Advances in Cell biology, vol. 32, 2005, issue 1

Lipotubuloids – Domains of Cytoplasm Rich in Lipid Bodies Entwined by Microtubule System, Active in Lipid Synthesis

Summary: Lipotubuloids were first described in Ornithogalum umbellatum and now they have been found in Helianthus albiflos. They are cytoplasmic domains containing a great number of lipid bodies surrounded by a network of microtubules, which join one lipid body with the others. This makes a lipotubuloid move as an integrated body. It is largely surrounded by a tonoplast since it invaginates into a cell vacuole. Lipotubuloids also contain ribosomes, endoplasmic reticulum and some mitochondria, Golgi structures and microbodies (glyoxisomes or peroxisomes) as well as autolytic vacuoles containing acid phosphatase and lipase, which appear at the final stages of lipotubuloid development. Lipotubuloids move in different directions with a progressive-rotary movement which is generated by autonomic drive. The peripheral speed of the rotating lipotubuloids is several times greater than that of the cytoplasmic motion. Moreover, 2,4-dinitrophenol-induced cyclose blockade does not stop the rotation of lipotubuloids which is continued and gradually terminated. Lipotubuloids are the site of active incorporations of 3H-palmitin acid into lipids. Dispersion of autoradiographic grains resulting from non-radioactive postincubation shows that lipids are used and metabolized by a cell since these grains do not disappear after extraction with use of lipid solvent. EM autoradiography silver grains localised at the surface of lipid bodies proves that it is the active site in lipid synthesis. Microtubules adjacent to lipid bodies probably take part in this synthesis.

Key words: lipotubuloid, lipid bodies, microtubules, lipid synthesis, autonomic rotary movement

PI(4,5)P₂, a Unique Plasma Membrane Lipid: Various Functions and Multiple Effector Proteins

Summary: Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] is a lipid of the inner leaflet of the plasma membrane. This lipid controls activity of distinct ion channels and pumps of the plasma membrane and is bound by numerous submembraneous proteins, including proteins of the actin cytoskeleton. In addition, hydrolysis and phosphorylation of PI(4,5)P₂ generate three second messengers, IP₃, DAG and PI(3,4,5)P₃. For all these reasons, PI(4,5)P₂ participates in signal transduction and controls many other processes including cell migration, exo-, endo- and phagocytosis and ion transport. This wide array of PI(4,5)P₂ functions suggests that many distinct pools of the lipid co-exist in the plane of the plasma membrane. They can be generated as a result of a local PI(4,5)P₂ synthesis and by a restricted lateral mobility of PI(4,5)P₂ in the membrane ensuing from sequestration of PI(4,5)P₂ by submembraneous proteins like MARKCS and GAP-43. PI(4,5)P₂ is synthesized mainly by phosphorylation of PI(4)P catalyzed by PIP5-kinases of type I. PIP5-kinase Ia associates with the plasma membrane due to binding to both the PI(4)P substrate and the PI(4,5)P₂ product of its activity. A line of data suggest that PI(4,5)P₂ and PIP5-kinase Ia are compartmentalized in plasma membrane microdomains enriched in sphingolipids and cholesterol, so-called rafts. Interactions of PI(4,5)P₂ with effector proteins are mediated either by unfolded sequences enriched in basic amino acids or by globular domains among which pleckstrin homology (PH) domain of phospholipase Cr1 displays especially high specificity toward PI(4,5)P₂. Recombinant proteins composed of the PH domain tagged with fluorescent proteins have been recently introduced as tools to study the dynamics of PI(4,5)P₂ in cells.

Key words: phosphatidylinositol-4,5-bisphosphate, pleckstrin homology domain, plasma membrane

Vanadium Derivatives as Compounds of High Biological Significance. Part I. Antidiabetic Activity

Vanadium Derivatives as Compounds of High Biological Significance.Part II. Effect on Neoplastic Cells

Key words: Vanadium derivatives, cancer cells, pro- and anticarcinogenic activity

Summary: The information published during the last years, concerning the recognition of hemoglobin-haptoglobin complex by cells has been presented. It is generally accepted that the major function of Hb-Hp formation is the clearance of free hemoglobin through endocytosis of the complex by specific receptors on liver parenchymal cells. The internalised complex is then rapidly degraded. The function of the receptor specific for the hemoglobin in parenchymal cells not has been elucidated to date. Molecular mechanisms involved in the removal of hemoglobin-haptoglobin complex have been elusive, until the recent identification of the macrophage-restricted CD163 antigen as the specific hemoglobin scavenger receptor (HbSR). CD163/HbSR has been reported to be expressed by macrophages accumulating during the down regulation of inflammatory reactions and during the wound healing process. Moreover, the megalin-cubilin complex appears to be essential in the scavenging of hemoglobin, which would otherwise be excreted by the kidneys. The objective of this article is to review the most important publications dealing with structure, regulation and function of receptors for hemoglobin.

Key words: hemoglobin receptor, monocyte/macrophage, hepatocytes, megalin-cubilin, haptoglobin

Receptors for Hemoglobin. Part II. Hemoglobin Scavenger Receptor as a Component of the Innate Immune Response to Infection

Summary: CD163 binds haptoglobin-hemoglobin (Hp-Hb) complexes, implicating this receptor as a hemoglobin scavenger receptor (HbSR). Hemoglobin clearance prevents excessive toxicity by removing redox-reactive iron from the circulation, and removes Hb as a potential source of iron, a normally limiting nutrient for pathogen growth. Soluble CD163/HbSR is cleaved from the surface of monocytes by pathogens at the same time that hemolysis and Hp-Hb complex formation is likely to occur. The consequences of an infection implicate additional functions for the HbSR beyond binding and internalizing Hp-Hb complexes, as this receptor is cleaved from the surface of monocytes during infections at a time when Hp-Hb complexes are forming, and complex clearance is imperative. The initial innate immune response to infection includes the release of proinflammatory cytokines to recruit new leukocytes to areas of inflamed tissue, in addition to the production of anti-inflammatory mediators such as IL-10, which help to limit damage to the local tissue environment. CD163/HbSR is involved in the resolution of inflammation; anti-inflammatory mediator IL-10 markedly increases expression of CD163/HbSR on the cell surface, and the pro-inflammatory cytokines TNF-a and IFN-g suppress CD163/HbSR expression. Decreased surface expression of CD163/HbSR may reduce the contribution of Hb internalization to intracellular iron pools, thereby limiting the growth of intracellular pathogens dependent on iron for replication.

Key words: hemoglobin, receptor CD163/HbSR, infection, innate immune response, haptoglobin.

Summary: Heat shock proteins (HSPs) are conservative group of proteins which are produced by prokaryotic and eukaryotic cells as response to external signals. HSPs are divided into six groups depending on their molecular weight. In infection with Chlamydia sp. and Chlamydophila sp. three groups are involved: HSP10, HSP60 and HSP70. The researchers showed, that HSPs are connected with primary and secondary infection with Chlamydia (C.) trachomatis and Chlamydophila (Cp.) pneumoniae. HSPs can stimulate cells of immunological system, what can cause increase of synthesis of proinflammatory cytokines (IFNg, TNFa, IL-1b, IL-6, IL-8, IL-10, IL-12, IL-23), which increase pathogenic effect of those bacteria what was demonstrated in experimental infections in animals. Cooperation of human and chlamydial HSP60 also cause the increase of pathogenic effect of i.e. Cp. pneumoniae, what was shown in coronary heart disease. On the other hand, detection of anti-HSP antibodies in serum can show infection with Chlamydia sp. or Chlamydophila sp., what can be important in disease, where those bacteria are cofactors. Heat shock proteins are responsible for pathogenic effect, and also may be used in diagnosis of those bacteria.

Key words: Toll-like receptor (TLR), pathogen associated with molecule patterns (PAMP), innate immunity, adaptive immunity, regulatory T lymphocyte (Treg), dendritic cell (DC)

Summary: Plasmodesmata are plasma membrane-lined channels that cross the cell walls of the neighboring cells. They allow passive movement of small molecules, but also intercellular transport of RNAs or proteins. Latest studies have revealed that plant cell differentiation depends on the movement of the proteins – transcriptional regulators between neighboring plant cells. Changes in the number of plasmodesmata or their diameter (what influence the permeability of plasmodesmata for different substances) are important factors in regulation of plant cells differentiation. The presented paper reviews the latest literature data on different signals transported symplastically and the role of symplasmic transport of proteins in the plant cell differentiation. The one part of results concerns the involvement of protein transport through plasmodesmata in root epidermis pattering on the example of Arabidopsis thaliana. It appeared that proteins like CPC and GL3/EGL3 play an important role in specification of plant cell fate. Namely, obtained results suggests that pattering of root epidermal cells into hair and non hair cells depends on the movement of CAPRICE and GLABRA3 transcriptional regulators between epidermal cells [13]. The authors showed evidence that WAREWOLF autoregulation does not contribute to the initial pattering of epidermal cell fates. In this model the active intercellular movements of proteins underline a mechanism for pattern formation of cells and this mechanism is based on the mutual support of two cells [13]. Initiation of cell differentiation in one row of cells influence the cells differentiation in neighboring cells by the intracellular movement of CPC and GL3/EGL3 proteins. What is important, proteins transported symplastically are involved in genes regulation in the target cell. Another aspect of involvement of proteins transported through plasmodesmata and their role in specification of cell differentiation concerns the mechanism of cells pattering in Arabidopsis leaves. Trichome initiation in the model plant Arabidopsis has been an important model for understanding cell fate and pattering [16]. It was postulated many years ago that trichome pattering is generated by intercellular communication. Results described in presented paper concerned the correlation between different classes of proteins which are involved in trichome cell specification [16]. The authors [16] analyzed a network of three classes of proteins consisting of bHLH and MYB transcription factors, and a WD40 repeat protein, TRANSPARENT TESTA GLABRA1 (TTG1), which act in concert to activate trichome initiation and patterning. Using YFP-TTG1 translational fusions, they showed that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using a conditional transgenic allele, the authors demonstrated that TTG1 directly targets the same genes as the bHLH protein GLABRA3 (GL3). In vivo binding of the R2R3-MYB protein GLABRA1 (GL1) to the promoters of GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) established that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, the authors confirmed that TTG1 associates with GL3 and GL1 in vivo, forming a complex. The loss of TTG1 and GL1 through mutation, affects the subcellular distribution of GL3. Using particle bombardment, it was shown that TTG1, GL3, GL1 and the homeodomain protein GL2 do not move between adjacent epidermal cells, while the R3-MYB, CPC, does move to neighboring cells. Presented data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf [16]. From results described in presented paper appeared that genetic control of cell fate and pattering must be support by mechanisms in which the transport of proteins through plasmodesmata play a key role and that such a mechanism can be a universal.

Key words: symplast, symplasmic transport, cell differentiation, mutual support mechanism

Summary: The paper describes an important biological process, i.e. autophagy – a conservative and phylogenetically old mechanism, which role is to eliminate damaged and (or) used parts of cell, in order to avoid a complete degradation of it. The process of autophagy, being one of four basic mechanisms of cell death, demands different genes and factors to run correctly. Within autophagy one may distinguish macroautophagy, microautophagy, specific autophagy and chaperone-dependent autophagy. Macrophagy relies on eliminating elements from the cell by forming autophagosome, microautophagy is a process of digesting smaller parts of the cell directly by the fusion with lysosome, specific autophagy takes place only in peroxysomes and chaperone-dependent autophagy is held only in the presence of heat-shock proteins (hsp). Autophagy plays a crucial role in innate and adaptive immunity. The participation of this process is registered in bacterial and viral infections, where TLR receptors are involved. Moreover, autophagy participates in physiology, for example stress and round-labour period and in pathology, like cancer, liver damage, muscle and nervous system dysfunction and Crohn's disease.

Summary: Doxorubicin (DOX) and other anthracycline derivatives are effective antineoplastic agents with an antitumor activity against many solid tumors and leukaemias. The clinical application of these drugs is, however, limited due to cumulative, dose-dependent side effects for example cardiotoxicity, myelosuppression or kidneys failure. Therefore, several strategies have been developed to reduce these effects and to improve the chemiotherapeutic potency of doxorubicin and other anthracyclines. One of the ways is using drug carriers, such liposomes, dendrimers, nanoparticles, antibodies and others. The drug delivery systems lead to increased intracellular concentration of the cytotoxic agents mainly in cancer cells and may help to overcome the chemoresistance of neoplastic cells. In this article we presented the most promising strategies based on protein or peptide carriers. Peptide linkers are a short sequences usually containing multiple lysine or arginine residues. They are linked to DOX via self-immolative spacer, which release the free drug spontaneously. The very useful spacer is p-aminobenzyloxycarbonyl (PABC) releasing the anticancer agent at low pH. It is very important that peptide linker has usually a specific structure and can serve as a substrate for tumor-associated overexpressing proteases like cathepsin, metalloproteinases, urokinase type plasminogen activator or prostate specific antigen. We presented here, for example, prodrugs which contain in their structure peptide spacer which is specific substrate for metaloproteinases, especially MMP2 and MMP9. These proteinases play a critical role in tumor progression, angiogenesis and metastasis. The protease activity of MMP2 and MMP9 was exploited to release the anticancer agent from carrier. Since MMPs are primarily secreted in the interstitial strome of tumor tissue, the drugs are selectively released in these tissues but not in normal cells. The second important strategy is using cellular receptors as targets for carrier-linked prodrug design. There are many tumor-associated receptors that have been investigated in receptor – targeting approaches, such as the transferrin receptor, integrins or asialoglycoprotein receptor. The av-integrins, cell adhesion molecules highly expressed on activated endothelial cells and tumor cells but not on normal ones, present an attractive target for tumor therapy. Prodrugs containing tumor-homing RGD (Arg-Gly-Asp) peptide have been shown to display high selectivity for binding to the integrin receptors. Asialoglycoprotein receptors are expressed on hepatocytes. These receptors have been also targeted using prodrugs. ASGP-R is a lectin mediating uptake of galactosyl terminating macromolecules which can be use as hepatotropic carrier of drugs like lactosaminated human albumin (L-HAS). We described also the receptor for peptide hormones such as luteinizing-hormone releasing hormone, somatostatin, bombesin or calcytionin. Prodrugs of DOX which contain ligands (peptides) with a high affinity to the hormone receptors are taken up by receptor-mediated endocytosis. The use of targeted cytotoxic peptide analogues could inhibit the growth of cancer and improves the tumor treatment outcome. The peptide prodrugs can be also conjugated with large impermeable macromolecules (for example albumin) to facilitate active drug entry. In these conjugates substrate peptide sequences bind with cysteine-34 position of endogenous or exogenous albumin via maleimide hydrazine spacer. An albumin preferentially accumulates in solid tumors due to vascular permeability of the blood vessels of tumor and impaired metabolic drainage. Besides, albumins are a major energy and nutrition source for tumor growth. Albumin prodrug may also contain acid-sensitive linkers that allow DOX to be realized either extra or intracellulary. Multidrug resistance (MDR) is a major problem in the treatment of malignant diseases. Glycoprotein P (P-gp) is one of the best-studied cell-membrane efflux pumps responsible for MDR in cancer. The very interesting strategy are prodrugs which include an inhibitor of glycoprotein P. This kind of conjugate may help to overcome the chemoresistance of neoplastic cells.

Summary: The physiological role of tyreoliberin is preservation of homeostasis among four systems: (i) the hypothalamic-hypophysiotropic neuroendocrine system, (ii) the brain stem/midbrain/spinal cord system, (iii) the limbic/cortical system, and (iv) the chronobiological system. Thus TRH by various cellular mechanisms regulates a wide range of biological processes (arousal, sleep, learning, locomotor activity, mood) and possess the potential for unique and widespread applicability in human illnesses. Since therapeutic usage of TRH is limited by its low pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in monotherapy or adjunctive therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of physiological role of TRH and describes its putative clinical indications in CNS maladies with underlined action of TRH analogues.

Key words: tyreoliberin, metabolism, neuromodulator, nootropic factor, analepsy, antidepressant, neuroprotection