Diffusion tensor imaging in the early phase of schizophrenia: what have we learned?

The dysconnectivity model suggests that disturbed integration of neural communication is central to schizophrenia. The integrity of macro-structural brain circuits can be examined with diffusion tensor imaging (DTI), an MRI application sensitive to microstructural abnormalities of brain white matter. DTI studies in first-episode schizophrenia patients and individuals at high-risk of psychosis can provide insight into the role of structural dysconnectivity in the liability, onset and early course of psychosis. This review discusses (i) views on the role of white matter abnormalities in schizophrenia, (ii) DTI and its application in schizophrenia, (iii) DTI findings in first-episode patients and subjects at high-risk of psychosis; their timing, anatomical location and early course, (iv) the hypothesized underlying pathological substrate and possible causes of DTI white matter alterations, including effects of adolescent cannabis use, and (v) some methodological issues and future recommendations. In summary, there is evidence that DTI abnormalities convey a liability for psychosis and additional abnormalities occur around onset of psychosis. However, findings in first-episode patients are less robust than in chronic patients, and progression of disturbances may occur in the early course of poor-outcome patients. In addition, acceleration of the normal aging process may occur. Adolescent cannabis use has specific effects on DTI measures. An unresolved issue is the underlying pathology of DTI abnormalities, and combining DTI with other MRI indices can provide more insight. More research is needed on which genetic and environmental factors play a role in the variability of current results.

Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranoside.

Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non-volatile S-linalyl-beta-D-glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Microprotoplast-mediated transfer of single specific chromosomes between sexually incompatible plants.

Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (Solanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotiana tabacum) and the wild tomato species Lycopersicon peruvianum is reported. The microprotoplasts contained one or a few chromosomes. Monosomic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of potato using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting enzyme mixture. The microprotoplasts were isolated from the micronucleated protoplasts by high-speed centrifugation. By using sequential filtration, small microprotoplasts containing one or few chromosomes were separated from the bigger subdiploid microprotoplasts. These small microprotoplasts were fused with recipient protoplasts of tobacco or tomato using polyethylene glycol. The selectable marker kanamycin resistance (KanR) and the reporter gene β-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion products and the isolation of hybrid calli. Several monosomic addition plants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus and one or two copies of the neomycin phosphotransferase (nptII) gene conferring KanR, and the complete set of chromosomes of tobacco or tomato, as revealed by genomic in situ hybridization and Southern blot hybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions, and some introgression plants showing integration of gus and nptII in the tomato genome, were recovered in the first backcross progeny. The potential value of MMCT for the transfer of economically important traits, genome analysis, and gene expression is discussed. Key words : chromosome transfer, microprotoplast fusion, monosomic-disomic additions, sexual transmission, DNA integration, alien gene expression.

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion.

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan(R)) and ß-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan(R) calli. A high frequency of plants regenerated from Kan(R) calli expressed both Kan(R) and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.

Interspecific transfer of isolated plant mitochondria by microinjection.

Data on isolation, purification and transfer of mitochondria from a cytoplasmic male sterile line of the "Ogura" type of Raphanus sativus to a male fertile line of Brassica napus are reported. Microinjection has been used for the transfer of the donor mitochondria to the recipient protoplasts. The injected protoplasts were identified and followed individually throughout their development using a computerized microscope stage which greatly enhanced the number of injections (five-fold). The transferred donor mitochondria were stably maintained during several successive cell divisions, revealing that they were viable and functional. Several calluses were obtained from injected protoplasts without using any selection pressure. Restriction fragment length analysis of seventeen calluses, using mitochondrial DNA probes, indicated that three contained the donor "Ogura" type mitochondria. No recombinant types of mitochondria have been observed. Flow cytometric and karyotype analyses of the calluses revealed the presence of similar amount of DNA and chromosome number as those of the recipient plants of B.napus. The application of microinjection for the manipulation of cytoplasmic composition is discussed.

Direct cell to cell transfer of organelles by microinjection.

A new technique for transfer of organelles to plant cells is presented. The organelles are removed from the donor protoplast by micromanipulation and microinjected directly into the acceptor cells. First results obtained by this technique for transfer of chloroplasts and fluorescently labelled mitochondria are presented.

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia.

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.

Flow-cytometric characterization and sorting of plant chromosomes.

Flow cytometric measurements of DNA frequency distribution were used to follow the synchronization process in suspension cells from Haplopappus gracilis (2n=4). Metaphase chromosomes were isolated from these synchronized cells and both the acro- and metacentric chromosomes were sorted by flow cytometry based on the different DNA contents. Possible applications of this procedure in fundamental genetics as well as practical plant breeding are discussed.

S-specific proteins in styles of self-incompatible Nicotiana alata.

A comparison of the stigma protein patterns of individual plants of the inbred- and cross-progenies in Nicotiana alata by isoelectric focusing revealed the presence of S-specific proteins. The S allele-protein relationship was found for three different S alleles. The S-specific proteins occurred in both stigma and stylar parts of the pistil whereas they were absent in leaves. In clone OWL the concentration of S-specific proteins in the stigma increased gradually during floral development. The shift from compatibility to incompatibility was not accompanied by an abrupt increase in concentration of the S-proteins.

Do S allele-specific peroxidase isoenzymes exist in self-incompatible Nicotiana alata?

In order to test Pandey's hypothesis that peroxidase isoenzymes determine S-gene specificity in Nicotiana alata, peroxidase isoenzymes in styles and pollen from various plants of an inbred- and a cross progeny were compared by means of starch gel electrophoresis and electrofocusing.No relation between the S-genotype and the peroxidase isoenzyme patterns of pollen or of styles could be established. The differences between the isoenzyme patterns of different S-genotypes were ascribed to differences in the genetic background of various plants that had the same S-genotype.