Cellular Malfunction Research Articles

Cell and Gene-Based Therapies for the Lysosomal Storage Diseases

Lysosomal storage disorders (LSD) are a group of approximately 40 genetic diseases that are caused by the deficiency of one or more lysosomal enzymes. The incidence of LSD is estimated to be approximately 1 in 7500 live births, which makes this one of the more prevalent groups of genetic diseases in humans. The loss in enzymatic activity leads to the accumulation of undegraded substrates within lysosomes, resulting in distension of the organelle and subsequent cellular malfunction. Although palliative treatments such as enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) have been shown to be effective for some of the LSD such as Gaucher, Fabry and MPS I, they are not available as yet, or ineffective, for a large number of other LSD patients. To fulfill this unmet medical need, gene therapy is being considered as an alternate or adjunctive therapy for this group of disorders. A goal of gene therapy for LSD is to introduce a normal copy of the DNA for the lysosomal enzyme into a depot organ such as the liver or muscle with the intent that this will lead to the sustained production and re-constitution of therapeutic levels of the enzyme in the affected tissues. Here, we review the utility of various gene therapy strategies under consideration for the treatment of the LSD, including viral and non-viral gene transfer approaches, as well as stem cell transplantation.

Experimental investigation of protein folding and misfolding

Newly synthesised proteins need to fold, often to intricate and close-packed structures, in order to function. The underlying mechanism by which this complex process takes place both in vitro and in vivo is now becoming understood, at least in general terms, as a result of the application of a wide range of biophysical and computational methods used in combination with the techniques of biochemistry and protein engineering. It is increasingly apparent, however, that folding is not only crucial for generating biological activity, but that it is also coupled to a wide range of processes within the cell, ranging from the trafficking of proteins to specific organelles to the regulation of cell growth and differentiation. Not surprisingly, therefore, the failure of proteins to fold appropriately, or to remain correctly folded, is associated with a large number of cellular malfunctions that give rise to disease. Misfolding, and its consequences such as aggregation, can be investigated by extending the types of techniques used to study the normal folding process. Application of these techniques is enabling the development of a unified description of the interconversion and regulation of the different conformational states available to proteins in living systems. Such a description proves a generic basis for understanding the fundamental links between protein misfolding and its associated clinical disorders, such as Alzheimer’s disease and Type II diabetes, and for exploring novel therapeutic strategies directed at their prevention and treatment on a rational basis.

Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli.

The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NB-prone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NB-prone neutral glycolipid alpha-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg(2+) was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg(2+) and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.

Impact of treatment modality, biochemical parameters and apoptosis on polymorphonuclear cell (PMN) function in patients with renal failure

It has been suggested that PMN apoptosis is increased in dialysis patients and may contribute to cellular dysfunction. We investigated the effect of treatment modality and biochemical parameters on PMN apoptosis and function. Blood was drawn from 17 controls, 17 patients with chronic renal failure (CRF; creatinine clearance 28 ± 14 ml/min/1.73 m2), 10 hemodialysis (HD) and 11 CAPD patients. Upon collection, whole blood aliquots were incubated in RPMI-1640 with propidium iodide (PI)-labeled Saureus (SA), PMA, fMLP or LPS for 30 min. Cells were then stained with DCFH-DA and analyzed by flow cytometry, in order to quantify phagocytosis and H2O2 release by PMN. After separation by gradient centrifugation, PMN were stained with Annexin-V and PI in order to quantify apoptosis by flow cytometry. The results were correlated with blood levels of urea, creatinine, bicarbonate, albumin and PTH. Results are presented as means ± SD. Among CRF and HD patients, there was an inverse correlation between apoptosis and SA- (r = 0.62, P = 0.01 and r = 0.89, P = 0.02, respectively) and LPS-stimulated H2O2 release (r = 0.68, P = 0.005 and r = 0.61, P = 0.058, respectively). No biochemical parameters correlated with apoptosis or cellular functions. In summary, PMN apoptosis contributes to cellular malfunction in uremia, but does not account for all the dysfunction. Hence, it is possible that other uremic toxins affect cell performance independently of apoptosis. Table

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture

Autoimmunity and cancer - Beneficial relationships

Autoimmune diseases are the consequence of the immune system attack on the self. Autoimmunity, sometimes, is reflected through the production of autoantibodies to various self antigens leading to cellular malfunction or destruction. In the current study we employ such autoantibodies for the treatment of cancer originated from the respective normal cells. This concept is wide and entails many autoimmune disorders and diversity of malignant conditions. To illustrate the therapeutic and practical potential we depicted four examples: vitiligo and melanoma - vitiligo is considered dermatologic autoimmune disorder presented as depigmented skin areas. The destruction of the pigmented cells (melanocytes) is mediated by autoantibodies. We have purified these anti-melanocyte antibodies from patients with vitiligo and showed their cytotoxic effect towards malignant melanoma cells in vitro and in vivo; anti-phospholipid syndrome (APLS) and cancer-cancer cells differ from normal cells by the expression of phosphatydilserine (PS) on their outer membrane surface. We have used such anti-PS autoantibodies, derived from patients with APLS, as effective treatment for melanoma in a murine system; Autoimmune hemolytic anemia (AIHA) and hematological malignancies - in patients with AIHA, the red blood cells (RBC) destruction is mediated by binding of autoantibodies to RBC. We have shown the specific binding of these anti-RBC autoantibodies to RBC and to malignant cells of the erythroid lineage. Thus, these autoantibodies may have diagnostic/therapeutic potential for conditions such as erythroleukemia and polycythemia vera; anti-lymphocyte antibodies and lymphoproliferative diseases - in SLE and other systemic autoimmune conditions autoantibodies directed against lymphocytes can be found. These cytotoxic antibodies can be targeted against lymphocytes of lymphoproliferative diseases such as chronic lymphocytic leukemia, or lymphoma. The diversity of autoimmune diseases provides a great source for human preformed highly specific autoantibodies which can be used as an effective immunotherapy by themselves, for diagnostic purposes, as a cell specific carrier conjugated to other cytotoxic agents and in combined therapy in the combat against cancer.

Human nucleotide excision repair syndromes: Molecular clues to unexpected intricacies

INTRODUCTION THE DEVELOPMENT and maintenance of life has critically depended on the evolution of mechanisms that ensure genetic integrity and stability. DNA, the vital carrier of genetic information, is continually subjected to undesired chemical alterations. Numerous environmental or endogenous compounds and various types of radiation, such as X-rays and ultra-violet (UV) light, induce a wide variety of lesions in the bases, sugars or phosphates that make up the DNA. Obviously, such lesions (adducts, crosslinks, breaks, etc.) interfere with the proper functioning of the genome. An intricate network of DNA repair systems constitutes the main barrier protecting against the deleterious consequences of DNA injury. This is illustrated by the phenotype of inherited defects in one of these repair pathways. Such disorders are invariably associated with a characteristic hypersensitivity to a specific class of genotoxic agent. In addition, the DNA lesions that persist lead to cellular malfunction and to enhanced mutagenesis, due to the increased probability that errors occur upon replication of a damaged template. Somatic mutagenesis is the initiator of multistep carcinogenesis. Rare inborn disorders with hallmarks characteristic of repair defects comprise the class of chromosomal instability syndromes. Well-known examples are Fanconi’s anaemia, ataxia telangiectasia and Bloom’s syndrome, all of which display different manifestations of cancer predisposition and increased sensitivity to specific mutagens. The prototype disorder of this type is, however, xeroderma pigmentosum (XP), in which a defect in the nucleotide excision repair pathway underlies the pronounced predisposition to skin cancer and characteristic photosensitivity. The molecular mechanism and biological relevance of this repair system constitutes the topic of this chapter. For a comprehensive review of DNA damage and the intricate network of DNA repair systems in general, the interested reader is referred to Friedberg PI. Nucleotide excision repair (henceforth abbreviated as NER) is one of the most versatile cellular repair mechanisms. This universal system eliminates a remarkably diverse array of structurally unrelated lesions, that range from W-induced photoproducts (cyclobutane pyrimidine dimers and 6-4 photoproducts) to bulky and small chemical adducts as well as intra-strand crosslinks. In this light, it is not surprising that the NER process entails multiple steps, and involves the concerted action of a number of proteins. The details of the reaction mechanism are best understood in the case of the UvrABC system in the

Membrane defect and energy status of rabbit skeletal muscle cells in sepsis and septic shock.

Measurements of the resting membrane potential in skeletal muscle were used to monitor cell function in live Escherichia coli-induced sepsis and septic shock. Depolarization of the cell membrane, indicative of cell swelling, occurred before the onset of deep hypotension, suggesting cellular injury as a primary cause rather than a result of shock. The normal values for high-energy phosphates found in skeletal muscle in the terminal stages of shock reduce the possibility of an energy deficit as one of the factors in the cellular membrane malfunctions in septic shock.

Evaluation of burn blister fluid.

Although edema is evident immediately after a burn, the diffusion of nutrient chemical constituents of the body is not impaired. Blister fluid, not unlike plasma or serum, contained all substances found in the body, including parenterally administered penicillin. The elevation of potassium and the cation to anion imbalance is primarily due to the Na/K cellular pump malfunction, and the destruction of the permeability of the cell membrane is most likely a direct result of complement and other cellular enzymes, which include the prostaglandins and thromboxanes. The elevated SGOT, CPK, and LDH indicated severe trauma to the cells in the immediate area of burn and possibly to the skeletal muscle. The presence of immunoglobulins indicated that high-molecular-weight proteins diffuse equally well during this edematous phase (IgM, 900,000; IgG, 190,000). Evidence of this nature strongly suggests that the integrity of the burn blister by maintained.