.

Lipid-Lowering Agents in Nonalcoholic Fatty Liver Disease and Steatohepatitis: Human Studies

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease which refers to the presence of hepatic steatosis without significant intake of alcohol. NAFLD is an asymptomatic disease that can progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD is currently the most common cause of incidental abnormal liver tests and elevated serum liver enzyme activities in the developed world. Obesity, diabetes, and other components of the metabolic syndrome are frequently associated with the NAFLD. The treatment of NAFLD focuses on life-style modifications. Statins, fibrates, and other lipid-lowering agents have been proposed as effective lipid-lowering treatments in patients with NAFLD/NASH. However, clinicians are concerned that hyperlipidemic patients with NAFLD/NASH who are treated with statins could develop transaminitis. We assessed the efficacy and safety of lipid-lowering agents for NAFLD/NASH by reviewing reports of human studies including pilot, prospective, preliminary, and post hoc analysis studies on online databases during the period of 1980 to December 2012. The results of studies provide compelling evidence that lipid-lowering agents are safe and efficacious in patients with NAFLD/NASH and that some of these agents can induce a reduction in the extent of the hepatic steatosis. Well-designed randomized controlled studies of adequate size and duration with histological endpoints are needed in order to establish a suitable lipid-lowering treatment for hyperlipidemic patients with NAFLD/NASH, and for nonhyperlipidemic patients with NAFLD/NASH with a high risk for cardiovascular disease.

Genetic Polymorphisms of Glutathione-S-transferase and Microsomal Epoxide Hydrolase in Egyptian Acquired Aplastic Anemia Patients

Exposure to various environmental toxins with a reduced ability to metabolize them may lead to acquired aplastic anemia (AA). Genetic polymorphism of the detoxifying enzymes, the glutathione-S-transferase (GST) and microsomal epoxide hydrolase (mEh), with alteration in their activities could explain the genetic interindividual risks for AA. We aimed to characterize the genetic polymorphisms of the GST and mEh and to test their impact on the susceptibility, disease severity, and prognosis in Egyptian patients with AA. The GST and mEh genotypes were determined by multiplex-polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism analysis, respectively, in 21 patients with AA and 20 healthy control subjects. The mEh functional phenotypes were assessed. The frequency of GST θ1-null genotype was found significantly higher in AA patients compared with the controls (odds ratio=2.8, 95% confidence interval = 1.1-7.8; P = 0.001). The frequency of heterozygous 139A--G of the mEh gene was significantly higher in AA patients compared with the controls (odds ratio=3.07, 95% confidence interval = 1.23-7.7; P = 0.018). Moreover, the patients with normal functional phenotype of the mEh had significantly favorable prognosis than those with abnormal enzyme activity (P = 0.027). Thus, the GST θ1-null genotype and the 139A--G mEh gene polymorphism may enhance the susceptibility to AA and provide an evidence of gene-environmental interaction.

Caloric restriction delays aging-induced cellular phenotypes in rhesus monkey skeletal muscle

Sarcopenia is the age-related loss of skeletal muscle mass and function and is characterized by a reduction in muscle mass and fiber cross-sectional area, alterations in muscle fiber type and mitochondrial functional changes. In rhesus monkeys, calorie restriction (CR) without malnutrition improves survival and delays the onset of age-associated diseases and disorders including sarcopenia. We present a longitudinal study on the impact of CR on early stage sarcopenia in the upper leg of monkeys from ~ 16 years to ~ 22 years of age. Using dual-energy X-ray absorptiometry we show that CR delayed the development of maximum muscle mass and, unlike Control animals, muscle mass of the upper leg was preserved in CR animals during early phase sarcopenia. Histochemical analyses of vastus lateralis muscle biopsies revealed that CR opposed age-related changes in the proportion of Type II muscle fibers and fiber cross-sectional area. In contrast the number of muscle fibers with mitochondrial electron transport system enzyme abnormalities (ETS ab) was not significantly affected by CR. Laser capture microdissection of ETS ab fibers and subsequent PCR analysis of the mitochondrial DNA revealed large deletion mutations in fibers with abnormal mitochondrial enzyme activities. CR did not prevent stochastic mitochondrial deletion mutations in muscle fibers but CR may have contributed to the maintenance of affected fibers.

Differential detection of IL-1 beta and TNF-alpha mRNA on lung macrophages from patients with pulmonary sarcoidosis

In pulmonary sarcoidosis or experimental granuloma formation, interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha) are considered to play important roles during inflammatory evolution. In order to examine whether IL-1 beta or TNF-alpha mRNA expression on lung macrophages relates to the disease activity or clinical course, ten cases with pulmonary sarcoidosis were divided into two groups: five cases who had a disease duration of more than 10 years (14.6 +/- 4.4 years; group A), and 5 cases with duration of less than 3 years (1.7 +/- 1.1 years; group B). All cases showed both abnormal radiographs and elevated serum angiotensin converting enzyme activities. We compared the 10 cases with 12 healthy individuals as normal control (6 nonsmokers: NS and 6 current smokers: S), and 5 cases with idiopathic pulmonary fibrosis (IPF) as disease control. Lavage macrophages were purified by rosette forming method and plastic adhesion was then performed for 1 hour. Thereafter mRNA was extracted by AGPC method and amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) (20 cycles). The results showed that IL-1 beta mRNA was detected in all materials studied, but TNF-alpha mRNA expression was different among the groups: 5/5 (100%) in group A, 1/5 (25%) in group B, 5/5 (100%) in IPF, and 12/12 (100%) in normal controls. The absence of detection of TNF-alpha mRNA (rapid down regulation) in pulmonary sarcoidosis may relate to spontaneous regression, because a substantial number of cases in group B showed spontaneous regression in their natural course.(ABSTRACT TRUNCATED AT 250 WORDS)

Metastasis associated lung adenocarcinoma transcript 1 is up-regulated in placenta previa increta/percreta and strongly associated with trophoblast-like cell invasion in vitro

Placenta previa increta/percreta (I/P) is a severe form of invasive placentation associated with massive peripartum hemorrhage, which often requires Cesarean hysterectomy. The pathogenesis of invasive placentation is multidimensional, involving decidual deficiency, endomyometrial damage and excessively deep trophoblast invasion into the uterus. In this study, annealing control primer-polymerase chain reaction (ACP-PCR) was used to identify differentially expressed genes, which may impair placentation resulting in placenta previa I/P. Placental tissues from I/P and non-increta/percreta (non-I/P) sites were concomitantly collected from patients undergoing Cesarean hysterectomy. After ACP-PCR experiments (three patients), the differentially expressed bands, consistently showing up- or down-regulated trends between each of the I/P and non-I/P tissue pairs, were cloned and sequenced. Human non-protein coding metastasis associated lung adenocarcinoma transcript 1 (MALAT-1) gene was identified. Real-time quantitative PCR (10 patients) confirmed significant overexpression of MALAT-1 in I/P samples (P = 0.005). To investigate the role of MALAT-1 gene in the regulation of trophoblast cell invasion, targeting of MALAT-1 mRNA expression with short interfering RNA (siRNA) in trophoblast-like BeWo, JAR and JEG-3 choriocarcinoma cells was performed. The invasion ability of these cells was significantly suppressed after siRNA silencing (P < 0.001), and this was not correlated with abnormal MMP-2 and MMP-9 enzyme activities. Our results suggest that MALAT-1 expression in placenta previa I/P is increased and its down-regulation inhibits trophoblast-like cell invasion in vitro. MALAT-1 might be involved in regulating trophoblast invasion during the development of advanced invasive placentation.

Generation of N‐ethyl‐N‐nitrosourea‐induced mouse mutants with deviations in plasma enzyme activities as novel organ‐specific disease models

Measurement of plasma enzyme activities is part of routine medical examination protocols and provides valuable parameters for the diagnosis of various organ diseases. In the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project, clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, alpha-amylase and creatine kinase. We identified a large number of animals that consistently exhibited altered plasma enzyme activities. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for each parameter. Breeding experiments in selected lines detected the linkage of the causative mutations to defined chromosomal regions. Subsequently, identification of the mutated genes was successfully carried out in chosen lines, resulting in a novel alkaline phosphatase liver/bone/kidney (Alpl) alteration in one line and the strong indication for a dystrophin (Dmd) alteration in another line. The mouse mutants with abnormal plasma enzyme activities recovered in the Munich ENU project are novel tools for the systematic dissection of the pathogenesis of organ diseases.

Case Report: Dystrophic Epidermolysis Bullosa: dental management and oral health promotion

Epidermolysis Bullosa (EB) is a group of rare genetically determined disorders characterised by the development of blisters following minor or insignificant trauma or traction to the skin or mucosal surfaces. It appears that the cause of EB is related to abnormal enzyme activity and collagen degradation, but the exact pathogenesis and aetiology remain obscure. There are three major forms -- simple, junctional, and dystrophic. Oral manifestations and dental involvement of EB vary in frequency, and severity according to the form. A 13-year old boy with a known diagnosis of Dystrophic Epidermolysis Bullosa (DEB) since birth was seen in our dental practice in 2004. Upon examination, the patient had multiple scars and blisters involving his neck, head, elbows and knees. Pseudosyndactyly was present on both hands. Intraoral examination revealed multiple carious lesions affecting almost the entire dentition, with severe gingival inflammation, mycrostomia, ankyloglossia and elimination of buccal and vestibular sulci. This was divided into four phases- (1) oral hygiene and dietary instructions, elimination of gingival inflammation, (2) restorative procedures, (3) extractions of the carious destroyed teeth, and (4) recall system. The patient was recalled on a schedule every three months with continued evaluation of oral status (Pl and GI indices). The preventive regimen included twice yearly topical fluoride application (Duraphat 2.26%). The final recommendations for our patient (except brushing twice a day), were to use daily fluoride mouthrinse (NaF 0.05%), to avoid cariogenic food products, and to visit a dietician to correct his diet. By applying present knowledge of DEB the oral health of these patients can be improved and eliminate complications arising from the oral condition. After three routine check-ups and due to the cooperation of this patient, his oral health status remains very satisfactory.