Sunday, June 26, 2011

Understanding of involvement of polyamines in plants

Polyamines (PAs) are naturally occurring polycationic aliphatic amines, which due to their ubiquity and versatility are involved in the regulation of various cellular and molecular processes. They are positively charged compounds with their charge distributed along the molecule. The common PAs, spermidine (SPD) and spermine (SPM) and their diamine precursor putrescine (PUT) play a critical role in the normal functioning of all cells. They are involved in the cellular functioning both at the molecular and physiological levels due to their association with various Plant macromolecules (DNA, RNA and proteins) and membranes as well as their high concentration in the cytosol thus behaving as osmolytes. The role of PAs is much better studied in animal systems than plants, though they have been suggested to have a role as new plant growth regulators either by mediating the plant hormone effects or independently signalling other responses.

PAs exist in three forms in the cell, viz. as free cations, covalently bound to low molecular weight phenolic compounds like hydroxycinnamic acids (conjugated form of PAs) and bound to marcomolecules or membranes (bound form of PAs). Though the major form is the free cationic form of PAs, there are instances when the amounts of conjugated form exceed the free form and these are known to be critical in certain physiological processes including seed germination, flower development, defence responses and stress reactions. Besides PUT, SPD and SPM, there are certain unusual PAs found in nature, e.g. thermo SPM which have been detected in bacteria residing in hot springs and they seem to be important in protecting the enzymes from heat denaturation and aminobutylhomo-SPD found in fast growing cells of root nodule bacteria Rhizobium. NorSPD and norSPM are found in thermophilic red algae, brown algae,and Chlamydomonas, Nitella and Chlorella. Similarly, some unusual PAs have been reported in plants, homo-SPD was first detected in sandalwood and also in mosses and ferns. In leguminous plants, other unusual PAs like canavalmine, homoagmatine, aminopropylcanavalmine and aminobutylcanavalmine have been detected. NorSPD and NorSPM have been detected in alfalfa grown under drought conditions and have been postulated to play a protective role under stress conditions. As a matter of fact it has been suggested that PA distribution, especially of SPM, may serve as a phylogenetic marker.

PAs have been demonstrated to be associated with regulation of somatic embryogenesis, root and shoot formation, flower and fruit development , stress responses and senescence. In fact, PAs may serve as ‘biomarkers’ for in vitro morphogenetic potential including plant regeneration via somatic embryogenesis. The multifaceted functions of PAs as well as the variations in their levels in response to changes in the physiological state, point towards their role as possible second messengers, though their high titres do not support the view. Various studies have been conducted to investigate the involvement of PAs in cell functioning, using mutants of PA biosynthetic genes and specific substrate-based inhibitors of PAs. Though much information could be generated regarding the involvement and possible mechanisms of action, no clear picture of their functioning emerged. Hence, transgenic plants expressing PA biosynthetic genes in constitutive and regulated manner were generated, with an aim to answer some of the queries regarding the functioning and role of PAs.

Tags: Bio Technology, Bio Genetics, Polyamines in Plants

Saturday, March 12, 2011

Different Mechanisms for Transporting DNA into the Cell

Following two basic mechanisms are assumed to contribute to the transport of the DNA into the cell.

1) An active, energy dependent uptake of the transfection complexes by a process called endocytosisd

2) “Passive” membrane fusion and release of DNA into the cytoplasm.

The compaction agent used in the first step largely determines which mechanism is more important in a given case. For polycationic molecules, a direct interaction (fusion) with the hydrophobic membranes is not likely. The most likely way for them to enter the cell would be by endocytosis. Cationic lipids, on the other hand, can potentially interact and fuse with the membrane. Experiments with synthetic membranes have demonstrated the fusogenic ability of liposomes formed by cationic lipids,but convincing data that this mechanism is also operative during transfection of living cells are still lacking. Other reports seem to indicate that liposomes also preferably enter the cell by endocytosis.

Endocytosis is a process by which cells take up extracellular molecules such as cholesterol via a receptor-mediated mechanism. Cholesterol, insoluble in aqueous solutions, naturally occurs in association with the so-called low-density lipoproteins (LDL). The uptake of cholesterol by the cells depends on receptors specific for LDL. In a first step the ligands bind to the receptor. Receptors occupied with ligands form clusters and induce the formation of a clathrin-coated pit. Clathrin induces the expansion of the pit. Such pits can subsequently enter the cell as a membrane-bound vesicle containing the ligand/cholesterol-complex. Inside the cell, the vesicle rapidly loses its clathrin coat. Vesicles containing receptor bound ligands undergo further changes. Protons are actively imported into the vesicle leading to a drop in pH from the physiological values of 7 to about 5. Under these mildly acidic pH conditions, receptor and ligand dissociate. Receptors are then recycled back to the membrane with the aid of a sorting vesicle. The ligand/cholesterol-complexes stay within the vesicle and are transported towards the so-called lysosome, an even more acidic vesicle containing digestive enzymes. In the case of the ligand LDL, the ligand/cholesterol complex is digested inside the lysosome into amino acids, cholesterol and fatty acids.

Receptor mediated endocytosis may easily be exploited for DNA transfer into a cell, but, if DNA ends up in a lysozyme, it will be degraded. In order to succeed with gene transfer, the DNA needs to escape the endosome before it is digested by lysosomal nucleases. This is possible, as demonstrated by a number of infectious viruses, which use endocytosis for the efficient transfer of their genetic material into certain target cells. Such viruses have special capsid proteins that allow them to escape the early endosome. The signal for their escape is triggered by the drop in pH. As soon as the pH in the endosome starts to decrease, the capsid proteins undergo a conformational change that enables them to fuse with the membrane of the early endosome. The result is a disruption of the vesicle and the release of the virion into the cytoplasm. A synthetic peptide derived from the capsid of the hepatitis A virus has recently been shown to mimic this endosome escape induced by low pH. Another, less efficient, way to escape the lysosome consists in the utilization of lysosome blocking agents such as chloroquine or - even simpler - in an osmotic shock enforced by exposing the cells to nontoxic and nonionic compounds but osmotically active molecules such as glycerol and DMSO.

Tuesday, January 11, 2011

How the compacted DNA gets Attached to the Cell Surface

If a foreign DNA sequence is to be introduced into a cell, it is obviously necessary that the two meet, i.e., that the compacted DNA somehow attaches to the cell surface within and for a reasonable amount of time.

Cell membranes consist of a lipid bilayer into which a number of complex (glyco)protein molecules are inserted or anchored. The dominant mechanism for interaction between the DNA complex and the negatively charged cell surface are electrostatic forces. The negative surface charge is in many cases provided by proteoglycan molecules carrying anionic sulfate groups, which are present on the surface of many cell types. Positively charged complexes may attach themselves to the cell surface via these molecules. The importance of this type of interaction to the success of a transfection has been demonstrated by the following experiment. It has been shown, that DNA charged cationic liposomes fail to transfect so-called Raji cells, which are proteoglycan negative, but transfect genetically modified, proteoglycan positive (syndecan-1), cells of the same cell line with good efficiency.

Electrostatic interaction with the proteoglycans, however, is not the only possibility for interaction between a DNA-carrying transfection agent and a cell surface. Many membrane proteins expose binding sites (receptors) for certain biochemical messenger molecules (ligands).

In general, such receptor proteins control the specific uptake of molecules and make the cell sensitive to hormones and other signal molecules. This natural mechanism can be subverted for DNA transfer. The receptor ligands can be used to increase transfection efficiency in general or they can be used to target the transfection complex to a specific cell or tissue type by evoking an interaction between the transfection complex and a cell-specific receptor. Targeting can, for example, be achieved by introducing ligands such as, insulin, transferrin, lactose, galactose, mannose, folate, poly(acrylic acid) or specific monoclonal antibodies or antibody fragments into the transfection complex. This addition has been shown to dramatically increase the efficiency of transfections with agents such as poly(lysine) or poly(ethyleneimine) for certain cell lines, which otherwise were difficult to transfect. It seems that the improvement is due to the ligand’s ability to subsequently induce receptor-mediated uptake of the DNA into the cell (endocytosis, see below). In addition, receptor mediated transfection can be blocked (controlled) if necessary by complementing the cell culture medium during the transfection with an excess of the free ligand.

Tags: Bio Technology, Bio Genetics, Gene Compaction