Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.     

Senescence-associated mRNAs that may participate in signal transduction and protein trafficking

The differential display technique was used to generate cDNA probes in order to identify mRNAs that are up-regulated during senescence of Arabidopsis leaves. Three mRNAs were examined that had not previously been associated with senescence. The steady-state levels of these mRNAs are detectable in small amounts in mature green leaves, but increase considerably as chlorophyll levels begin to decline. This relationship to senescence occurs under natural circumstances as well as when senescence is accelerated by leaf detachment in the dark or by addition of 1-aminocyclopropane-1-carboxylic acid (ACC). Retardation of senescence by benzyladenine slows the increase of the mRNAs. One of these mRNAs appears to code for a protein (Sec 13) that may be involved in vesicle formation at the endoplasmic reticulum. Another mRNA codes for a protein with WD‐repeat motif whose function is as yet unidentified, and the third codes for a putative calcium-dependent protein kinase. A fourth cDNA has also been cloned by subtractive hybridization from senescing Arabidopsis leaves that encodes vacuolar-processing enzyme ( γ VPE). Incubation of detached leaves in darkness also caused an abrupt elevation in the steady-state levels of the γVPE , similar to that of the senescing attached leaves. The possible functions of the gene products and their involvement in cellular and biochemical processes during senescence are discussed.     

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.     

Mechanism of transport and storage of neurotransmitters

This review will focus on the bioenergetics, mechanism, and molecular basis of neurotransmitter transport. As indicated in the next section, these processes play an important role in the overall process of synaptic transmission. During the last few years, direct evidence has been obtained that these processes are coupled chemiosmotically, i.e., the accumulation of neurotransmitters is driven by ion gradients. Two types of neurotransmitter transport systems have been identified: sodium-coupled systems located in the synaptic plasma membrane of nerves (and sometimes in the plasma membrane of glial cells) and proton-coupled systems which are part of the membrane of intracellular storage organelles. From a bioenergetic point of view, the sodium-coupled systems are especially interesting, since it has recently been discovered that many systems require other ions in addition to sodium. It has now been demonstrated in several cases that, besides sodium ions, these additional ions, such as chloride and potassium, serve as additional coupling ions. These systems will be reviewed here in considerable detail with emphasis on the role of the additional ions. In the second part of the review we shall focus on neurotransmitter transport into storage organelles. Although both sodium and proton coupled systems have been reviewed in the past, there has been a shift from a kinetic and thermodynamic to a biochemical approach. In fact, a few transporters have been identified and functionally reconstituted. These developments have of course been incorporated in this review.     

An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

Molecular Exploration of the First-Century Tomb of the Shroud in Akeldama,Jerusalem

The Tomb of the Shroud is a first-century C.E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected.