Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Waste paper derived three-dimensional carbon aerogel integrated with ceria/nitrogen-doped reduced graphene oxide as freestanding anode for high performance and durable microbial fuel cells

Bioprocess and Biosystems Engineering - Despite the green energy generation with low cost compared to conventional fuel cells, microbial fuel cells (MFCs) still suffer with anode related...     

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

Exploration of green integrated approach for effluent treatment through mass culture and biofuel production from unicellular alga,Acutodesmus obliquus RDS01

Abstract

This study deals with the open pond (OP) pilot scale treatment of cassava effluent and enhancement of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) enzyme through CO₂ utilization by the microalga, Acutodesmus obliquus RDS01. The cassava effluent treatment (ET) revealed maximum reduction of ammonia (96.8%), calcium (94.6%), chloride (95.2%), chlorine (98.5%), inorganic phosphate (94.6%), magnesium (96.8%), nitrate (96.89%), organic carbon (95.9%), organic phosphorus (96.3%), potassium (97.9%), sodium (97.1%), and sulfate (95.4%) on 15^th day using A. obliquus. The microalga produced highest RuBisCO enzyme activity (90%), CO₂ utilization efficiency (95%), biomass (8.9 gL^?1), lipid (176.65?mg mL^?1), carbohydrate (96.78?mg mL^?1), biodiesel (4.1?mL g^?1), and bioethanol (3.7?mL g^?1) during OP treatment. The isolated RuBisCO gene (rbcL) was used to construct the protein model by homology modeling. The microalgal-lipid content was analyzed through thin layer chromatography, the biodiesel produced was analyzed using Fourier-transform infrared spectroscopy and gas chromatography mass spectrometry (GCMS). The bioethanol production was confirmed by high performance liquid chromatography and GCMS analyses. A. obliquus produced of 98.75% biodiesel and 96.83% bioethanol in the OP pilot scale treatment A. obliquus. Overall, the microalga A. obliquus could act as an effective CO₂ capturing and bioremediation agent in the cassava ET, and also for the biodiesel and bioethanol can be produced.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

Synthesis and evaluation of anti-HIV activity of isatin beta-thiosemicarbazone derivatives

On the basis of pharmacophoric modelling studies of existing NNRTIs, a series of isatin beta-thiosemicarbazone derivatives was synthesized and evaluated for their anti-HIV activity in HTLV-III(B) strain in the CEM cell line. Three compounds showed significant anti-HIV activity, whereupon compound 6 was found to be the most active compound with an EC(50) value of 2.62 microM and a selectivity index of 17.41, while not being cytotoxic to the cell line at a CC(50) value of 44.90 microM. Other tested compounds exhibited marked activity below their toxicity threshold.     

Perlecan and its immunoglobulin like domain IV are abundant in vitreous and serum of the chick embryo.

Perlecan is a highly conserved heparan sulfate proteoglycan in cartilage and basement membranes. We identified chick perlecan and a 90 KD perlecan fragment in vivo using a newly generated monoclonal antibody. Chick perlecan is, like its human and mouse homologue, a hybrid heparan sulfate/chondroitin sulfate proteoglycan with a core protein of 400 KD. Analysis of the 90 KD fragment by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Capillary LC nano Electrospray Ionization tandem MS (LC nano ESI MS/MS) showed that it belonged to domain IV of the perlecan core protein. We found that full-length perlecan and its domain IV fragment are abundant in embryonic vitreous body and serum. Their expression in vitreous and serum is greatly down-regulated shortly after hatching of the chick. We speculate that the abundance of perlecan in the embryonic circulation and vitreous reflects the ongoing formation of new BMs in the expanding vascular system and the growing retina. In addition, we found that perlecan as a substrate does not support, rather inhibits neurite outgrowth.     

Chromosome numbers and meiotic analysis in the pre-breeding of <Emphasis Type="BoldItalic">Brachiaria decumbens</Emphasis> (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.