Micropropagation and molecular characterization of Thymus sibthorpii Benth. (Lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern

Thymus sibthorpii Benth. (Lamiaceae), with accession number 01,1796-22, is a biotype of native Greek thyme with ascending stems and potential use as a new medicinal-aromatic crop and ornamental plant. An efficient and reliable protocol for in vitro clonal propagation of T. sibthorpii from nodes and meristem tip explants was developed. Shoot proliferation succeeded on a new basal medium (BB) without plant growth regulators, as prior experiments with 6-benzyladenine generated hyperhydricity. Eight different basal media were compared; on two formulations using the new BB 5.9 and 5.6 shoots per explant were produced. Regenerated single shoots were rooted in the BB medium, supplemented with 5 μM of indole-3-butyric acid, and produced 3.1 roots along with 2.5 adventitious shoots. Three types of acclimatization were assessed: in vitro, using two different systems (no significant differences); ex vitro, using eight soil substrates under greenhouse and outdoor nursery conditions (in two of them, 100% of plantlets survived); and in field cultivations, established at eight geographically distant areas of Greece (100% survival rate at all locations). Molecular characterization of T. sibthorpii was evaluated with one nuclear ribosomal DNA and seven chloroplast DNA markers, followed by DNA sequence comparisons with a total of 30 different Thymus species, subspecies, and varieties. The trnH/psbA, trnL/trnF, and matK genes were the most efficient markers for molecular characterization of T. sibthorpii. The molecular markers rpoC1 and petB/petD did not match to any Thymus species and therefore, these DNA sequences provide new sequence information for entire Thymus taxa.

     

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.     

Supernumerary marker chromosome 5 diagnosed by M-FISH in a child with congenital heart defect and unusual face

We describe a female patient with a small supernumerary marker chromosome (sSMC) present in mosaic and characterized in detail by fluorescence in situ hybridization (FISH) using all 24 human whole chromosome painting probes, multicolor banding (MCB) and subcentromere specific multicolor FISH (subcenM-FISH). The sSMC was demonstrated to be derived from chromosome 5 and the karyotype of our patient was as follows: 47,XX,+mar.ish r(5)(::p13.2 approximately p13.3-->q11.2::) [60%]/46,XX [40%]. Partial trisomy for the proximal 5p and q chromosomal regions is a rare event. A critical region exists at 5p13 for the phenotype associated with duplication 5p. As far as we know, eight similar cases have been published up to now. We describe a new case which, to our knowledge, is the first characterized in such detail. The role of uniparental disomy (UPD) in cases of SMC is also discussed.     

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.     

Micropropagation of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. ‘Malagouzia’ and ‘Xinomavro’

The effects of six basal media on in vitro shoot proliferation of the greek grapevines Vitis vinifera L. cv. ‘Malagouzia’ and ‘Xinomavro’ were investigated. Galzy and Zlenco proved to be the most effective for ‘Malagouzia’ and ‘Xinomavro’, respectively. If only BA was present in the medium, shoot development was poor and the plantlets were chlorotic. When the medium was supplemented with BA and NAA, growth was enhanced. The best ratio (in μM) of growth regulators was 0.5/0.3 for ‘Malagouzia’, and 0.1/0.03 for ‘Xinomavro’, which resulted in the highest number of microshoots per explant and greatest proliferation rate. The development of ‘Malagouzia’ and ‘Xinomavro’ explants at 21±2 and 26±2°C was also investigated, revealing the higher temperature to be more effective. Regarding rooting, 0.5 μM IBA improved root formation at 26°C for ‘Malagouzia’ and 0.5 μM IBA at 21°C for ‘Xinomavro’. Moreover, 0.5 μM IBA resulted in a higher rooting percentage (>95%) and proved to be more beneficial for the overall morphological appearance of the plantlets of ‘Malagouzia’. After acclimatization, survival of microshoots cultivated in media with IBA was higher than those in NAA.     

Metal biosorption-flotation. Application to cadmium removal

Biosorption, using suspended non-living biomass, and flotation (for consequent solid/liquid separation of the metal-loaded biomass) have been studied in the laboratory as a possible combined process, for the removal of toxic metals (i.e. cadmium) from dilute aqueous solutions. The various parameters of the process were investigated in depth, including re-use of biosorbent. A filter aid (contained in the biomass industrial waste used) was found not really to interfere. -potential measurements of the aforementioned system were also carried out. Promising results were obtained during continuous-flow experiments. A flotation residence time of 4 min was achieved. Metal removal and suspended biomass recovery were generally over 95%.     

An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity

Artemis is required for V(D)J recombination and the repair of a subset of radiation-induced DNA double strand breaks (DSBs). Artemis-null patients display radiosensitivity (RS) and severe combined immunodeficiency (SCID), classified as RS-SCID. Strongly impacting hypomorphic Artemis mutations confer marked infant immunodeficiency and a predisposition for EBV-associated lymphomas. Here, we provide evidence that a polymorphic Artemis variant (c.512C > G: p.171P > R), which has a world-wide prevalence of 15%, is functionally impacting. The c.512C > G mutation causes an ～3-fold decrease in Artemis endonuclease activity in vitro. Cells derived from a patient who expressed a single Artemis allele with the polymorphic mutational change, showed radiosensitivity and a DSB repair defect in G2 phase, with Artemis cDNA expression rescuing both phenotypes. The c.512C > G change has an additive impact on Artemis function when combined with a novel C-terminal truncating mutation (p.436C > X), which also partially inactivates Artemis activity. Collectively, our findings provide strong evidence that monoallelic expression of the c.512C > G variant impairs Artemis function causing significant radiosensitivity and a G2 phase DSB repair defect. The patient exhibiting monoallelic c.512C > G-Artemis expression showed immunodeficiency only in adulthood, developed bilateral carcinoma of the nipple and myelodysplasia raising the possibility that modestly decreased Artemis function can impact clinically.     

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

A quantitative kinetic scheme for 70 S translation initiation complex formation

Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50 S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and fMet-tRNA(f)(Met). According to this scheme, an initially formed labile 70 S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30 S and 50 S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA(fMet), which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA(fMet). The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the non-hydrolyzable analog GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70 S formation, but perturbs fMet-tRNA(fMet) positioning in the P-site. In contrast, thiostrepton severely retards stable 70 S formation, but allows normal binding of fMet-tRNA(fMet)(prf20) to the P-site.     

The translational fidelity function of IF3 during transition from the 30 S initiation complex to the 70 S initiation complex

IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.