Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI₂ inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID₅₀) at 10 nM. 6_β-PGI₁ also inhibited the action of histamine (ID₅₀ 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6_β-PGI₁ inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6_β-PGI₁ stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.     

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non‐livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1–C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7‐C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection.     

Modular evolution and the origins of symmetry: reconstruction of a three-fold symmetric globular protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.     

Age-related diffuse chronic telogen effluvium- type alopecia in female squirrel monkeys (Saimiri boliviensis boliviensis)

We investigated the diffuse alopecia affecting some female squirrel monkeys (Saimiri boliviensis boliviensis) housed in a breeding facility. We randomly selected 100 female and 10 male animals and performed a complete physical exam and a hair assessment on all animals; blood tests, trichograms, hair density; and skin biopsies in representative cases; and a dominance behavioral assessment of 50 animals. Hair coat was normal in 35 female monkeys and all 10 male animals. Of the 65 females with diffuse alopecia, 17 had mild, 22 moderate, and 26 severe hair loss. The alopecia group had a mean age of 9.6 +/- 0.6 years, whereas that of the normal group was 4.7 +/- 0.6 years (P < 0.05). The parity in the alopecia group was 4.2 +/- 0.6 but 2.0 +/- 0.6 (P < 0.05) in the normal group. There were no statistically significant differences in body weight, hemoglobin, blood urea nitrogen, serum glucose, liver aspartate aminotransaminase, or free thyroxine. The trichogram demonstrated 20.8% +/- 1.6% (mean +/- standard error) of telogen hairs in the alopecia group compared with 9.5% +/- 2.8% of the control group (P < 0.05). The hair density in the alopecia group was 52.8 +/- 4.1/cm2 and 79.6 +/- 14.3/cm2 in the control group. Skin biopsies in affected monkeys demonstrated increased telogen follicles, with no fibrosis or inflammation. There were no statistically significant differences in the dominance behavioral analysis. The findings are consistent with chronic telogen effluvium (CTE). A number of organic, behavioral, and dominance-related stress causes of CTE were excluded. CTE appears to be predominantly age-related in this population. CTE in female squirrel monkeys may serve as an animal model for human diffuse alopecia.     

Dynamic Disturbance Processes Create Dynamic Lek Site Selection in a Prairie Grouse

It is well understood that landscape processes can affect habitat selection patterns, movements, and species persistence. These selection patterns may be altered or even eliminated as a result of changes in disturbance regimes and a concomitant management focus on uniform, moderate disturbance across landscapes. To assess how restored landscape heterogeneity influences habitat selection patterns, we examined 21 years (1991, 1993–2012) of Greater Prairie-Chicken (Tympanuchus cupido) lek location data in tallgrass prairie with restored fire and grazing processes. Our study took place at The Nature Conservancy’s Tallgrass Prairie Preserve located at the southern extent of Flint Hills in northeastern Oklahoma. We specifically addressed stability of lek locations in the context of the fire-grazing interaction, and the environmental factors influencing lek locations. We found that lek locations were dynamic in a landscape with interacting fire and grazing. While previous conservation efforts have treated leks as stable with high site fidelity in static landscapes, a majority of lek locations in our study (i.e., 65%) moved by nearly one kilometer on an annual basis in this dynamic setting. Lek sites were in elevated areas with low tree cover and low road density. Additionally, lek site selection was influenced by an interaction of fire and patch edge, indicating that in recently burned patches, leks were located near patch edges. These results suggest that dynamic and interactive processes such as fire and grazing that restore heterogeneity to grasslands do influence habitat selection patterns in prairie grouse, a phenomenon that is likely to apply throughout the Greater Prairie-Chicken’s distribution when dynamic processes are restored. As conservation moves toward restoring dynamic historic disturbance patterns, it will be important that siting and planning of anthropogenic structures (e.g., wind energy, oil and gas) and management plans not view lek locations as static points, but rather as sites that shift around the landscape in response to shifting vegetation structure. Acknowledging shifting lek locations in these landscapes will help ensure conservation efforts are successful by targeting the appropriate areas for protection and management.     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.     

Evidence that residues −15 to −46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator

We previously identified three well-dispersed mutations, E⁹⁷⁸-K, F⁹⁸⁹-L and D¹⁰⁰⁹-R within the haemolysin A signal region, located at positions –46, –35 and –15, with respect to the C-terminus, respectively. Each mutation reduces the efficiency of secretion two- to threefold leaving 30–45% of the wild-type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild-type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild-type HlyA co-expressed In the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3′-end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HiyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition for steps     

Evaluation of the involvement of nitric oxide and substance P in reducing baroreflex gain in the genetically hypertensive (GH) rat

The attenuation of baroreflex gain associated with hereditary hypertension could involve abnormal signalling by nitric oxide or substance P. Baroreflex gain was measured in age-matched male genetically hypertensive (GH) and nonnotensive (N) anaesthetised rats from heart rate changes in response to i.v. phenylephrine or sodium nitroprusside. In subgroups of these animals, nitric oxide synthesis was inhibited using NG-nitro-L-arginine methyl ester (L-NAME, 30 mg x kg(-1) i.v.), substance P transmission was blocked using the antagonist SR 140333 (360 nmoles x kg(-1) i.v.) or substance P release was inhibited with resiniferatoxin (4 doses of 0.3 microg x kg(-1) i.v. at 4 min intervals). Baroreflex gain was markedly reduced in GH compared to N animals (N -0.37 +/- 0.04 beat x min(-1) x mm Hg(-1), GH -0.17 +/- 0.02 beat x min(-1) x mm Hg(-1), p < 0.0001). Inhibition of nitric oxide synthase increased baroreflex gain in each strain, but the inter-strain difference in gain persisted (post-treatment N -0.57 +/- 0.07 beat x min(-1) x mm Hg(-1), GH -0.24 +/- 0.05 beat x min(-1) x mm Hg(-1) (p < 0.001). Blockade of receptors or inhibition of substance P release did not affect gain in either strain. Nitric oxide, but not substance P, appears to play an inhibitory role in the rat arterial baroreflex. Impairment of baroreflex gain in GH rats is not secondary to altered nitric oxide signaling.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.