Raman microspectroscopic discrimination of TCam-2 cultures reveals the presence of two sub-populations of cells

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells’ homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.     

Morphological and histological impacts of the laurina mutation on fructification and seed characteristics in Coffea arabica L.

Key message

The comparison between the cultivar Bourbon and its mutant, the Bourbon pointu, of Coffea arabica led to five novel findings on fruit development and three main impacts of the mutation.

Abstract

Coffea arabica ‘Laurina’ (Bourbon pointu) is a natural mutant of Coffea arabica ‘Bourbon’. Relative to the ‘Bourbon’ cultivar, it is characterized by internode dwarfism, a Christmas tree shape, and lower caffeine content. The effects of the laurina mutation on fructification over time, the fruit structure and seed characteristics were studied here. Fruits of ‘Bourbon’ and ‘Bourbon pointu’ were monitored. The trees were grown in the same plot and flowered on the same day. Harvesting was done every 2 weeks from the 6th to the 26th week after flowering. Histological observations were carried out using multiphoton and conventional microscopes. The measurements concerned the fruit, parchment and seed. Five novel findings on fructification development were obtained: (1) a sigmoid model and non-linear regression efficiently described the phenomenon; (2) a precise relationship was defined between the qualitative stages of fructification and quantitative observations, thus revealing key weeks in this process; (3) the parchment had a mesocarpic origin; (4) a meristematic zone was present close to the parchment; and (5) an endocarp with three cell layers was visible in young fruits. Three effects of the laurina mutation were highlighted: (1) fruit growth ended 1 week earlier in ‘Bourbon’, but without difference in fruit length. In contrast, fruits were wider on average in ‘Bourbon’; (2) the parchment of narrow seeds in ‘Bourbon pointu’ was thicker than in other ‘Bourbon pointu’ and ‘Bourbon’ seeds; and (3) the narrow seed frequency in ‘Bourbon pointu’ depended on environmental conditions.     

Diversity in Expression Patterns and Functional Properties in the Rice HKT Transporter Family

Plant growth under low K⁺ availability or salt stress requires tight control of K⁺ and Na⁺ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K⁺ Transporters), permeable either to K⁺ and Na⁺ or to Na⁺ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na⁺ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na⁺ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na⁺-K⁺ symport, Na⁺ uniport, or inhibited states, depending on external Na⁺ and K⁺ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K⁺ and Na⁺ accumulation in monocots.Although it is not clear what levels of Na⁺ are toxic in the plant cell cytosol and actually unacceptable in vivo, the hypothesis that this cation must be excluded from the cytoplasm is widely accepted. The most abundant inorganic cation in the cytosol is K⁺, in plant as in animal cells. This cation has probably been selected during evolution because it is less chaotropic than Na⁺ (i.e. more compatible with protein structure even at high concentrations; Clarkson and Hanson, 1980). Its selection might also be due to the fact that in primitive cells, which originated in environmental conditions (seawater) where Na⁺ was more abundant than K⁺, a straightforward process to energize the cell membrane was to accumulate the less abundant cation and to exclude the most abundant one.In the cell, K⁺ plays a role in basic functions, such as regulation of cell membrane polarization, electrical neutralization of anionic groups, and osmoregulation. Concerning the latter function, K⁺ uptake or release is the usual way through which plant cells control their water potential and turgor. Although toxic at high concentrations, Na⁺ can be used as osmoticum and substituted for K⁺, mainly in the vacuole, when the plant is facing low K⁺ conditions and Na⁺ is available in the soil solution. This use of Na⁺, however, requires a tight regulation of K⁺ and Na⁺ transport and compartmentalization that becomes crucial in conditions of high Na⁺ concentrations in the soil solution. Control of Na⁺ and K⁺ uptake, long-distance transport in the xylem and phloem vasculatures, accumulation in aerial parts, and compartmentalization at the cellular and tissue levels have actually been shown to be essential in plant adaptation to salt stress (Greenway and Munns, 1980; Flowers, 1985; Hasegawa et al., 2000; Mühling and Läuchli, 2002). Thus, accumulation of Na⁺ as osmoticum during K⁺ shortage or plant adaptation to salt stress requires integration at the whole plant level of Na⁺ and K⁺ membrane transport system activities (Apse et al., 1999; Shi et al., 2002; Qi and Spalding, 2004; Ren et al., 2005; Maathuis, 2006; Pardo et al., 2006; Horie et al., 2007).This report concerns transport systems named HKT upon first identification (for High-Affinity K⁺ Transporters) that are active at the plasma membrane and permeable to either K⁺ and Na⁺ or to Na⁺ only (Schachtman and Schroeder, 1994; Rodríguez-Navarro and Rubio, 2006). Several members of the HKT family have already been shown, by genetic approaches, to play important roles in plant salt tolerance (Berthomieu et al., 2003; Ren et al., 2005; Huang et al., 2006; Byrt et al., 2007) or growth in conditions of K⁺ shortage (Horie et al., 2007). In Arabidopsis (Arabidopsis thaliana), the HKT family comprises a single member, AtHKT1;1, which is permeable to Na⁺ only (Uozumi et al., 2000) and contributes to Na⁺ removal from the ascending xylem sap and recirculation from the leaves to the roots via the phloem vasculature (Berthomieu et al., 2003; Sunarpi et al., 2005). Interestingly, the HKT family comprises a much larger number of members in rice (Oryza sativa), with seven to nine genes depending on the cultivar (Garciadeblás et al., 2003). In line with previous reports using rice as a model species to decipher the roles that HKT transporters can play in the plant, we have analyzed the expression patterns of three rice HKT genes, OsHKT2;1, OsHKT1;1, and OsHKT1;3, and investigated the functional properties of these transporters after heterologous expression, revealing new patterns of expression for HKT transporters and striking functional diversity.     

GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex

Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser¹⁶³ and Ser¹⁶⁷. The importance of these phosphorylation events was determined by mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser¹⁶³ and Ser¹⁶⁷ in GAP45 controls the final step in assembly of the myosin XIV motor complex.     

Dimerization of plant defensin NaD1 enhances its antifungal activity

The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys⁴ as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys⁴ that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys⁴ mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.     

A Randomised Placebo-Controlled Trial to Differentiate the Acute Cognitive and Mood Effects of Chlorogenic Acid from Decaffeinated Coffee

In the current study, sixty healthy older adults aged 50 years or older, and who were light to moderate coffee drinkers, were administered 6g of a decaffeinated green coffee blend (NESCAFÉ Green Blend coffee; GB) or 540mg pure chlorogenic acids (CGA) or placebo in a double-blind acute cross-over design, with cognitive and mood assessments pre-dose, 40-mins and 120-mins post-dose. The primary outcome measure was accuracy in Rapid Visual Information Processing (RVIP). Secondary cognitive outcome measures included RVIP reaction time as well as Inspection time (IT), Jensen Box decision/reaction times, serial subtraction and N-Back working memory. Secondary mood measures included Bond-Lader and caffeine Research visual analogue scales (VAS). No significant treatment effects were found for the primary outcome measure, although significant effects were found amongst secondary measures. Overall, CGA in isolation was not found to significantly improve cognitive function relative to placebo whereas the GB was found to improve sustained attention as measured by the N-Back task in comparison to placebo overall (t=2.45,p=.05), as well as decision time on a 2-choice reaction time task (Jensen box) in comparison to placebo at 40 minutes post-dose (t=2.45,p=.05). Similarly, GB was found to improve alertness on both the Bond-Lader at 120 minutes relative to CGA (t=2.86, p=0.02) and the caffeine Research VAS relative to CGA (t=3.09, p=0.009) and placebo (t=2.75,p=0.02) at 120 minutes post-dose. Both the GB and CGA were also found to significantly improve symptoms of headache at 120 minutes relative to placebo (t=2.51,p=0.03 and t=2.43,p=.04 respectively), whilst there was a trend towards a reduction in jitteriness with GB and CGA in comparison to placebo at 40 minutes post-dose (t=2.24,p=0.06 and t=2.20,p=0.06 respectively). These findings suggest that the improvements in mood observed with GB, but not the improvements in cognitive function, are likely to some extent to be attributable to CGAs.Trial Registration: Australia New Zealand Clinical Trials Registry ACTRN12611000067976 www.anzctr.org.au     

The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta

Background and Aims

Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta.

Methods

Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine–glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin–Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions.

Key Results and Conclusions

The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by budding from the chloroplast and bound by a membrane resulting from the fusion of both chloroplast envelopes. The shuttle conveys numerous tannosomes through the cytoplasm towards the vacuole in which it is then incorporated by invagination of the tonoplast. Finally, shuttles bound by a portion of tonoplast aggregate into tannin accretions which are stored in the vacuole. Polymerization of tannins occurs inside the tannosome regardless of the compartment being crossed. A complete sequence of events apparently valid in all studied Tracheophyta is described.     

Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

Plant defensin AhPDF1.1 is not secreted in leaves but it accumulates in intracellular compartments

? Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. ? Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. ? All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. ? These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level.     

Relationship between Optical Coherence Tomography and Electrophysiology of the Visual Pathway in Non-Optic Neuritis Eyes of Multiple Sclerosis Patients

Purpose

Loss of retinal ganglion cells in in non-optic neuritis eyes of Multiple Sclerosis patients (MS-NON) has recently been demonstrated. However, the pathological basis of this loss at present is not clear. Therefore, the aim of the current study was to investigate associations of clinical (high and low contrast visual acuity) and electrophysiological (electroretinogram and multifocal Visual Evoked Potentials) measures of the visual pathway with neuronal and axonal loss of RGC in order to better understand the nature of this loss.

Methods

Sixty-two patients with relapsing remitting multiple sclerosis with no previous history of optic neuritis in at least one eye were enrolled. All patients underwent a detailed ophthalmological examination in addition to low contrast visual acuity, Optical Coherence Tomography, full field electroretinogram (ERG) and multifocal visual evoked potentials (mfVEP).

Results

There was significant reduction of ganglion cell layer thickness, and total and temporal retinal nerve fibre layer (RNFL) thickness (p<0.0001, 0.002 and 0.0002 respectively). Multifocal VEP also demonstrated significant amplitude reduction and latency delay (p<0.0001 for both). Ganglion cell layer thickness, total and temporal RNFL thickness inversely correlated with mfVEP latency (r = −0.48, p<0.0001 respectively; r = −0.53, p<0.0001 and r = −0.59, p<0.0001 respectively). Ganglion cell layer thickness, total and temporal RNFL thickness also inversely correlated with the photopic b-wave latency (r = −0.35, p = 0.01; r = −0.33, p = 0.025; r = −0.36, p = 0.008 respectively). Multivariate linear regression model demonstrated that while both factors were significantly associated with RGC axonal and neuronal loss, the estimated predictive power of the posterior visual pathway damage was considerably larger compare to retinal dysfunction.

Conclusion

The results of our study demonstrated significant association of RGC axonal and neuronal loss in NON-eyes of MS patients with both retinal dysfunction and post-chiasmal damage of the visual pathway.