Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Guard cells emit an alkali-induced, blue fluorescence upon excitationby ultraviolet radiation (emission maximum energy at 365 nm).Fluorescence emission of guard cells was brighter than thatof the neighbouring epidermal cells in a number of wild andcultivated plants including conifers, but the relative fluorescenceintensity and quality was species-dependent. Three representativeplants possessing stomatal complexes which differed morphologicallywere studied: Olea europaea, Vicia faba and Triticum aestivum.Immersing leaves of these plants in chloroform for 30 s (therebyremoving epicuticular waxes) significantly reduced the intensityof the fluorescence emitted by guard cells. This indicates thatguard cell fluorescence could be due to either an increasedconcentration of fluorescing compounds (probably wax-bound phenolics),or a thicker cuticular layer covering the guard cells. Giventhat the alkali-induced blue fluorescence of the guard cellsis a common characteristic of all plants examined, it couldbe used as a rapid and convenient method for in situ measurementsof the number, distribution and size of stomatal complexes.The proposed experimental procedure includes a single coatingof the leaf surface by, or immersion of the whole leaf in, a10% solution of KOH for 2 min, washing with distilled water,and direct observation of the leaf surface under the fluorescencemicroscope. Fluorescence images were suitable for digital imageanalysis and methodology was developed for stomatal countingusing Olea europaea as a model species. Copyright 2001 Annalsof Botany Company Cuticle, epicuticular waxes, fluorescence microscopy, image analysis, phenolics, stomata     

Retinoids inhibit human epidermal keratinocyte RNase P activity

Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.     

Ontogeny of intrinsic innervation in the human thymus and spleen.

The ontogeny of the innervation of human lymphoid organs has not been studied in detail. Our aim was to assess the nature and distribution of parenchymal nerves in human fetal thymus and spleen. We used the peroxidase immunohistochemical technique with antibodies specific to neuron-specific enolase (NSE), neurofilaments (NF), PGP9.5, S100 protein, and tyrosine hydroxylase (TH) and evaluated our results with image analysis. In human fetal thymus, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerves were identified associated with large blood vessels from 18 gestational weeks (gw) onwards, increasing in density during development. Their branches penetrated the septal areas at 20 gw, reaching the cortex and the corticomedullary junction between 20 and 23 gw. Few nerve fibers were seen in the medulla in close association with Hassall's corpuscles. In human fetal spleen, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were localized in the connective tissue surrounding the splenic artery at 18 gw. Perivascular NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were seen extending into the white pulp, mainly in association with the central artery and its branches, increasing in density during gestation. Scattered NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers and endings were localized in the red pulp from 18 gw onward. The predominant perivascular distribution of most parenchymal nerves implies that thymic and splenic innervation may play an important functional role during intrauterine life.     

Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function

Phospholamban (PLN) is a key regulator of Ca(2+) homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca(2+)-ATPase, (SERCA2a), resulting in alterations of its Ca(2+)-affinity. To identify additional proteins that may interact with PLN, we used the yeast-two-hybrid system to screen an adult human cardiac cDNA library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding partner. The minimal binding regions were mapped to amino acid residues 203-245 for HAX-1 and residues 16-22 for PLN. The interaction between the two proteins was confirmed using GST-HAX-1, bound to the glutathione-matrix, which specifically adsorbed native PLN from human or mouse cardiac homogenates, while in reciprocal binding studies, recombinant His-HAX-1 bound GST-PLN. Kinetic studies using surface plasmon resonance yielded a K(D) of approximately 1 muM as the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by cAMP-dependent protein kinase reduced binding to HAX-1, while increasing concentrations of Ca(2+) diminished the PLN/HAX-1 interaction in a dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects from hypoxia/reoxygenation-induced cell death. These findings suggest a possible link between the Ca(2+) handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.     

Intensive allochthonous inputs along the Ganges River and their effect on microbial community composition and dynamics

Little is known about microbial communities in the Ganges River, India and how they respond to intensive anthropogenic inputs. Here we applied shotgun metagenomics sequencing to study microbial community dynamics and function in planktonic samples collected along an approximately 700 km river transect, including urban cities and rural settings in upstream waters, before and after the monsoon rainy season. Our results showed that 11%–32% of the microbes represented terrestrial, sewage and human inputs (allochthonous). Sewage inputs significantly contributed to the higher abundance, by 13-fold of human gut microbiome (HG) associated sequences and 2-fold of antibiotic resistance genes (ARGs) in the Ganges relative to other riverine ecosystems in Europe, North and South America. Metagenome-assembled genome sequences (MAGs) representing allochthonous populations were detectable and tractable across the river after 1–2 days of (downstream) transport (> 200 km apart). Only approximately 8% of these MAGs were abundant in U.S. freshwater ecosystems, revealing distinct biodiversity for the Ganges. Microbial communities in the rainy season exhibited increased alpha-diversity and spatial heterogeneity and showed significantly weaker distance-decay patterns compared with the dry season. These results advance our understanding of the Ganges microbial communities and how they respond to anthropogenic pollution.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Reprogramming of the MicroRNA Transcriptome Mediates Resistance to Rapamycin

The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17–92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17–92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.