Green Fluorescent Protein (GFP) Structure and Properties

Summary: In the last decade Green Fluorescent Protein (GFP) form the jellyfish Aequorea victoria has become a commonly used tool in cell and molecular biology. Its advantages are high stability of its tertiary structure and the fact that its chromophore is formed by autocatalytic cyclization of amino acids that does not require any cofactors apart from oxygen. This work describes major structural and photochemical features of this protein and gives an overview of mutations which accelerate GFP folding and maturation of its chromophore as well as influence the spectral characteristic of the protein.

Key words: green fluorescent protein (GFP), Aequorea victoria, fluorescence,  protein chromophore, enhanced green fluorescent protein (EGFP).
 

Application Of Green Fluorescent Protein (GFP) in Biological Research
 

Summary: The unique properties of Green Fluorescent Protein (GFP) are the main reason of its common use as a tool in cell and molecular biology. This work gives an overview of some of the major applications of GFP such as protein tagging, monitoring of gene expression and protein interactions as well as use in biological screening. The recent discoveries of fluorescent proteins from many Anthozoa species expand the applicabilities of already know techniques and enable creation of new ones, which will be useful in high-throughput cytological and proteomic experiments.

Key words: green fluorescent protein (GFP), Aequorea victoria, fusion tag, reporter gene, FRAP, FRET, pH indicator, calcium indicator, biological screening.
 

Plant Blue - UV a Light Receptors Mediating in Phototropic Reactions, Photomorphogenesis and Circadian Clock Entrainment

Summary: Many important aspects of plant growth and development are regulated by specific blue (390–500 nm) and UV-A (320–390 nm) light. Molecular genetic and cell biological studies using Arabidopsis thaliana as a model system have identified three classes of blue-light/UV-A photoreceptors – phototropins, cryptochromes and flavoproteins of Zeitlupe family. Phototropins (phot1 and phot2) are the light-activated, FMN-binding serine/threonine protein kinases that mediate in phototropism, chloroplasts migration, stomatal opening and rapid inhibition of stem growth. Cryptochromes (Cry1, Cry2 and Cry3) are the blue light receptors containing FAD and a pterine as chromofore that mediate light control of stem elongation, leaf expansion, photoperiodic flowering, and circadian clock. Ztl, Fkf1 and Lkp2 are members of new blue-light photoreceptors family. All three proteins contain PAS/LOV domain that binds FMN, F-box domain and six KELCH repeats. Last results suggest that flavoproteins of Ztl family mediate in resetting of the circadian clock by targeting oscillator components for degradation in a light-dependent manner.

Key words: phototropins, cryptochromes, flavoproteins of Zeitlupe family, circadian clock
 

Endothelial Nitric Oxide Synthase. Part. I. Gene Structure and Protein Product

Summary: TEndothelial nitric oxide synthase (eNOS) is one out of the three known enzymes capable of producing nitric oxide, a labile molecule that plays important biological role in cardiovascular system. The eNOS gene is composed of 26 exons and spans 22 kb fragment on chromosome 7. Characterization of the 5’-flanking genomic region revealed that eNOS promoter is lacking of TATA box and contains a great number of consensus sequences for the variety of transcription factors indicating the possibility of dynamic and complex regulation of expression. The eNOS exhibits considerable polymorphism within introns, exons and promoter region. In some studies it was tried to link these allelic variation with the risk of coronary artery disease (CAD) and hypertension. Active endothelial nitric oxide synthase is composed of two identical subunits, each consisting the following distinct domains: C-terminal reductase domain exhibiting considerable aminoacid sequence homology with cytochrome P450 reductase, small calmoduline (CaM) binding domain, oxygenase domain and N-terminal fragment where acylation (mirystylation, palmitylation) takes place. The enzyme contains relatively tightly bound cofactors tetrahydrobiopterin (BH₄), FAD, FMN and haem. FAD and FMN transfer electrons from NADPH to the haem. The precise role of BH₄ remains unclear. Endothelial NOS activity strictly depends on the presence of Ca²⁺/calmoduline complex which upon binding to the enzyme relieves it from the negative influence of an inhibitory insert localized in the FMN binding domain. Nitric oxide is produced in the two-step reaction with arginine and O₂ serving as substrates, and NADPH as a source of electrons. The products of eNOS catalytic activity are claimed to be nitric oxide and citruline. Reaction mechanism is not fully understood and requires further investigation. Many important questions concerning the enzyme structure, function and inhibition still remain non-answered.

Key words: nitric oxide, endothelial nitric oxide synthase, calmoduline, calcium  ions.
 

Endothelial Nitric Oxide Synthase. Part II. Functional Aspects

Summary: Some of the key functions of endothelium are dependent on the endothelial nitric oxide synthase (eNOS) activity, thus suggesting a role of this enzyme in pathological conditions such as coronary artery disease (CAD), essential hypertension, hypercholesterolemia and atherosclerosis. To evaluate the proposed role of eNOS, its activity and expression has been widely analyzed. Endothelial NOS was first identified in endothelial cells of arteries and veins, further investigations demonstrated its presence in the other cell types including neurons, platelets, myocytes, Sertoli and Leydig cells. Enzyme has been shown to be targeted to plasmalemmal caveolae. These specialized invaginations of plasma membrane seem to play a major role in extracellular signal transduction as they contain a great variety of signal molecules. Endothelial NOS associates with caveolin-1, a member of transmembrane family proteins which renders enzyme inactive. NO generated by eNOS is a crucial activator of soluble cGMP cyclase. The activity of cGMP cyclase gives rise to the secondary messenger (cGMP) which activates protein kinase G (PKG). A great variety of proteins which are substrates for PKG phosphorylation determines the complex cell response observed after eNOS activation. Endothelium-derived NO is a physiologically important vasodilator, an inhibitor of platelet aggregation and adhesion, and a modulator of myocyte contractility. It has also been shown that functional eNOS is absolutely required for angiogenesis and vessel remodeling. Since NO overproduction might be harmful for adjacent tissues, enzyme activity is subjected to complex and subtle control mechanisms. The most basic control factor is Ca²⁺/calmodulin complex which upon binding to the enzyme dissociates caveolin and renders eNOS full activity. Additional regulatory mechanisms include phosphorylation, depalmitylation and Hsp90 association. Endothelial nitric oxide synthase, although previously regarded as a constitutive factor, turned out to be the enzyme which expression is dynamically regulated by growth factors, hormones, cytokines and physical stimuli. To determine the precise role for eNOS in pathogenesis of hypertension, CAD or atherosclerosis remains to be elucidated.

Key words: nitric oxide, endothelial nitric oxide synthase, cyclic GMP, protein kinase G, angiogenesis, cardiovascular disease.
 

Morphology and Function of Sperm Mitochondria and Male Fertility. Part I. Normal Morphology and Function of Sperm Midpiece

Summary: The sperm mitochondria are the unique organelles somewhat different from the somatic mitochondria. They undergo morphological, biochemical and molecular changes connected with the expression of the structural and enzymatic specific proteins which may be recognized as markers of their maturation during spermatogenesis. The markers among others include testicular cytochrome –cyt c_T, LDH-C₄, sulphydryl oxidase, LON protease and hsp60. Aktivin A – product of Sertoli cells – influences the morphological changes of spermatyd mitochondria during spermiogenesis, causing the appearance of a condensed type mitochondria. In turn, spergen-1 protein might be responsible for assembly and adhesion of sperm mitochondria around the appearing flagellum of elongating spermatyd. The mature mitochondria are elongated and crescentic and display condensed type structure with many tightly packed mitochondrial cristae and characteristic intracristal spaces. Each mitochondrium is surrounded by a unique protein capsule containing structural selenoproteins, sulphoproteins (SMPC) and enzymatic protein (PHGPx) displaying antyoxidative ability. The surface of sperm mitochondria reveals domain specific structural specializations involved in connecting of adjacent mitochondria. The structural specializations of terminal as well as lateral surfaces of adjacent mitochondria enable the stable and helical connection of mitochondria. The joined mitochondria are assembled into an organized sheath. The mitochondrial sheath surface facing the outer dense fibers is joined to them by submitochondrial network of midpiece cytoskeleton complex. The molecular structure of specific mitochondrial proteins and connections of adjacent mitochondria result in mitochondrial sheath resistant to the action of physical and chemical factors. It makes the mitochondrial sheath act as the organellum providing ATP for sperm movement.

Key words: spermatozoa, mitochondria,mitochondrial proteins, spermiogenesis.
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Morphology and Function of Sperm Mitochondria And Male Fertility. Part II. Morphological and Functional Defects  of Sperm Midpiece Mitochondria

Summary: The male infertility (asthenozoospermia) is very often connected with the morphological and functional defects of sperm midpiece. The mitochondrial defects can be identified with precise and comprehensive diagnostic methods. They include: 1) cytochemical screening test for mitochondrial oxidoreductases/NADH, 2) cytofluorometric evaluation of mitochondrial membrane potential, 3) fluorescence studies using mitochondrial probes, 4) electron-microscopic assessment, 5) biochemical studies revealing disorders of mitochondrial respiratory chain and LDH-C₄ activity, 6) immunocytochemical studies to display ubiquitinated sperm mitochondria or spermatozoa with lower expression of mitochondrial keratinous capsule proteins and 7) genetic studies showing  mitochondrial genome mutations. Many morphological and functional defects of mitochondrial sheath result from the disturbances of their midpiece morphogenesis during spermiogenesis. These defects can be connected with spermatids apoptosis, genetic disorders of their nuclear (t haplotype) or mitochondrial genome and can develop in the epididymis. The mitochondrial defects decrease the sperm movement and finally the sperm ability to fertilize egg cell. Moreover, in some cases of conception (in vitro and in vivo) they may deactivate their own elimination (ubiquitination of mitochondria) in fertilized egg cell. Sometimes in idiopathic infertility it may trigger the spontaneous, pre-implantation embryo abortions. The mitochondrial diagnostics which enables the discovering of the causes of asthenozoospermia is of considerable prognostic and predictive value for in vitro and in vivo fertilization.

Key words:  spermatozoa, mitochondria, mitochondrial DNA, asthenoteratozoospermia.
 

The Role of Toll-Like Receptors in Immunity

Summary: Toll receptors belong to highly conserved in the evolution receptor family, from insects to mammals. They play an important role in innate immunity. They were uncovered and characterized in a fly Drosophila melanogaster, in which they participate in embryogenesis and immunity versus pathogens. Homologous receptors were detected in mammals, including man, known as a Toll-like receptors (TLRs). As in Drosophila, TLRs of mammals contain extracellular domain with multiple leucine-rich repeats (LRR), transmembrane portion and cytoplasmic domain, homologous with that of type I interleukin 1 receptor (IL-1RI). TLRs are expressed on monocytes, macrophages, granulocytes but also on lymphocytes, epithelial cells and endothelia. Soluble forms of these receptors are also known, found in secretions. Ten types of human TLRs have been described so far. Their ligands are only partially known. Most of them are microbial origin (egzogenous), but also from the host (endogenous). TLRs recognize, in the case of egzogenous ligands, so called pathogen associated molecular patterns (PAMPs). Endogenous ligands include proteins and peptides formed during cell or tissue damage. Binding of ligand to receptor results in a fast activation of innate immunity, initiation of inflammatory response due to induction of proinflammatory cytokines, increased expression of MHC antigens, costimulatory molecules on antigen-presenting cells (APC). This leads to better antigen processing and presentation and in effect, proper activation of aquired (adaptive) immunity. Precise knowledge of effector mechanisms and signal transduction due to ligand binding to TLRs may be of value in exploration of recognition self-nonself, activation of inflammatory process and also the role of innate immunity on induction, specificity and type of adaptive immunity.

Keywords: Toll-like receptors (TLRs), innate immunity, pathogen associated molecular patterns (PAMPs), bioinformatics

Brain Capillaries. Ultrastructural Manifestation of Angiogenesis

Summary: The morphological features of microvessels in barrier competent and barrier free regions of brain are described. Some examples of new vessel formation are presented on ultrastructural level. New vessel formation comprises two different mechanisms: endothelial sprouting and intussusceptive microvascular growth. Recently, intensive studies are evaluated on endothelial progenitors participation in new vessel formation. We present samples of new formed blood capillaries. Immunocytochemical ultrastructural studies additionally confirmed our morphological observations.

Key words:  brain capillaries, ultrastructure, vasculogenesis, angiogenesis, progenitor endothelial cell.
 

The Channels of the Mitochondrial Protein Import Apparatus

Summary: Channels are crucial for protein import into mitochondria as they enable protein translocation across mitochondrial membranes and their insertion into these membranes. The outer mitochondrial membrane contains the TOM complex channel (the channel of the translocase of the outer membrane) and the TOB complex channel (the channel of the complex supporting the topogenesis of mitochondrial outer membrane b-barrel proteins), in which the b-stranded membrane proteins can fold. The inner mitochondrial membrane contains two channels of distinct specificity. They are present within the TIM complexes (the translocase of the inner membrane), namely TIM23 and TIM22. Undoubtedly, the channels are fundamental checkpoints in protein import into mitochondria, which means that they could be regarded as therapeutic goals in  mitochondrial diseases.

Key words: : the TOM complex channel, the TIM complex channels, the VDAC channel, the TOB complex channel, protein import.
 

Mechanisms of Resistance in Leukemia Cells Mediated by the Oncogenic Tyrosine

Summary: Chromosom Filadelfia (Ph) jest wynikiem translokacji chromosomalnej prowadzącej do transformacji nowotworowej komórek krwiotwórczych. Powodem jest aktywność onkogennej kinazy tyrozynowej BCR/ABL powstającej z fuzji genów w wyniku przeniesienia fragmentu chromosomu 9 do chromosomu 22 [t(9;22)]. Istnieje cała rodzina fuzyjnych kinaz tyrozynowych (FTKs) spokrewnionych z BCR/ABL, takich jak: TEL/ABL, TEL/JAK2, TEL/TRKC(L), TEL/PDGFâR oraz NPM/ALK, które indukują zarówno ostre, jak i przewlekłe białaczki szpikowe oraz chłoniaki. FTKs aktywują szlaki wewnątrzkomórkowego przekazywania sygnału stymulujące komórki do podziałów, chronią je przed apoptozą i aktywują mechanizmy prowadzące do lekooporności. Różne FTKs mają zdolność aktywacji czynników transkrypcyjnych STAT. STAT5 jest niezbędny do zależnej od FTKs regulacji RAD51 w mechanizmie naprawy przez rekombinację homologiczną (HRR). Ponadto podczas terapii lekowej komórki zmienione przez FTKs wykazują opóźnienie cyklu komórkowego w punkcie kontrolnym G2/M. Odnajduje się w nich również podwyższony poziom białek antyapoptotycznych. Najprawdopodobniej współdziałanie wszystkich tych procesów leży u podstaw zjawiska lekoopornosci i stanowi główny problem skutecznej terapii białaczek.

Key words:  BCR/ABL, fuzyjne kinazy tyrozynowe, lekooporność, białaczki.
 

Molecular and Physiological Bases of Circadian Clock in Plants

Summary: The circadian clock forms one of the most fascinating adaptations to life on earth. Owing to the internal clock organisms can not only react to the periodical sequence of day and night but can make measure daylenght, which is an indicator of the seasons. Endogenous clock creates about 24-h rhythms. The clock is set mainly by the alternations in light and temperature conditios at dawn and dusk. The machinery responsible for generating circadian rythms is composed of three major components: an input pathway by which environmental cues act to synchronize the clock; the endogenous oscillator; and the output pathway as the rhythmic of phsiological and developmental processes controlled by the clock. Light forms the dominant signal in resetting the clock. A close link between the photoreceptors and the clock’s components has been demonstrated for different experimental models. In plants both of the photoreceptor molecules families – phytochromes (phy) and cryptochromes (cry) participate in light signal transduction to the oscillator. The ZTL, FKF1 and LKP2 proteins family (which contain LOV domain-, Kelch repeat- and F-box) and the novel nuclear proteins GI and ELF3, have also been implicated in light input to the clock. Molecular studies of the central oscillator from several different organisms (ex. Arabidopsis thaliana, Neurospora crassa, Drosophila melanogaster) indicate that it works as feedback loop consisting of positive and negative elements. Proteins encoded by clock genes act as negative elements that repress their own expression by blocking transcriptional activators that act as positive elements. In plants putative elements of the oscillator are: TOC1 or APRR1 protein, an element of two – component response regulator system and two related MYB transcription factors – LHY and CCA1. It was demonstrated that the circadian clock controlls many different physiological and developmental processes in plants a.o. cells elongation, stomatal opening, leaf movement and flowering.

Key words: Arabidopsis thaliana, circadian clock, circadian rhythms, oscillator, photoreceptors, plants