Some Anti-Inflammatory Cytokines - Properties and Mechanism of Action

Summary: Classification of cytokines is based on structure of active proteins and their receptors but it takes into account also their biological activities. Anti-inflammatory cytokines include IL-10 and related to each other IL-4 and IL-13. Transduction of signal from receptors of these cytokines requires protein kinases JAK, TYK and several transcription factors belonging to STAT family. The above-mentioned three cytokines inhibit synthesis of pro-inflammatory cytokines (IL-1, TNFa, IL-8) in the blood cells stimulated with endotoxin, but they may enhance production of pro-inflammatory cytokines in other types of cells. The mechanism of paradoxical effect of anti-inflammatory cytokines has not been so far fully elucidated.
 

Key Words: proinflammatory and anti-inflammatory cytokines, IL-10, IL-4, IL-13, TNFa, IL-1, IL-6, signal transduction, transcription factors

Some Inhibitors of Signal Transduction in The Down-Regulation of Cytokine Synthesis

Summary: Acute phase response caused by infections, tumor growth and injuries results in the release of secondary mediators � cytokines, which serve to restore the organism's homeostasis. Overexpression of the cytokines can lead to many diseases or even death. Therefore, substances with the ability to block cytokines' synthesis are being constantly searched for. The aim of this paper is to characterize action of rooperol, SB203580 and parthenolide � the three signal transduction inhibitors, which, on the one hand, could be tools in the investigation of the molecular mechanisms of cell metabolism regulation and, on the other hand, might be useful as antiinflammatory drugs.

Key Words: acute phase response, rooperol, SB203580, parthenolide, cytokines, p38 kinase

Leptin � Signal Transduction and Interaction with Cytokines

Summary: Leptin, the protein product of the ob gene, is secreted mainly by adipose tissue and plays an important role in the control of food intake, body weight and energy expenditure. The leptin receptors OB-R belong to the class I cytokine receptor family and are produced in several alternatively spliced izoforms. Only the full-length isoform, the OB-RL is believed to be involved in leptin signaling and is consider to be the functional receptor. OB-R is known to act through Janus kinases (JAK) and signal transducers of transcription STAT. Initially most investigations focused on the effects of leptin on the central nervous system. However different leptin receptor isoforms are not only confined to the brain but exhibit widespread distribution including liver, lungs, kidneys, pancreas, small intestine. In this context leptin is a good example of functional pleiotropy as it seems to be involved in quite diverse physiological function such as energy balance, reproduction and immunology.

Słowa kluczowe: leptin, leptin receptor, Janus kinase (JAK), transcription factors STAT, SOCS proteins, proinflammatory cytokines, antyinflammatory cytokines
 

Receptors for Advanced Glycosylation Endproducts

Summary: Advanced glycation end products (AGE) are formed as a result of nonenzymatic glycosylation of proteins, known as Maillard reaction. They modify cell function by interaction with three specific cell surface receptors: RAGE � receptor for advanced glycosylation endproducts, MSR � scavenger receptor, and AGE-R1-R2-R3 � complex receptor in endothelium. The interaction of AGE-RAGE is responsible for oxidative stress and activation of nuclear factor NFkB through Ras/MAPK pathway. Amphoterin and amyloid-b are the other ligands for RAGE. The MSR receptor mediates phagocytosis and degradation of AGE, and macrophage adhesion. As a proinflammatory factor, AGE is associated with development of many diseases, like atherosclerosis or nephropathy.

Key Words: advanced glycation endproducts, AGE, RAGE, scavenger receptor, glycation

The Influence of Cellular Redox State on the Activation of Transcription Factors and Gene Expression

Summary: Reactive oxygen species (ROS) have accompanied living organisms for over 2 billion years. Because they constitute a serious threat to all components of the cell, during the course of evolution some defence systems evolved to detoxify them. At the same time some enzymes appeared, that play a role as producers of ROS upon special stimuli.The recent years' experiments have demonstrated, that ROS thus produced could take part in intracellular signalling. The paper is concerned with the role of ROS in pathways leading to the activation of transcription factors and gene expression. The special emphasis will be laid on the activation of NF-kB and AP-1, transcription factors, that play a key role in the activation of a number of genes involved in inflammation and immune response.

Key Words: reactive oxygen species, oxidative stress, antioxidants, transcription factors, gene expression
 

Contact Guidance of Animal Cells � Clinical Application

Summary: Reactions of cells to the topography and chemical properties of the substratum and mechanisms of contact guidance are outlined. Responses to the physical and chemical properties of the substratum include cell orientation, changes in cell shape and cell motility, reorganisation of the cytoskeleton, activation and inactivation of gene expression and activation of proteins involved in signal transduction. Contact guidance phenomenon is discussed in terms of application of patterned substrata as a scaffold for regenerating tissue in clinical practice.

Key Words: contact guidance, cell movement, tissue engineering
 

Assays For Analysis of Tumour Invasion

Summary: The paper reviews some qualitative and quantitative experimental assays for the analysis of tumour cell invasion. The current state of development of in vivo videomicroscopy, which permits direct observation of steps in formation of metastasis is discussed. Various invasion assays in vitro are also reviewed. The 2- and 3-dimensional confrontation models for cancer invasion, transmigration in two-compartment assays, organotypic cultures, and cell motility assays in dense cultures and 3-dimensional collagen gel lattices are compared. The limitations and advantages of the presented methods are considered. 

Key Words: Invasion assays, videomicroscopy, animal models, cell motility, models of tumour cell invasion in vitro
 
 

Cell Cocultures in Skin Reconstruction for Clinical Applications

Summary: The ability of carrying out large-scale cultures from small human tissue biopsies and to modificate of their behaviour in vitro led to the increase an interest in practical use of these achievements in biology and medicine. Conventional methods of skin wound healing are not sufficiently efficient and there is a need to construct skin equivalents. A full thickness skin equivalent has been constructed and it may substitute epidermis. The last has a lot of advantages but also disadvantages, which stimulate further research. Our experience in this field concerns the use of cultured autologous keratinocytes for healing of trophic leg ulcers. Reconstructed skin is used not only in medicine but also in basic research for example in analysis of toxicity of various substances and to study the tumour invasiveness. Reconstructed in vitro skin equivalents appear to be efficient substitutes enabling wound healing, but their large-scale use is limited because of high costs of their production.

Key Words: wound healing, keratinocyte, skin substitute, in vitro culture
 

EGF-Like Growth Factors and Their Contribution in Epidermis Regeneration

Summary: One of important activity areas of EGF growth factors activity is epidermal and epithelial cells differentiation and growth regulation. In recent ten years, there is a great deal of publications related to EGF-like polypeptides and their participation in specific receptors (ErbB) activation. Little, however, is known about physiological regulation of keratinocytes growth, differetiation and apoptosis by EGF-like peptides. Basic informations concerning structure and function of mammalian EGF-like growth factors, ErbB receptors and their cooperation in normal and pathological growth of epidermal cells are described.

Key Words: epidermal growth factors, epidermis regeneration
 

Targeted Gene Delivery by Receptor-Mediated Endocytosis for Gene Therapy

Summary: Receptor-mediated endocytosis (RME) is a type of delivery system by which therapeutic agents can be specifically transported to their targets. In this paper methods of delivery of macromolecules via RME are discussed. This review focuses on molecular conjugate vectors as a delivery system of therapeutic genes to targeted cells. Characteristic of DNA-binding agents, together with further factors such as cell-specific ligands, endosomal escape and nuclear localisation are discussed. Advantages and disadvantages of molecular conjugates and future perspectives of application of these vectors for gene therapy are considered. 

Key Words: gene therapy, gene delivery, non-viral vectors, molecular conjugates, receptor-mediated endocytosis
 

The Significance of Anti-Gal Antibodies

Summary: Human, ape and Old World monkey sera contain unique anti-Gal antibodies directed against Gala1-3Galb1-4GlcNAc epitope (a-gal). It is the most common antibody specificity within the sera of these animals. Anti-Gal antibodies are involved in many processes of immune defence but they might be also involved in pathogenesis of some disorders. Anti-Gal might be the first line of defence against some parasites, viruses and bacteria. They activate complement cascade on the surface of some cancer cells and regulate senescent or pathological erythrocytes clearance. Elevated levels of anti-Gal antibody were shown in many autoimmunological disorders. For example in Graves' disease anti-Gal antibodies activate thyrocytes. Anti-Gal antibodies are also the most important barrier to successful transplantation of organs obtained from mammals such as pig to human. Most common xenoreactive antibodies responsible for hyperacute organ rejection are in fact anti-Gal antibodies.

Key Words: epitope (a-gal), natural antibodies, complement, transplantation
 

Catecholamines in the Immune System � Identification and Function

Summary: The search for the possible connection between nervous-, and immune systems has been conducted since early 90-ties. A suggestion that the immune response might be controlled by the brain, initiated the search for the specific substances involved in this process. It has been found that glucocorticoids, opiates, polypeptides, pituitary hormones, and catecholamines influence activity of the immune system. Catecholamines and their metabolites are also synthesized in the peripheral blood lymphocytes and in the T-, and B-cells clones. This finding has urged us to perform quantitative studies on the catecholamines level in lymphocytes from healthy and leukemic patients. This work reviews the actual knowledge on catecholamines in the immune system and describes techniques for their identification and quantitation.

Key Words: Capillary electrophoresis, Catecholamines, Lymphocytes, Mass Spectrometry, High performance liquid chromatography

Chicken Egg Riboflavin-Binding Protein � Protein Structure And Ligand-Binding Mechanism

Summary: The structure and ligand-protein interaction of chicken egg riboflavin-binding protein, a representative transport protein and simple flavoprotein, is extensively studied. This work reviews the current progress of this field over last fifteen years, starting from the study establishing the protein primary structure. This protein is highly homologous to the folate-binding proteins and forms a unique fold, consisting of two domains: a ligand-binding domain cross-linked with multiple disulphide bridges and a separate phosphorylated motif which is responsible for protein transport to the oocyte. Riboflavin is bound into a hydrophobic pocket, the izoalloxazine rings stacked in between tryptophan-156 and tyrosine-75 and the rybityl chain anchored with multiple hydrogen bonds. Recent studies specified the structure of the oligosaccharide moiety and presented general pathways of the protein folding.

Key Words: riboflavin transport, protein folding, folate-binding proteins

Thiamine Biosynthesis and Bioactivation

Summary: The biosynthesis of thiamine (vitamin B1) is performed in microorganisms and plants. First step leads to the formation of two precursors: 5-(2-hydroxyethyl)-4-methyltiazole and 4-amino-5-hydroxymethyl-2-methylopyrimidine. They are phosphorylated and then combined by the key enzyme, thiamine phosphate pyrophosphorylase. Thiamine phosphate is dephosphorylated; free thiamine is then converted by thiamine pyrophosphokinase into a metabolically active coenzyme, thiamine pyrophosphate.

Key Words: vitamine B1, thiamine biosynthesis, thiamine phosphate pyrophosphorylase, thiamine pyrophosphokinase
 

Hypothetical Function of Thiamine in Nervous System

Summary: Thiamine deficiency in animals predominantly results in malfunctions or degradation of both central and peripheral nervous systems. These effects are partly due to the universal metabolic function of thiamine diphosphate, the cofactor of key enzymes involved in the carbohydrate metabolism and energy supply to the cell. However, many recent observations seem to indicate that thiamine derivatives, mainly thiamine triphosphate may play an additional non-cofactor role, specific to the nervous tissue. Three hypotheses attempt to interpret the neurochemical role of thiamine triphosphate on the molecular level. First two assume the action of this thiamine compound upon the ligand-gated Na⁺  channels or the voltage-gated Cl^� channels. The recent hypothesis suggests that thiamine triphosphate in involved in the neurotransmission mechanism serving as a specific phosphate donor in the phosphorylation of some regulatory proteins. 

Key Words: thiamine deficiency, thiamine triphosphate, pyrithiamine, oxythiamine, regulatory protein phosphorylation

Thiamine Transport and Metabolism in Saccharomyces Cerevisiae Cells

Summary: This review presents selected problems of thiamine transport and metabolism in Saccharomyces cerevisiae cells with focus on the proteins that are unique in this microorganism. The protein, encoded by THI6 gene, combines in one molecule two enzymatic activities, necessary at two steps of thiamine biosynthesis. Another enzyme, the periplasmic acid phosphatase (PHO3 gene product), exihibits the additional thiamine binding property. It probably dephosphorylates thiamine phosphate taken up from the medium. Free thiamine crosses the cell membrane via the membrane-bound transporter encoded by THI10 gene.

Key Words: membrane transport, periplasmic space, thiamin phosphates
 

Transport Proteins in The Periplasmic Space of Gram-Negative Bacteria

Summary: Proteins, that specifically bind diverse compounds operate in the periplasmic space of gram-negative bacteria. They share the uniform plan of structure and the model of action. These proteins serve as the primary receptors in the transport process across the inner cellular membrane. The periplasmic binding proteins cooperate with the ABC transporters, which ubiquitously occur in procaryotic and eucaryotic organisms. They have also additional functions: of the chaperones for periplasmic proteins and of the primary receptors for chemotaxis processes. The periplasmic binding proteins have some properties suggesting their further functions, still to be recognized.

Key Words: periplasmic binding proteins, ABC transporters, periplasmic transport proteins
 

Antimicrobial Peptides � Structure and Function

Summary: Antimicrobial peptides are part of defense system mainly in plants and animals. In spite of great diversity of origin and amino acid composition, almost all of them are cationic (due to presence excess Arg and Lys residues) and molecules form amphipathic structures. Antimicrobial peptides can be divided into several main groups based on their three-dimensional structure: 1. Linear, forming  a-helices; 2. Antiparallel b-sheets stabilized by intramolecular disulfide bonds; 3. a-helical and b-sheet mixed structure with disulfide bonds; 4. Cyclic structures; 5. Linear, with unusually  high content of certain amino acid, often forming extended helices. Antimicrobial activity of these peptides is very broad, including bacteria, fungi, some protozoa and even cancer cells. They are selectively toxic to microorganisms. Owing to the increasing resistance of bacteria to conventional antibiotics, antimicrobial peptides seem to be promising source of antibiotics in future.

Key Words: antimicrobial peptides, antibacterial, peptide antibiotics
 

Mode Of Action Of Antimicrobial Peptides

Summary: Plants and animals synthesise antimicrobial peptides constitutively or in response to microorganism infection and they are an important component of the organisms innate defence system. This paper focuses on the recent knowledge and views concerning interactions of antimicrobial peptides with structures of the microorganism's cells. There are described several suggested models of mechanisms of action: membrane pore formation (barrel stave� mechanism), carpet like�, wormhole�, and ion channels formation. Additionally, there are alternative mechanisms of action including interaction with DNA or influence on protein synthesis. Moreover, this paper explains the selectivity of these peptides and describes their physiological role and attempts of clinical applications.

Key Words: antimicrobial peptides, cell membrane, ion channel
 

Molecular Simulation � Studies of The Cholesterol-Phospholipid Interactions

Summary: A 5-ns molecular dynamics simulation of the fully hydrated, liquid-crystalline bilayer membrane built of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) was performed. The primary aim of the analyses of generated trajectories was to investigate the atomic-level mechanisms of the Chol ordering and condensing effect. A strong increase in the chain order in the DMPC-Chol membrane as compared to the pure DMPC membrane, was observed. Alkyl chains of PCs, which were the nearest neighbours of the smooth face of Chol (a face), were the most ordered. For these molecules, a decrease in the number of gauche conformation/myristoyl chain as well as in the tilt of the chain, were observed. PCs neighbouring the a face of Chol were less ordered than PCs neighbouring the a face. For the Chol condensing effect, an increase in the van der Waals interactions among PCs chains was responsible; interactions between hydrocarbon chains and cholesterol rings were week. 

Key Words: Phosphatidylcholine, cholesterol, membrane order

Oriented Crystallization of Metalloporphyrins: Effect of Magnetic Field and Lipid Membranes

Summary: Copper(II) tetraphenylporphyrin (CuTPP) has been found to partition favorably into lipid bilayer membranes. Electron paramagnetic resonance (EPR) spectra of oriented fluid-phase dimyristoylphosphatidylcholine (DMPC) membranes indicate that for a low (Ł1/100) CuTPP/DMPC molar ratio the plane of CuTPP is preferentially perpendicular to the membrane surface while for a high (ł1/25) CuTPP/DMPC molar ratio the plane of the molecule is parallel to the bilayer surface. Additional saturation-recovery EPR measurements, as well as computer simulations of EPR spectra lead to the conclusion that the change of the CuTPP orientation at high CuTPP/DMPC molar ratio is coupled with self-association by planar stacking of CuTPP molecules near the lipid bilayer center. An additional factor that can induce oriented crystallization of CuTPP is a strong (ł1000 G) magnetic field. Crystals formed in oversaturated solutions are oriented with planes of molecules parallel to the magnetic field. The presence of the magnetic field also increases the rate of CuTPP crystallization. The relevance of these findings to porphyrin photodynamic therapy is discussed 

Key Words: porphyrin aggregation; porphyrin-membrane orientation; lipid bilayer; porphyrin phototherapy
 

Molecular Simulation Studies of The Interfacial Region in Lipid Bilayers

Summary: The structure and the dynamics of the membrane/water interface in pure dimyristoylphosphatidylcholine (DMPC) and DMPC-cholesterol (DMPC-Chol) bilayers were studied using a molecular dynamics (MD) simulation method. In this region, an extended network of interactions among polar groups of lipid and water is formed. In the DMPC bilayer, these interactions involve 1) water cross-bridges between negatively charged oxygen atoms of PCs, which link 76% of DMPC  molecules in the membrane, 2) charge pairs between positively and negatively charged groups of PCs, which link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together link 98% of all membrane phospholipids at any instant. At the DMPC-Chol bilayer interface, in addition to the PC-PC links, 1) direct hydrogen bonds, 2) water bridges, and 3) charge pairs between PC polar groups and the Chol are formed. PC-PC and PC-Chol links involve at any instant 97.5% of DMPC and 70% of Chol molecules. Average lifetimes of PC-PC and PC-Chol associations are 1 ns or more. These associations are dynamic and they temporarily break and re-form several times during their lifetime.

Key Words: phosphatidylcholine, cholesterol, hydrogen bonds, water bridges, charge pairs, molecular dynamics
 

Grzegorz JEZIERSKI, Marta PASENKIEWICZ-GIERULA

Simulated Annealing Method � an Application to Investigate Structure and Dynamics of Proteins

Summary: Simulated annealing is a method applied to solve structures of biomolecules, as well as for modelling the structure and dynamics of these compounds. This method is a variation of a molecular dynamics method, and as such, it assumes classical treatment of atoms. Additional techniques, such as increasing temperature in a model system, which allow for better penetration of configurational space, are applied. The simulation of the influence of an aminoacid exchange in antitrypsin on the structure in the aminoacid's vicinity and on the dynamics of the protein has been investigated. Advantages and disadvantages of the method have been discussed; examples of applications of the method in drug design are given, eg. simulation of protein folding. This paper describes original and cited results, published mainly during the last decade and reflecting new trends in the field of protein simulation by means of computer simulation.

Key Words: molecular modelling, molecular dynamics simulation, drug design, protein structure, a₁-antitrypsin, y and f angles
 

Reflections on Biomedicine

Summary: Present status in European medical biotechnology was summarised after 20 years of international cooperation in 1999. European Federation of Biotechnology (EFB) has impact in many disciplines, especially in medical science. Present genetic engineering in biomedicine is integrated with biopharmacy for specific therapy using  nucleotide and protein sequence. The biopharmaceutics are various recombinant peptides, monoclonal antibodies and molecular probes, used in molecular diagnostic and for gene therapy.

Key Words: biopharmaceutics, gene therapy, Single Nucleotide Sequences, genomics, proteomics