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141.
The retinitis pigmentosa GTPase regulator (RPGR) and nephrocystin-4 (NPHP4) comprise two key partners of the assembly complex of the RPGR-interacting protein 1 (RPGRIP1). Mutations in RPGR and NPHP4 are linked to severe multisystemic diseases with strong retinal involvement of photoreceptor neurons, whereas those in RPGRIP1 cause the fulminant photoreceptor dystrophy, Leber congenital amaurosis (LCA). Further, mutations in Rpgrip1 and Nphp4 suppress the elaboration of the outer segment compartment of photoreceptor neurons by elusive mechanisms, the understanding of which has critical implications in uncovering the pathogenesis of syndromic retinal dystrophies. Here we show RPGRIP1 localizes to the photoreceptor connecting cilium (CC) distally to the centriole/basal body marker, centrin-2 and the ciliary marker, acetylated-α-tubulin. NPHP4 abuts proximally RPGRIP1, RPGR and the serologically defined colon cancer antigen-8 (SDCCAG8), a protein thought to partake in the RPGRIP1 interactome and implicated also in retinal–renal ciliopathies. Ultrastructurally, RPGRIP1 localizes exclusively throughout the photoreceptor CC and Rpgrip1nmf247 photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1nmf247 mice without RPGRIP1 expression lack NPHP4 and RPGR in photoreceptor cilia, whereas the SDCCAG8 and acetylated-α-tubulin ciliary localizations are strongly decreased, even though the NPHP4 and SDCCAG8 expression levels are unaffected and those of acetylated-α-tubulin and γ-tubulin are upregulated. Further, RPGRIP1 loss in photoreceptors shifts the subcellular partitioning of SDCCAG8 and NPHP4 to the membrane fraction associated to the endoplasmic reticulum. Conversely, the ciliary localization of these proteins is unaffected in glomeruli or tubular kidney cells of Rpgrip1nmf247, but NPHP4 is downregulated developmentally and selectively in kidney cortex. Hence, RPGRIP1 presents cell type-dependent pathological effects crucial to the ciliary targeting and subcellular partitioning of NPHP4, RPGR and SDCCAG8, and acetylation of ciliary α-tubulin or its ciliary targeting, selectively in photoreceptors, but not kidney cells, and these pathological effects underlie photoreceptor degeneration and LCA.  相似文献   
142.
A major challenge in the production of metabolites by plant cells is the separation and purification of a desired product from a number of impurities. An important application of plant cell culture is the biosynthesis of the anticancer agent paclitaxel. Liquid–liquid extraction plays a critical role in the recovery of paclitaxel and other valuable plant‐derived products from culture broth. In this study, the extraction of paclitaxel and a major unwanted by‐product, cephalomannine, from plant cell culture broth into organic solvents is quantified. Potential solvent mixtures show varying affinity and selectivity for paclitaxel over cephalomannine. The partition coefficient of paclitaxel is highest in ethyl acetate and dichloromethane, with measured values of 28 and 25, respectively; however, selectivity coefficients are less than 1 for paclitaxel over cephalomannine for both solvents. Selectivity coefficient increases to 1.7 with extraction in n‐hexane, but the partition coefficient decreases to 1.9. Altering the pH of the aqueous phase results in an increase in both recovery and selectivity using n‐hexane but does not change the results for other solvents significantly. The addition of extractants trioctylamine (TOA) or tributylphosphate (TBP) to n‐hexane gives significantly higher partition coefficients for paclitaxel (8.6 and 23.7, respectively) but no selectivity. Interestingly, when 20% hexafluorobenzene (HFB) is added to n‐hexane, the partition coefficient remains approximately constant, but the selectivity coefficient for paclitaxel over cephalomannine improves to 4.5. This significant increase in selectivity early in the purification process has the potential to simplify downstream processing steps and significantly reduce overall purification costs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 990–997, 2012  相似文献   
143.

Background

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.

Methodology/Findings

The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.

Conclusions

We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.  相似文献   
144.
Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.  相似文献   
145.
146.
Predictive classification of major structural families and fold types of proteins is investigated deploying logistic regression. Only five to seven dimensional quantitative feature vector representations of tertiary structures are found adequate. Results for benchmark sample of non-homologous proteins from SCOP database are presented. Importance of this work as compared to homology modeling and best-known quantitative approaches is highlighted.  相似文献   
147.
Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlAm) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlAm), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlAm significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.  相似文献   
148.
We characterized the passive structural and active neuromuscular control of pharyngeal collapsibility in mice and hypothesized that pharyngeal collapsibility, which is elevated by anatomic loads, is reduced by active neuromuscular responses to airflow obstruction. To address this hypothesis, we examined the dynamic control of upper airway function in the isolated upper airway of anesthetized C57BL/6J mice. Pressures were lowered downstream and upstream to the upper airway to induce inspiratory airflow limitation and critical closure of the upper airway, respectively. After hyperventilating the mice to central apnea, we demonstrated a critical closing pressure (Pcrit) of -6.2 +/- 1.1 cmH(2)O under passive conditions that was unaltered by the state of lung inflation. After a period of central apnea, lower airway occlusion led to progressive increases in phasic genioglossal electromyographic activity (EMG(GG)), and in maximal inspiratory airflow (Vi(max)) through the isolated upper airway, particularly as the nasal pressure was lowered toward the passive Pcrit level. Moreover, the active Pcrit fell during inspiration by 8.2 +/- 1.4 cmH(2)O relative to the passive condition (P < 0.0005). We conclude that upper airway collapsibility (passive Pcrit) in the C57BL/6J mouse is similar to that in the anesthetized canine, feline, and sleeping human upper airway, and that collapsibility falls markedly under active conditions. Active EMG(GG) and Vi(max) responses dissociated at higher upstream pressure levels, suggesting a decrease in the mechanical efficiency of upper airway dilators. Our findings in mice imply that anatomic and neuromuscular factors interact dynamically to modulate upper airway function, and provide a novel approach to modeling the impact of genetic and environmental factors in inbred murine strains.  相似文献   
149.
Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 102–105 colony forming units g−1 fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 105 – 4.0 × 107 colony forming units g−1 tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.  相似文献   
150.
Citrus limonoids were demonstrated to possess potential biological activities in reducing the risk of certain diseases. Limonoids are present in citrus fruits in the form of aglycones and glucosides. At present, limonoid aglycones and limonoid glucosides are extracted in multiple steps using different solvents. In order to understand their potential bioactivity, it may be beneficial to isolate and purify these compounds using environment friendly methods. A new method of extraction and purification of limonoids was established using a hydrotrope polystyrene adsorbent resin. Extraction of aglycones and glucosides was achieved in a single step, using an aqueous solution of sodium cumene sulphonate (Na-CuS). Sour orange (Citrus aurantium L.) seed powder was extracted with 2 M Na-CuS solution at 45 degrees C for 6 h. The filtered extract was diluted with water and loaded on an SP 700 adsorbent column. The column was washed with distilled water to remove the hydrotrope and then eluted using water and methanol in different compositions to obtain three compounds. The structures of the isolated compounds were confirmed by NMR spectroscopy as deacetyl nomilinic acid glucoside (DNAG), deacetyl nomilin (DAN) and limonin (LIM).  相似文献   
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