Molecular biological approaches to plant nutrition

In the last decade, understanding of ion transport has grown sufficiently to pose sensible questions about the molecular nature of the processes and their regulation. Techniques for identifying and cloning genes and for genetic transformation provide the means for answering these questions.Transport of ions across membranes is obviously a major aspect of mineral nutrition since it occurs during initial absorption, compartmentation and mobilisation of nutrients. Here, we will briefly review the types of transport protein involved and show how molecular biology and recombinant DNA technology have revealed something of their structure. Strategies used to identify the genes for transporters are discussed and reference is made to areas in which the availability of cloned genes will facilitate future studies.Mineral nutrition involves, however, more than membrane transport. The absorption rates of major nutrients are quite strictly regulated by biochemical factors which vary with the rate at which nutrients are used in growth. Nitrogen, sulphur and phosphate nutrition in micro-organisms are regulated by the interaction of various DNA-binding proteins with the promoter regions of genes for key enzymes in the assimilatory pathways and the specific ion permeases. The expression of the regulatory protein or its activity can be modified by metabolites, such as glutamine. Some evidence supports the idea that higher plants also have groups of genes with a common regulation of expression.An attempt is made to identify some reasonable objectives, which should increase understanding of the regulation of nutrient transport.Abbreviations and conventions cDNA complementary strand of DNA prepared from a messenger RNA - NR nitrate reductase - NiR nitrite reductase - P_i inorganic phosphate - PPase pyrophosphate - RUBISCO rubulose bisphosphate carboxylase oxygenase. Genes are referred to as follows e.g. cys-3 and scon-1. The protein products of these genes, where they have no familiar name are referred to as CYS3 and SCON-1 respectively     

Immunocytochemistry of the olfactory marker protein.

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.     

The olfactory system of crustaceans as a model for ecologo-toxicological studies

Based on our own studies and on literature data, there are considered peculiarities of structural-functional organization of the crustacean olfactory system and effects of pollutants on it. The behavioral reaction changes based on chemoreception in the polluted aquatic environment are described. Usefulness of study of the crustacean olfactory system is substantiated as a perspective object of ecologo-toxicological investigations.     

Molecular genetic approaches to microtubule-associated protein function

Protein function in vivo can be studied by deleting (knock-out) the gene that encodes it, and search for the consequences. This procedure involves different technologies, including recombinant DNA procedures, cell biology methods and histological and immunocytochemical analysis. In this work we have reviewed these procedures when they have been applied to ascertain the function of several microtubule-associated proteins. These proteins have been previously involved, through in vitro experiments, in having a role in the microtubule stabilization. Here, we will summarize the generation and characterization of different microtubule-associated protein knock-out mice. Special attention will be paid to MAP1B knock-out mice. Amongst the different MAPs knock-out mice these show the strongest phenotype, the most likely for being MAP1B, the MAP that is expressed earliest in neurogenesis. Molecular genetics could be considered as a valid and useful procedure to truly establish the in vivo functions of a protein, although it is necessary to be aware of possible artifacts such as the generation of some kinds of RNA alternative splicing. To avoid this the best strategy to be used must consider the deletion of the exon that contains the functional domains of the protein.     

Molecular genetics of cell migration: Dictyostelium as a model system

A central unresolved issue in modern cell biology concerns how eukaryotic cell migration is achieved. Although the underlying mechanics of cell locomotion appear similar in cells ranging from amoebae to leukocytes, the organisms that have been historically studied have not been amenable to the techniques of modern molecular genetics. The recent development of high-efficiency gene targeting technology for Dictyostelium discoideum, coupled with the classic cell migration behavior of this organism, offers an opportunity to resolve many of the controversial issues concerning cell locomotion.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Molecular genetic approaches to developing quality protein maize

Since its development more than two decades ago, Quality Protein Maize (QPM) has been adopted for cultivation in many regions of the developing world. Given the potential benefits of widespread use of QPM, research to better understand the genetic and biochemical mechanisms responsible for its altered kernel texture and protein quality is important. Recent investigations into the improved protein quality of the opaque2 mutant and the genetic mechanisms that can suppress its starchy kernel phenotype provide new insights to support the continued improvement of QPM. Chief among these developments are the use of transgenic approaches to improve nutritional quality and the discovery that an important component of modified endosperm texture in QPM is related to altered starch granule structure.     

Molecular dynamics simulation study of a two-dimensional fluid mixture system: a model for biological membranes

The computer simulation technique of molecular dynamics was applied to a model two-dimensional fluid mixture system to examine the short-range ordering of lipid and protein molecules in biological membranes. The model system consists of small disks (lipids) and large disks (proteins) with a radius ratio of 6, constrained to move in a plane. The particles interact with pairwise additive repulsive short range potentials, so as to simulate hard disks. Periodic boundary conditions are assumed in order to minimize boundary effects. For values of the number density of the small disks and of the temperature appropriate for a lipid membrane, the fraction, f, of small disks 'next to' at least one large disk was computed by molecular dynamics. This was done as a function of concentration and for several definitions of 'next to'. The molecular dynamics results show that, at moderately low mole fractions of the large disks, the calculated values of f deviate noticeably from the linear relation which would be expected in the absence of protein-protein proximity effects. The results are discussed in terms of current models of lipid-protein ordering in biological membranes.     

Olfactory marker protein immunohistochemistry and the anterograde transport of horseradish peroxidase as indices of damage to the olfactory epithelium

The present study compared the relative effectiveness of wheatgerm agglutinin--horseradish peroxidase (WGA--HRP) and olfactory marker protein (OMP) in detecting the presence of intact olfactory axons in glomeruli of the main olfactory bulb (MOB) in the rat. The olfactory epithelium was damaged by i.p. injections of the toxin 3-methyl indole and, after 5 or 6 days, the olfactory sac was injected with a 1% WGA--HRP solution. No anterograde labeling was observed in the dorsal and ventromedial quadrants of the MOB in the WGA--HRP material. However, in the same cases OMP immunostaining was observed throughout the MOB. In other rats the rostral olfactory epithelium was aspirated unilaterally and after 3, 11 and 16 days the olfactory sacs were injected with WGA--HRP and rats were perfused 1 day later. In these cases WGA--HRP reaction product was absent in the dorsolateral quadrant of the MOB on the aspirated side in all animals, but OMP immunostaining could be detected in the 4 and 12 day survival animals but not in the 17 day survival rat. These findings indicate that anterograde transport of WGA--HRP accurately reflects the presence of intact axons en route to the MOB. In contrast, OMP immunostaining persists in axon terminals severed from their parent cell body for at least 12 days and is a less useful marker of intact olfactory axons in experiments using short survival times.     

Telomere length as a biological marker in malignancy

Telomere maintenance is important for tumor cell growth and survival. Telomere length (TL) is determined by the balance between positive and negative factors impacting telomere homeostasis. In the last decade, TL has emerged as a promising clinical marker for risk and prognosis prediction in patients with malignant disorders. Tumor TL, as well as TL in healthy tissues such as peripheral blood, may carry valuable information for future treatment strategies. Here we discuss the present status of TL as a biological marker in cancer patients.     

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19–37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 nM. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 377–390, 1998     

Use of yeast as a model system to investigate protein conformational diseases

Protein conformational diseases arise when a cellular protein adopts an aberrant shape that either directly or indirectly alters the physiology of its host cell. Notable conformational diseases include cystic fibrosis, Huntington’s disease, the prion-related diseases, Alzheimer’s disease, and antitrypsin deficiency. In principle, the severity and progression of conformational diseases can be altered by cellular factors that recognize and attempt to ameliorate the harmful effects of the disease-causing, misshapen protein. To better define the mechanistic underpinnings of cellular factors that mediate quality control, and to understand why a single misfolded protein can impact cell viability, specific proteins that cause each of the diseases listed above have been expressed in a model eukaryote, the yeast Saccharomyces cerevisiae. In this review, we describe what has been learned from these studies, and speculate on future uses of yeast expression systems.     

Molecular mechanisms of exocytosis: The adrenal chromaffin cell as a model system

1. The release of neurotransmitters, hormones, and enzymes involves exquisitely regulated events which ultimately result in the fusion of the secretory vesicle with the cell's plasma membrane, releasing the vesicle contents into the extracellular space. 2. The biochemical and cellular mechanisms mediating exocytosis have been extensively studied in a model system of primary cultured adrenal chromaffin cells. 3. This paper briefly reviews current understanding, and directions of future studies in exocytosis using this model system.     

Identification of members of the Bex gene family as olfactory marker protein (OMP) binding partners

Olfactory marker protein (OMP) expression is a hallmark of mature vertebrate olfactory receptor neurons (ORNs). Evidence for OMP function derives from altered behavioral and electrophysiological activities of OMP-KO mice. The molecular basis for the altered phenotype following the deletion of OMP is still unclear. Recent structural studies predict the involvement of OMP in protein-protein interaction. Here we report the identification of an OMP partner, Bex2, by phage-display screening of an olfactory mucosal cDNA-library. In situ hybridization demonstrates cellular co-localization of OMP mRNA with mRNAs for Bex1, Bex2, and Bex3 in ORNs of olfactory tissue of the mouse. The OMP/Bex interaction has been confirmed by demonstrating the chemical cross-linking of recombinant rat OMP with a synthetic peptide derived from the Bex amino acid sequence. The subcellular localization of Bex and OMP proteins was evaluated in transfected HEK293 cells. Bex is visualized in the nucleus and cytoplasm. Following co-transfection we observed the unexpected presence of some OMP in the nucleus along with Bex. Together, these data argue convincingly that we have identified Bex as an OMP partner whose further characterization will provide insight to the role of OMP and to the mechanism of the OMP/Bex interaction in ORN differentiation and function.