Morphology of nectaries and biology of nectar production in the distylous species Fagopyrum esculentum

Background and Aims

The mechanisms of floral nectar production in buckwheat (Fagopyrum esculentum, Polygonaceae), a distylous pseudo-cereal, have received relatively little attention, prompting an investigation of the factors that regulate this process. The aim was to perform a refined study of the structures that secrete nectar and of the internal and external parameters influencing nectar volumes and sugar concentrations.

Methods

In order to control environmental parameters, plants were cultivated in growth rooms under controlled conditions. The structure of nectaries was studied based on histological sections from flowers and flower buds. Nectar was extracted using glass micropipettes and the sugar concentration was measured with a hand refractometer. Sugar concentration in the phloem sap was measured using the anthrone method. To test the influence of photosynthesis on nectar production, different light and defoliation treatments were applied.

Key Results

Unicellular trichomes were located in the epidermis at the ventral part of eight nectary glands situated on the flower receptacle alternately with stamens. Vascular bundles consisting of both phloem and xylem were identified at the boundary between a multilayered nectary parenchyma and a sub-nectary parenchyma with chloroplasts. A higher volume of nectar in thrum morphs was observed. No other difference was found in morphology or in sugar supply to inflorescences between morphs. Nectar secretion was strongly influenced by plant age and inflorescence position. Nectar volumes were higher in the upper inflorescences and during the flowering peak. Light had a dual role, (1) acting directly on reproductive structures to trigger flower opening, which conditions nectar secretion, and (2) stimulating photosynthetic activity, which regulates nectar accumulation in open flowers.

Conclusions

In buckwheat, nectar is secreted by trichomes and probably proceeds, at least in part, from phloem sap. Nectar secretion is strongly influenced by floral morph type, plant age, inflorescence position and light.Key words: Buckwheat, distyly, Fagopyrum esculentum, inflorescence position, morph comparisons, nectary histology, nectar sugar concentration, nectar volume, light intensity, organ biomass, phloem sap, plant age     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Retinal Pathology of Pediatric Cerebral Malaria in Malawi

Introduction

The causes of coma and death in cerebral malaria remain unknown. Malarial retinopathy has been identified as an important clinical sign in the diagnosis and prognosis of cerebral malaria. As part of a larger autopsy study to determine causes of death in children with coma presenting to hospital in Blantyre, Malawi, who were fully evaluated clinically prior to death, we examined the histopathology of eyes of patients who died and underwent autopsy.

Methodology/Principal Findings

Children with coma were admitted to the pediatric research ward, classified according to clinical definitions as having cerebral malaria or another cause of coma, evaluated and treated. The eyes were examined by direct and indirect ophthalmoscopy. If a child died and permission was given, a standardized autopsy was carried out. The patient was then assigned an actual cause of death according to the autopsy findings. The eyes were examined pathologically for hemorrhages, cystoid macular edema, parasite sequestration and thrombi. They were stained immunohistochemically for fibrin and CD61 to identify the components of thrombi, β-amyloid precursor protein to detect axonal damage, for fibrinogen to identify vascular leakage and for glial fibrillary acidic protein to detect gliosis. Sixty-four eyes from 64 patients were examined: 35 with cerebral malaria and 29 with comas of other causes. Cerebral malaria was distinguished by sequestration of parasitized erythrocytes, the presence and severity of retinal hemorrhages, the presence of cystoid macular edema, the occurrence and number of fibrin-platelet thrombi, the presence and amount of axonal damage and vascular leakage.

Conclusions/Significance

We found significant differences in retinal histopathology between patients who died of cerebral malaria and those with other diagnoses. These histopathological findings offer insights into the etiology of malarial retinopathy and provide a pathological basis for recently described retinal capillary non-perfusion in children with malarial retinopathy. Because of the similarities between the retina and the brain it also suggests mechanisms that may contribute to coma and death in cerebral malaria.     

Detection of silent cells,synchronization and modulatory activity in developing cellular networks

Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature “silent” cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity‐based pixel correlations to identify cellular‐based regions of interest (ROIs) and coincident cell activity. However, using activity‐based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell‐dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi‐automatically detect cells within developing neuronal networks that were imaged using calcium‐sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 357–374, 2016