Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.     

Impact of habitat modification on the distribution and abundance of fruit flies (Diptera: Tephritidae) in Southeast Queensland

Loss of rainforest because of agricultural and urban development may impact the abundance and diversity of species that are rainforest natives. Tropical fruit flies are one group of such organisms indigenous to rainforests. In southeast Queensland, a region subject to rapid urbanization, we assessed the impact of habitat disturbance on the distribution and abundance of native fruit flies. Data on four species (Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera chorista, and Dacus aequalis) were gathered and analyzed over 6 months in three habitat types: suburbia, open sclerophyll forest, and rainforest. We also analyzed the data at a combined "dacine fruit fly" level incorporating all fruit fly species trapped over the period of study (as might occur in a biodiversity assessment): these included the four species already named and Bactrocera melas, Bactrocera bryoniae, Bactrocera newmani, and Dacus absonifacies. Analysis at the species level showed that the polyphagous pest species responded differently to the monophagous species. Bactrocera tryoni, which has more exotic than native hosts, was positively affected by transformation of natural habitat into suburbia whereas B. neohumeralis, which has nearly identical numbers of native and exotic hosts, was found equally across habitat types. Bactrocera chorista and Dacus aequalis, each monophagous on a species-specific rainforest host plant, were most abundant in rainforest. The analysis based on the combined data suggests that replacing rainforest with suburbia has a neutral, or even positive, effect on the abundance of fruit flies as a whole. At the species level, however, it can be seen that this is an erroneous conclusion biased by the abundance of a single pest species. Our discussion raises the issue of analyses at supraspecific levels in biodiversity and impact assessment studies. Received: March 6, 2000 / Accepted: June 19, 2000     

LR8 expression in fibroblasts of healthy and fibrotic human tissues

LR8 gene was first reported in a subpopulation of cultured human lung fibroblasts expressing the receptor for C1q-globular domain, and it was not detectable in cultured endothelial cells and smooth muscle cells. LR8 mRNA levels were higher in fibrotic lungs. In this study we assessed LR8 production in human tissues and determined if the distribution of fibroblasts producing LR8 is affected in fibrosis. Normal and fibrotic tissue sections from human liver, lung and kidneys were immunostained with antibodies to LR8 and examined for the presence of fibroblasts staining positively and negatively. The cells were also examined for co-expression of α-smooth muscle actin (SMA), a marker for myofibroblasts. The results showed that LR8 was expressed by fibroblasts, smooth muscle cells, endothelial cells, bile duct cells, pulmonary alveolar cells and distal and proximal kidney tubule cells. Connective tissues of normal and fibrotic tissues contained fibroblasts staining positively and negatively with anti- LR8 antibody. The number of LR8-positive cells was higher in fibrotic tissues, but differences were not statistically significant. Fibroblasts producing both LR8 and SMA were present in higher numbers in fibrotic tissues as compared to normal tissues and the differences were statistically significant (p<0.05). Our results show that fibroblast subtypes differing in LR8 expression are present in human tissues, and that in fibrotic tissues cells co-expressing LR8 and SMA are present. Our results indicate that LR8 expressing cells may participate in the early stages of fibrotic diseases and that fibroblasts expressing LR8, not LR8 negative cells, have potential to become myofibroblasts in fibrotic tissues.     

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.     

RdgB proteins: functions in lipid homeostasis and signal transduction

The RdgBs are a group of evolutionarily conserved molecules that contain a phosphatidylinositol transfer protein (PITP) domain. However in contrast to classical PITPs (PITPalpha) with whom they share the conserved PITP domain, these proteins also contain several additional sequence elements whose functional significance remains unknown. The founding member of the family DrdgB alpha (Drosophila rdgB) appears to be essential for sensory transduction and maintenance of ultra structure in photoreceptors (retinal sensory neurons). Although proposed to support the maintenance of phosphatidylinositol 4, 5 bisphosphate [PI (4, 5) P(2)] levels during G-protein coupled phospholipase C activity in these cells, the biochemical mechanism of DrdgB alpha function remains unresolved. More recently, a mammalian RdgB protein has been implicated in the maintenance of diacylglycerol (DAG) levels and secretory function at Golgi membranes. In this review we discuss existing work on the function of RdgB proteins and set out future challenges in understanding this group of lipid transfer proteins.     

Emerging perspectives on multidomain phosphatidylinositol transfer proteins

The phosphatidylinositol transfer protein domain (PITP_d) is an evolutionarily conserved protein that is able to transfer phosphatidylinositol between membranes in vitro and in vivo. However some animal genomes also include genes that encode proteins where the PITP_d is found in cis with a number of additional domains and recent large scale genome sequencing efforts indicate that this type of multidomain architecture is widespread in the animal kingdom. In Drosophila photoreceptors, the multidomain phosphatidylinositol transfer protein RDGB is required to regulate phosphoinositide turnover during G-protein activated phospholipase C signalling. Recent studies in flies and mammalian cell culture models have begun to elucidate functions for the non-PITP_d of RDGB and its vertebrate orthologs. We review emerging evidence on the genomics, functional and cell biological perspectives of these multi-domain PITP_d containing proteins.     

Identification of acid-stress-tolerant proteins from promising cyanobacterial isolates

Cyanobacterial cultures were isolated from acidic (pH 4.9–6.2) rice grown soils in Tamil Nadu, India. The predominant genera were Anabaena (50%), Westiellopsis (17.5%), Nostoc (15%), Oscillatoria (5%) and others that were unicellulars (12.5%) viz., Microcystis, Calothrix and Phormidium. The levels of tolerance to acidity varied among these strains, which were tested and authenticated for their acid tolerance capacity under both in vitro and pot culture conditions. Westiellopsis sp. was found to predominate from pH 4.9 to pH 6.2, indicating its adaptability. Cultures tolerant to acidic conditions were characterized for growth, biomass production and biochemical constituents. Under acidic conditions, Westiellopsis sp. showed pronounced chlorophyll a content, phycobilin pigment content, ammonia excretion and nitrogenase activity compared to normal conditions. Molecular characterization, particularly isozyme and random amplified polymorphic DNA (RAPD) analysis, were also carried out. Three strains of Westielliopsis sp. strains were selected, of which two were able to grow at an acidity level of pH 4.0, while one strain was able to sustain growth at an acidity level of 5.0. These three cultures, along with acid susceptible strains of Westielliopsis sp. and Anabaena sp. PCC 7120 (standard check) were subjected to acid shock for different time intervals. Protein profiling of both the acid-tolerant and acid-susceptible strains was carried out with samples collected at different time intervals. Based on the presence/absence of protein bands in the tolerant/susceptible strains, some low- and medium-molecular weight proteins can be linked to conferring acid tolerance. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.