Laboratory Investigation (April 2013) – Listarfish

Ryanodine receptor 2 (cardiac)- New markers for preventing and treating heart attack, angina pectoris and various types of arrhythmia.

The official name of this gene is “ryanodine receptor 2 (cardiac).” This protein is part of a family of ryanodine receptors, which form channels that transport positively charged calcium atoms (calcium ions) within cells.

One such group of proteins is known as ryanodine receptor 2 (cardiac),  (intracellular calcium release channels), which are the major channels through which Ca(2+) ions leave the sarcoplasmic reticulum, leading to cardiac muscle contraction.

Channels made with the ryanodine receptor 2 protein are found in heart (cardiac) muscle cells called myocytes. These channels are embedded in the outer membrane of a cell structure called the sarcoplasmic reticulum, which acts as a storage center for calcium ions. The RYR2 channel controls the flow of calcium ions out of the sarcoplasmic reticulum.

For the heart to beat normally, the cardiac muscle must tense (contract) and relax in a coordinated way. This cycle of muscle contraction and relaxation results from the precise control of calcium ions within myocytes. In response to certain signals, the RYR2 channel releases calcium ions from the sarcoplasmic reticulum into the surrounding cell fluid (the cytoplasm). The resulting increase in calcium ion concentration triggers the cardiac muscle to contract, which pumps blood out of the heart. Calcium ions are then transported back into the sarcoplasmic reticulum, and the cardiac muscle relaxes. In this way, the release and reuptake of calcium ions in myocytes produces a regular heart rhythm.

The RYR2 gene mutations responsible for ARVC (arrhythmogenic right ventricular cardiomyopathy) change single amino acids in the ryanodine receptor 2 protein.

Cytogenetic Location: 1q43. Molecular Location on chromosome 1: base pairs 237,205,701 to 237,997,287

The RYR2 gene is located on the long (q) arm of chromosome 1 at position 43. More precisely, the RYR2 gene is located from base pair 237,205,701 to base pair 237,997,287 on chromosome 1.

In fact the Metabolic Syndrome is characterized by a combination of obesity, hypertension, insulin resistance, dyslipidemia, and impaired glucose tolerance. This multifaceted syndrome is often accompanied by a hyperdynamic circulatory state characterized by increased blood pressure, total blood volume, cardiac output, and metabolic tissue demand.

One of the main and frequent complications seen in metabolic syndrome is cardiovascular disease. The primary endpoints of cardiometabolic risk are coronary and peripheral arterial disease, myocardial infarction, congestive heart failure, arrhythmia, and stroke.

Alterations in expression and/or functioning of several key proteins involved in regulating and maintaining ionic homeostasis can cause cardiac disturbances.

Nature – Progress and challenges in translating the biology of atherosclerosis.

MutaCHIP ARTERO - New test for the Analysis of Genetic Predisposition of Atherosclerotic Cardiovascular Diseases.

The MutaCHIP® ARTERO is a biomolecular test for the analysis of mutations in marker genes, which can lead to atherosclerotic cardiovascular diseases. The analyzed gene variations are associated with genes, which regulate blood pressure, lipid balance, nutrient metabolism and oxidative stress.

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrotic elements in the arteries. The early atherosclerotic lesions are made up of subendothelial accumulations of fat-containing macrophages, so called foam cells. These lesions, also called “fatty streaks”, can be detected in the aorta and with increasing age also in the coronary and cerebral arteries. The “fatty streaks” are the precursors of advanced lesions, which are characterized by the accumulation of lipid-rich necrotic debris and smooth muscle cells. These plaques can become more complex by calcium inclusion, ulcer formation and bleedings. Although these accumulation can become large enough to inhibit the blood flow, the most important clinical complication is an acute blockage caused by the formation of a thrombus or blood clot, which can lead to a myocardial infarction or stroke. 

Risk factors of atherosclerosis are environmental factors, like nutrition rich in fat, nicotine consumption, lack of movement and infection. In addition, there a several factors with a strong genetic component like increased LDL/VLDL (low density and very-low density lipoproteins), decreased HDL (high density lipoprotein), high blood pressure, increased haemostatic factors, increased homocysteine, diabetes, adiposity and gender.

Genomic-epidemiologic studies have identified candidate genes showing a significant or suggestive disease association. The following single nucleotide polymorphisms (SNP) and variations can be associated with the development of atherosclerosis and its resulting complications.

By genotyping those patients can be identified, who have an increased risk to develop atherosclerosis or its resulting complications. This will allow an early prevention and intervention. In addition, the differentiation of the varying forms of the disease is possible, which will allow an individualized therapy.

LISTARFISH Investigation 2013

HUMAN CLUSTERIN – a marker for the study of morphologically abnormal spermatozoa and fertility of men.

Clusterin, (Apolipoprotein) is a highly conserved disulfide-linked secreted heterodimeric glycoprotein of 75-80 kDa. A heterodimeric glycoprotein found at several sites in the human male reproductive tract, could be a marker of morphologically abnormal spermatozoa.

The protein is constitutively secreted by a number of cell types including epithelial and neuronal cells and is a major protein in physiological fluids including plasma, milk, urine, cerebrospinal fluid and semen.

Due to its wide breath of tissue distribution many diverse physiological functions have been attributed to Clusterin including sperm maturation.

Recent study demonstrate, that serum clusterin level increases significantly in diabetic type II patients and in patients with developing coronary heart disease, or myocardial infarction. These date raise the possibility that elevated clusterin levels in serum may represent a strong indication of vascular damage.

Metabolic disorders such as diabetes mellitus and obesity may compromise sperm quality and fertility of men; however, little evidence specifically links hypertension with the impairment of male reproductive function.

Clusterin is a protein encoded by the CLU gene and it is expressed in most human tissue types, especially when under stress, where it helps protects cells.  They found significantly elevated levels of clusterin protein and its mRNA precursor in men with dysfunctional sperm compared to men with healthy spermatozoa.

Clusterin levels were closely correlated with sperm quality, such that the higher the expression of CLU, the lower the sperm count and less motile the cells.

Increased clusterin expression is also associated with increased DNA fragmentation in the spermatozoa of infertile men.

Clusterin normally prevents proteins from precipitating inside sperm cells and stops abnormal sperm cells from clumping together.

The new findings suggest that increased expression of the protein may be indicative of spermatozoa under stress and could be a useful marker for quality sperm (normozoospermia,  asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia) and fertility of men.

Molecular mechanisms of cytostatic drugs, tested for sensitivity and resistance, in individual chemotherapy: efficacy and toxicity.

Summary
The TAO is a tool for targeted therapy in oncology: a therapy tailored to the individual patients suffering from tumors, with fewer side effects.

The purposes of the TAO is to improve the consequences of chemotherapy for cancer, thereby develop a new treatment of chemotherapy individual.The TAO will seek to identify, through a molecular mechanism of sensitivity and / or resistance, an effective and safe drug for a patient suffering from a tumor.The TAO is based on the complex interactions between patient characteristics, pathophysiology of the disease and drug metabolism.The genetic analysis, of a patient with cancer, will allow to identify a targeted chemotherapy, through monoclonal antibodies and biological modifiers.For this he was made a new plan for cancer research, based primarily on the availability of colorimetric techniques and their high rates, to avoid the results of false negatives and false positives.These accidents can be reduced by using the techniques of molecular biology and genetics, which are targets of new components sensitive to chemotherapy. Similar mechanisms can be sought between the MDR1 protein, the protein of LRP, glutathione transferase, gene amplification etc..In parallel, also other mechanisms, tumor infiltration, can be located through neovascularization factors that are considered necessary for the ability of the development of cancer metastasis.Resistance mechanisms and therapeutic efficacy of chemotherapy drugs, will be identified by the genomic and proteomic and molecular biology techniques (micro-array, m-antisense RNA, PCR, RT-PCR, Southern and Northern blot etc.) On sequence of samples before and after chemotherapy, also including in vitro studies of cell lines of human tumors.These mechanisms will be validated by analysis of peripheral blood (venous) or biopsies of patients with cancer.The result of these activities will be validated clinically and functionally, the mechanisms of individual chemotherapy.

Problem
The resistance to chemotherapy is still one of the biggest problems in oncology and that contributes to the death of many patients suffering from cancer. Today, despite great effort has been made ​​progress in pharmacological research, to solve this problem negative. Not only do you want to efficiently overcome the state of resistance to chemotherapy, but you want to have a successful response to chemotherapy: thanks to the usefulness of new diagnostic tools for individual chemotherapy.

Accordingly knowing the mechanisms of resistance, there has been the need to improve the developing of predictive tests and modulate the resistance, can be improved therapeutic treatments in oncology.  As we know, cancer chemotherapy is frequently associated, severe, side effects and toxicity. Today the new tools of molecular biology and genomics are needed to prevent adverse events from chemotherapy.

Purpose
The purpose of the TAO to develop a means to a tumor response to treatment with chemotherapy individual, is to improve the health conditions of the patient.

Expectations of results

The new knowledge of the TAO will lead to new tools for targeted treatment, without any consequences toxicity to chemotherapy. These diagnostic expertise, can be used to identify and prepare new potential modulators based on the mechanisms of the test sensitivity and resistance of drugs. This new information will help your doctor to carefully targeted cancer therapy

Potential Applications

Today, medical oncology, you can use predictive tests that profit to cancer therapy. The new information on molecular mechanisms and targeted genomic resistance, leading to the development of modulators and / or identification of drug-resistant. The use of the new technique of TAO, is the effective tool for targeted treatment of cancer drugs.

Summary
The TAO is a tool for targeted therapy in oncology: a therapy tailored to the individual patients suffering from tumors, with fewer side effects.

The purposes of the TAO is to improve the consequences of chemotherapy for cancer, thereby develop a new treatment of chemotherapy individual.

The TAO will seek to identify, through a molecular mechanism of sensitivity and / or resistance, an effective and safe drug for a patient suffering from a tumor.

The TAO is based on the complex interactions between patient characteristics, pathophysiology of the disease and drug metabolism.

The genetic analysis, of a patient with cancer, will allow to identify a targeted chemotherapy, through monoclonal antibodies and biological modifiers.

For this he was made a new plan for cancer research, based primarily on the availability of colorimetric techniques and their high rates, to avoid the results of false negatives and false positives.
These accidents can be reduced by using the techniques of molecular biology and genetics, which are targets of new components sensitive to chemotherapy.

Similar mechanisms can be sought between the MDR1 protein, the protein of LRP, glutathione transferase, gene amplification etc..

In parallel, also other mechanisms, tumor infiltration, can be located through neovascularization factors that are considered necessary for the ability of the development of cancer metastasis.
Resistance mechanisms and therapeutic efficacy of chemotherapy drugs, will be identified by the genomic and proteomic and molecular biology techniques (micro-array, m-antisense RNA, PCR, RT-PCR, Southern and Northern blot etc.) On sequence of samples before and after chemotherapy, also including in vitro studies of cell lines of human tumors.

These mechanisms will be validated by analysis of peripheral blood (venous) or biopsies of patients with cancer.

The result of these activities will be validated clinically and functionally, the mechanisms of individual chemotherapy.

Problem
The resistance to chemotherapy is still one of the biggest problems in oncology and that contributes to the death of many patients suffering from cancer. Today, despite great effort has been made ​​progress in pharmacological research, to solve this problem negative. Not only do you want to efficiently overcome the state of resistance to chemotherapy, but you want to have a successful response to chemotherapy: thanks to the usefulness of new diagnostic tools for individual chemotherapy.

Accordingly knowing the mechanisms of resistance, there has been the need to improve the developing of predictive tests and modulate the resistance, can be improved therapeutic treatments in oncology.

As we know, cancer chemotherapy is frequently associated, severe, side effects and toxicity. Today the new tools of molecular biology and genomics are needed to prevent adverse events from chemotherapy.

Purpose
The purpose of the TAO to develop a means to a tumor response to treatment with chemotherapy individual, is to improve the health conditions of the patient.

Expectations of results

The new knowledge of the TAO will lead to new tools for targeted treatment, without any consequences toxicity to chemotherapy.

These diagnostic expertise, can be used to identify and prepare new potential modulators based on the mechanisms of the test sensitivity and resistance of drugs.

This new information will help your doctor to carefully targeted cancer therapy

Potential Applications

Today, medical oncology, you can use predictive tests that profit to cancer therapy. The new information on molecular mechanisms and targeted genomic resistance, leading to the development of modulators and / or identification of drug-resistant.

The use of the new technique of TAO, is the effective tool for targeted treatment of cancer drugs.

Abstract

Epigenetics acts as an interface between environmental/exogenous factors, cellular responses, and pathological processes. Aberrant epigenetic signatures are a hallmark of complex multifactorial diseases (including neoplasms and malignancies such as leukemias, lymphomas, sarcomas, and breast, lung, prostate, liver, and colorectal cancers). Epigenetic signatures (DNA methylation, mRNA and microRNA expression, etc) may serve as biomarkers for risk stratification, early detection, and disease classification, as well as targets for therapy and chemoprevention. In particular, DNA methylation assays are widely applied to formalin-fixed, paraffin-embedded archival tissue specimens as clinical pathology tests. To better understand the interplay between etiological factors, cellular molecular characteristics, and disease evolution, the field of ‘molecular pathological epidemiology (MPE)’ has emerged as an interdisciplinary integration of ‘molecular pathology’ and ‘epidemiology’. In contrast to traditional epidemiological research including genome-wide association studies (GWAS), MPE is founded on the unique disease principle, that is, each disease process results from unique profiles of exposomes, epigenomes, transcriptomes, proteomes, metabolomes, microbiomes, and interactomes in relation to the macroenvironment and tissue microenvironment. MPE may represent a logical evolution of GWAS, termed ‘GWAS-MPE approach’. Although epigenome-wide association study attracts increasing attention, currently, it has a fundamental problem in that each cell within one individual has a unique, time-varying epigenome. Having a similar conceptual framework to systems biology, the holistic MPE approach enables us to link potential etiological factors to specific molecular pathology, and gain novel pathogenic insights on causality. The widespread application of epigenome (eg, methylome) analyses will enhance our understanding of disease heterogeneity, epigenotypes (CpG island methylator phenotype, LINE-1 (long interspersed nucleotide element-1; also called long interspersed nuclear element-1; long interspersed element-1; L1) hypomethylation, etc), and host–disease interactions. In this article, we illustrate increasing contribution of modern pathology to broader public health sciences, which attests pivotal roles of pathologists in the new integrated MPE science towards our ultimate goal of personalized medicine and prevention.

Keywords: CIMP; genetics; hypermethylation; molecular pathological epidemiology; omics; personalized therapy; unique tumor principle

Eoin P Cummins1, Glen A Doherty1 and Cormac T Taylor1

Abstract

Inflammatory bowel disease (IBD) is a common and debilitating clinical disorder comprising ulcerative colitis and Crohn’s disease. IBD occurs when inappropriate immunological activity in the intestinal mucosa results in epithelial barrier dysfunction leading to exposure of the mucosal immune system to luminal antigenic material. This in turn results in the cycles of inflammation and further barrier dysfunction which underlie disease progression. Although significant therapeutic advances have been made over the last decade, current immunosuppressive and anti-inflammatory treatments for IBD have significant limitations due to lack of treatment response in some patients and adverse effects, including increased risk of infection and malignancy. Recent studies using experimental models of IBD have identified that intracellular hydroxylases, a group of enzymes responsible for oxygen sensing and activation of adaptive transcriptional responses to hypoxia may represent a new class of therapeutic targets in IBD.

Hydroxylase inhibitors are effective in ameliorating symptoms of colitis at least in part through the promotion of intestinal epithelial barrier function. The mechanism of this protection is due to activation of hypoxia-sensitive transcription factors, including the hypoxia-inducible factor (HIF) and nuclear factor kappa-B (NF-κB), which activate specific epithelial barrier-protective transcriptional programs.

In this review, the mechanism(s) of action and the therapeutic potential of small molecule hydroxylase inhibitors for the treatment of IBD will be discussed.

Keywords:  Crohn’s disease; DMOG; epithelial barrier; therapeutics; ulcerative colitis

Jung-Chien Cheng, Hui-Pin Cheng, I-Ching Tsai and Meei Jyh Jiang

Reactive oxygen species (ROS) mediates the aberrant contractility in hypertension. Abnormal contractility occurs in atherosclerotic vessels but changes in proteins that regulate contractility remain poorly understood. Myosin phosphatase (MP) activity, which regulates smooth muscle relaxation, is regulated by the phosphorylation of its regulatory subunit, MP targeting subunit 1 (MYPT1). In the present study, we examined the roles of ROS in MP subunit expression both in cultured human aortic smooth muscle cells (HASMCs) and during atherosclerosis progression in apolipoprotein E-knockout (apoE-KO) mice. Furthermore, the effect of decreased MYPT1 on actin cytoskeleton and cell migration activity was assessed in HASMCs. Short hairpin RNA-mediated knockdown of MYPT1 increased stress fibers and attenuated platelet-derived growth factor-induced cell migration in HASMCs. Superoxide anion-inducing agent LY83583 downregulated MYPT1 mRNA and protein levels, but did not affect the phosphorylation of MYPT1 and catalytic subunit of MP, PP1δ. The LY83583-induced decrease in MYPT1 was abolished by co-treating with superoxide dismutase or by inhibiting NADPH oxidase with diphenyleneiodonium. Treatment of peroxynitrite, but not hydrogen peroxide (H₂O₂), downregulated MYPT1 protein expression and induced MYPT1 phosphorylation without affecting mRNA levels. Co-treatment with a proteasome inhibitor, MG-132, eliminated peroxynitrite-induced MYPT1 downregulation. In apoE-KO mice, MYPT1 protein, but not mRNA, levels were markedly decreased in 16-week- and 24-week-old mice. Oral estrogen treatment, which was previously shown to decrease aortic ROS levels, upregulated aortic MYPT1 expression. Moreover, reduction in MYPT1 expression correlated with increased aortic sensitivity toward vasoconstrictors.

These results suggested that during atherosclerosis progression oxidative stress mediates the downregulation of MYPT1, which may inhibit smooth muscle cell migration and contribute to the aberrant contractility.

Extra chromosome 21 removed from Down syndrome cell line

By Leila Gray

University of Washington scientists have succeeded in removing the extra copy of chromosome 21 in cell cultures derived from a person with Down syndrome, a condition in which the body’s cells contain three copies of chromosome 21 rather than the usual pair.

A triplicate of any chromosome is a serious genetic abnormality called a trisomy. Trisomies account for almost one-quarter of pregnancy loss from spontaneous miscarriages, according to the research team. Besides Down syndrome (trisomy 21), some other human trisomies are extra Y or X chromosomes, and Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13), both of which have extremely high newborn fatality rates.

In their report appearing in the Nov. 2 edition of Cell Stem Cell, a team led by Dr. Li B. Li of the UW Department of Medicine described how they corrected trisomy 21 in human cell lines they grew in the lab. The senior scientists on the project were gene therapy researchers Dr. David W. Russell, professor of medicine and biochemistry, and Dr. Thalia Papayannopoulou, professor of medicine.

The targeted removal of a human trisomy, they noted, could have both clinical and research applications.

In live births, Down syndrome is the most frequent trisomy. The condition has characteristic eye, facial and hand features, and can cause many medical problems, including heart defects, impaired intellect, premature aging and dementia, and certain forms of leukemia, a type of blood cancer.

IHSR – Tight Junction Research 

“QK305®” a important regulator of intestinal function and tight junction integrity.

The intestinal epithelium is a single layer of columnar epithelialcells that separates the intestinal lumen from the underlyinglamina propria. The intestinal epithelial cells are mainly absorptiveenterocytes (over 80%) but also include enteroendocrine,goblet, and Paneth cells. These epithelial cells are tightlybound together by intercellular junctional complexes thatregulate the paracellular permeability and are crucial for theintegrity of the epithelial barrier. Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the fence function and  the barrier (or gate) function.

In this study investigated the protective effect of the“QK305®”, against the tight junction disruption and hyperpermeability. Concurrently, the increased activities of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were observed, enhanced TJ integrity through the cytoskeletral association and expression of TJ proteins in human intestinal.

The“QK305®” are against the tight junction disruption and hyperpermeability. In fact, the actual characteristic of the “QK305®” is  an efficient medication in the maintenance of gut homeostasis by limiting the penetration of luminal bacteria and dietary allergens, yet allowing antigen sampling for the generation of toleranceand pathogens entering the mucosal tissues, potentially causing diseases (increased intestinal permeability is implicated in autoimmune, inflammatory, degenerative diseases such as cancer, diabetes, cardiovascular and atopic diseases, which can manifest both locally – within the intestinal mucosa – and systemically).

Laboratory Research  April 2013 

HUMAN FETUIN A – a new marker for the type 2 diabetes. 

Two studies report about a new and independent marker that is associated with type 2 diabetes. The protein that is called Fetuin-A is produced in the liver and secreted to the blood stream also indicates a higher risk of developing diabetes disease.

The liver-secreted protein Fetuin-A induces peripheral insulin resistance in vitro. In a pilot study, we observed that higher Fetuin-A levels were associatedwith diabetes mellitus in older persons. However, this finding has not been confirmed in large cohorts. We sought to confirm the association of Fetuin-A with incident diabetes mellitus in older persons to determine whether the association differs by age, sex, and race and among persons with cardiovascular diseases (CVD).

Higher Fetuin-A is associated with incident diabetes mellitus in older persons regardless of sex, race, or prevalent CVD status. Biologically, studies have demonstrated that Fetuin-A is the major calcification inhibitor found in circulation, where it interferes with calcium salt  precipitation.

The association may be attenuated in those ≥ 75 years of age.

Fetuin-A, also known as alpha-2-HS glycoprotein, is a 59 kDa glycoprotein that  consists of two amino-terminal cystatin domains and a smaller carboxyl-terminal  domain. Fetuin-A is synthesized by the liver and secreted into blood stream, where its  concentration in adult mammals ranges from 0.5 – 1.5 g/L. Fetuin-A occurs in high  serum concentration during fetal life and involves in protease inhibitory activities and development-associated regulation of calcium metabolism.

Type 2 diabetes mellitus has become a global epidemic that affects not only elder people but also younger especially obesed individuals. Nowadays the risk factors for developing diabetes are said to be gender, age, waist measurement, body mass index or other more or less contributing factors.

“Diabetes” and  “Journal of American Medical Association” demonstrated that a protein produced in the liver is highly associated with the risk of suffering from type 2 diabetes.

Fetuin-A is secreted in the blood stream when the body becomes unable to produce insulin in the needed amount. Its main function is regarded as a potent inhibitor of systemic calcification.

But previous studies have found an association between higher Fetuin-A levels and insulin resistance, too.

Independet from age people with a high Fetuin-A level (average 304 mg/ml) had a 75 per cent higher risk to develop diabetes than those with a lower level (average 158 mg/ml). 

Particulary in persons with a heightened blood glucose level a distincitiv association with a high fetuin-A level attested.

Association of Fetuin-A with incident diabetes mellitus in community-living older adults: the cardiovascular health study. Higher Fetuin-A is associated with incident diabetes mellitus in older persons regardless of sex, race, or prevalent CVD status. The association may be attenuated in those≥75 yeats of age.