Immune responses in hibernating little brown myotis (Myotis lucifugus) with white-nose syndrome.

White-nose syndrome (WNS) is a fungal disease responsible for decimating many bat populations in North America. Pseudogymnoascus destructans (Pd), the psychrophilic fungus responsible for WNS, prospers in the winter habitat of many hibernating bat species. The immune response that Pd elicits in bats is not yet fully understood; antibodies are produced in response to infection by Pd, but they may not be protective and indeed may be harmful. To understand how bats respond to infection during hibernation, we studied the effect of Pd inoculation on the survival and gene expression of captive hibernating Myotis lucifugus with varying pre-hibernation antifungal antibody titres. We investigated gene expression through the transcription of selected cytokine genes (Il6, Il17a, Il1b, Il4 and Ifng) associated with inflammatory, Th1, Th2 and Th17 immune responses in wing tissue and lymph nodes. We found no difference in survival between bats with low and high anti-Pd titres, although anti-Pd antibody production during hibernation differed significantly between infected and uninfected bats. Transcription of Il6 and Il17a was higher in the lymph nodes of infected bats compared with uninfected bats. Increased transcription of these cytokines in the lymph node suggests that a pro-inflammatory immune response to WNS is not restricted to infected tissues and occurs during hibernation. The resulting Th17 response may be protective in euthermic bats, but because it may disrupt torpor, it could be detrimental during hibernation.

Micro-CT evaluation of rheumatoid arthritis mouse model disease progression: Manual tracings versus semi-automated routines.

PURPOSE: The primary goal of this study was to demonstrate the value of micro-CT imaging in a rheumatoid arthritis (RA) mouse model. The secondary goal was to assess whether manual correction of the articular surface regions of interest (ROI) identification of the semi-automated methods may result in more effective assessment of bone volume and density loss. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) was induced in six DBA/1J mice at 12 weeks of age and three other DBA/1J identical mice served as controls. Micro-CT images were acquired at baseline and at four, seven, and nine weeks post-induction. Disease was monitored via ROI analysis, and ROIs were first generated using semi-automated techniques. These ROIs were manually manipulated so that a variety of edge irregularities were corrected. Effort was focused on the proximal and distal humerus and the distal femur. ROI volume and density were calculated, and data were compared. A histologic analysis of the study mice was also performed after the last time frame. RESULTS: There was a significant difference between the volume data comparison between the manually manipulated data and the semi-automated routine data across all time frames and across both humeri and femurs. There was no significant difference in densities calculated in Hounsfield units across any of the time frames, humeri or femurs, except for one time frame. CONCLUSION: Our findings suggest that the manual correction technique of semi-automated data can be used to quantify and evaluate bone volume, density, and joint surface architecture changes in a RA mouse model.

A high throughput in vitro analytical approach to screen for oxidative stress potential exerted by nanomaterials using a biologically relevant matrix: human blood serum.

Limited studies have shown that selected nanomaterials (NMs) impart various forms of toxicity in biological systems; however, a common metric to screen for potential toxicity is needed. This study optimized and utilized a 'Ferric reducing ability of serum (FRAS)' assay as a screening tool to quantitate the degree of oxidative damage induced by NMs on human blood serum. Antioxidants in blood protect against oxidative damage caused by free radicals via chemical quenching and will decrease when exposed to oxidatively stressful materials. Using this approach, the antioxidant capacity of NM treated serum was significantly decreased by nano-silver, a series of nano-carbon blacks, fullerene soot, and nano-TiO(2) (anatase, p<0.05), but not with nano-alumina, fullerite, purified fullerene, fine TiO(2) (rutile) and Min-U-Sil 5. Particle surface area and not biological particle size was highly associated with the degree of oxidative stress observed. This approach appears responsive to multiple determinants of oxidative damage, including particle chemistry, surface area and impurities, and may be a valid screening method to determine oxidative damage imparted by nanomaterials.

Apple juice prevents oxidative stress and impaired cognitive performance caused by genetic and dietary deficiencies in mice.

Increased oxidative stress contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer s disease. Dietary supplementation with fruits and vegetables that are high in antioxidant potential have in some cases compensated for dietary and/or genetic deficiencies that promote increased oxidative stress. Herein, we demonstrate that apple juice concentrate, administered ad libitum in drinking water, can compensate for the increased reactive oxygen species and decline in cognitive performance in maze trials observed when normal and transgenic mice lacking apolipoprotein E are deprived of folate and vitamin E. In addition, we demonstrate that this protective effect is not derived from the sugar content of the concentrate.

Integrated analysis of sequence evolution and population history using hypervariable compound haplotypes.

We have examined compound haplotypes from a highly informative region of human chromosome 16, in which information from the rapid evolution of a highly unstable minisatellite is integrated with data on the longer-term evolution of this segment from 10 flanking substitutional polymorphisms. Combined with sequence data from non-human primates, analysis of relationships between these compound haplotypes allows the reconstruction of a rooted network of the evolutionary pathways between them. Most relationships can be explained via simple substitutional mutations, although the origins of some haplotypes involve recurrent events at a hotspot for substitutional mutation and/or gene conversion. For compound haplotypes including the minisatellite array, the network found in a range of world-wide populations constitutes a highly informative data set for the analysis of population history (437 different compound haplotypes were discriminated among 658 studied). Since the mutation rates and processes of the minisatellite array are known from direct studies, ages for individual lineages have been estimated using associated minisatellite diversity. These analyses suggest that the higher information content and sampling depth of these compound haplotypes may allow more precise calibration of lineage ages than is possible using coalescent analysis of DNA sequence. Using this method we have dated the oldest Eurasian lineage as 52,000-66,000 years and the oldest European specific lineage as 37,600-56,200 years.

Studies of cholesterol and bile acid metabolism, and early atherogenesis in hamsters fed GT16-239, a novel bile acid sequestrant (BAS).

The purpose of this study was to compare the efficacy of GT16-239, an alkylated, cross-linked poly(allylamine) bile acid sequestrant with cholestyramine on cholesterol and bile acid metabolism, and early aortic atherosclerosis in hypercholesterolemic male F1B Golden Syrian hamsters. In this controlled study, 42 hamsters were divided into six groups and were fed a chow-based hypercholesterolemic diet supplemented with a 10% oil blend (55% coconut/45% corn), 0.1% cholesterol (w/w) (control) and either 0.9 or 1.2% cholestyramine or 0.2, 0.4 or 0.6% GT16-239 for 13 weeks. Laboratory analyses included evaluating plasma lipoprotein cholesterol and triglyceride concentrations, hepatic HMG-CoA reductase and 7 alpha-hydroxylase activities, fecal excretion of bile acids and neutral sterols, hepatic cholesterol concentrations, and early atherosclerosis (aortic fatty streak area). Relative to the control diet, the 0.6% GT16-239 versus the 1.2% cholestyramine significantly inhibited the elevation of plasma lipoprotein total cholesterol (TC) (-69% vs -40%), high density lipoprotein-cholesterol (HDL-C) (-49% vs -30%), and non-HDL-C (-81 vs -48%) concentrations; increased the activities of both HMG-CoA reductase (1492% vs 62%) and 7 alpha-hydroxylase (175% vs 86%); lowered the concentration of hepatic cholesteryl ester (-94% vs -59%); increased fecal cholesterol concentration (+28% vs -10%); and decreased aortic fatty streak area (-100% vs -86%). Unexpected findings of this comparison were increased fecal concentrations of cholic acid (533%) and chenodeoxycholic acid (400%) and the reduction in lithocholic acid (-50%) in the 0.6% GT16-239 compared to the 1.2% cholestyramine group. In summary, GT16-239 had a greater impact on cholesterol metabolism and early atherosclerosis in hypercholesterolemic hamsters than cholestyramine.

Effects of specific fatty acids (8:0, 14:0, cis-18:1, trans-18:1) on plasma lipoproteins, early atherogenic potential, and LDL oxidative properties in the hamster.

Although comparative studies of the cholesterolemic properties of trans fatty acids relative to cis-unsaturates and saturates have been conducted in humans and animals, there is no recent information relating these lipid responses to susceptibility to atherosclerosis. Therefore, hamsters were fed diets containing equivalent amounts of cholesterol (0.12% wt/wt) and test fats (20% wt/wt) for 8 weeks. Each test fat contained between 50-52% of the-total triacylglycerols as a single fatty acid, i.e., 8:0, 14:0, 18:0, cis-18:1, or trans-18:1 while the balance consisted of 16:0, cis-18:1 and 18:2 that were the same for all groups. Plasma total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) levels were not different for 8:0, cis-18:1, and trans-18:1, whereas 14:0 caused a significant rise in plasma TC, LDL-C, and HDL-C. LDL oxidation measurements showed that the lag phase of conjugated diene formation was longest for the trans-18:1 and cis-18:1 groups while rate of conjugated diene formation was lowest for the trans-18:1 and cis-18:1 groups. The trans-18:1- and cis-18:1-fed animals had significantly higher levels of LDL alpha-tocopherol relative to the 8:0- and 14:0-fed animals. Aortic fatty streak formation was highest for the 14:0- and 8:0-fed animals and lowest for the trans-18:1. In conclusion, the plasma lipid and antioxidant properties of trans-18:1 and cis-18:1 were comparable while the trans-18:1-fed hamsters had the least amount of early atherosclerosis. In addition, 8:0-fed animals unexpectedly had early atherosclerosis formation similar to the 14:0-fed animals.

Suppression of TGA mutations in the Bacillus subtilis spoIIR gene by prfB mutations.

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.

Regulation of plasma lipoprotein levels by dietary triglycerides enriched with different fatty acids.

Saturated vegetable oils (coconut, palm, and palm kernel oil) containing predominantly saturated fatty acids, lauric (12:0) or myristic (14:0 and palmitic (16:0), raise plasma total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in animals and humans, presumably by decreasing LDL receptor activity and/or increasing LDL-C production rate. Although stearic acid (18:0) is chemically a saturated fatty acid, both human and animal studies suggest it is biologically neutral (neither raising nor lowering) blood cholesterol levels. Although earlier studies indicated that medium chain fatty acids (8:0-10:0) were also thought to be neutral, more recent studies in animals and humans suggest otherwise. Unsaturated vegetable oils such as corn, soybean, olive, and canola oil, by virtue of their predominant levels of either linoleic acid (18:2) or oleic acid (18:1), are hypocholesterolemic, probably as a result of their ability to upregulate LDL receptor activity and/or decrease LDL-C production rate. Whether trans fatty acids such as trans oleate (t18:1), in hydrogenated products such as margarine, are hypercholesterolemic remains controversial. Studies in humans suggest that their cholesterol-raising potential falls between the native nonhydrogenated vegetable oil and the more saturated dairy products such as butter. Assessment of the magnitude of the cholesterolemic response of trans 18:1 is difficult because in most diet studies its addition is often at the expense of cholesterol-lowering unsaturated fatty acids, making an independent evaluation almost impossible.

Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters.

Conjugated linoleic acid is a collective term used to designate a mixture of positional and geometric isomers of linoleic acid in which the double bonds are conjugated. Unlike linoleic acid, there is a paucity of information regarding the effect of dietary conjugated linoleic acid on plasma lipoproteins and aortic atherosclerosis. Therefore, fifty hamsters were divided into five groups of ten and fed 0 (Control), 0.06 (LOW), 0.11 (MEDIUM), and 1.1 (HIGH) en% conjugated linoleic acid or 1.1 en% linoleic acid. Blood samples were taken at 4, 8 and 11 weeks for plasma lipid analyses and for plasma tocopherol assay at sacrifice. Animals fed the conjugated linoleic acid-containing diets collectively had significantly reduced levels of plasma total cholesterol, non-high density lipoprotein cholesterol, (combined very low and low density lipoprotein) and triglycerides with no effect on high density lipoprotein cholesterol, as compared to CONTROLs. Linoleic acid-fed animals relative to CONTROLs also had reduced plasma total cholesterol, non-high density lipoprotein cholesterol and triglycerides, but only the latter was statistically significant. Compared to the CONTROL group, plasma tocopherol/total cholesterol ratios determined from plasma pools for the LOW, MEDIUM and HIGH conjugated linoleic acid and linoleic acid groups were increased by 48%, 48%, 86% and 29%, respectively, suggesting a tocopherol-sparing effect, at least for the conjugated linoleic acid treatment. Morphometric analysis of aortas revealed less early atherosclerosis in the conjugated linoleic acid and linoleic acid-fed hamsters compared to the CONTROL group.

Ribosome regulation by the nascent peptide.

Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene. The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome. The peptides are therefore cis-acting negative regulators of translation. The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome. A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria. Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA. Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions. Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation. Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection. Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA. uORFs associated with several eukaryotic genes inhibit downstream translation. When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon. This sets up a blockade to ribosome scanning which minimizes downstream translation. Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the known examples the active amino acid sequences function after their emergence from the ribosome, cis control of translation by the nascent peptide is gene specific; nearly all such regulatory peptides exert no obvious trans effects in cells. The in vitro biochemical activities of the cat/cmla leader peptides on ribosomes and rRNA suggest a mechanism through which the nascent peptide can modify ribosome behavior. Other cis-acting regulatory peptides may involve more complex ribosomal interactions.

Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.

Anti-peptidyl transferase leader peptides of attenuation-regulated chloramphenicol-resistance genes.

The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose the inducer, Cm, converts a peptide-paused ribosome to the stalled state. We discuss the idea that cooperativity between leader peptide and inducer is necessary for ribosome stalling and may link the activation of a specific drug-resistance gene with a particular antibiotic.

The cis-effect of a nascent peptide on its translating ribosome: influence of the cat-86 leader pentapeptide on translation termination at leader codon 6.

Inducible cat genes from Gram-positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabilizes a downstream stem-loop structure that normally sequesters the ribosome-binding site for the cat structural gene. The five-amino-acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase in vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis-effect on its translating ribosome in vivo. Converting leader codon 6 to the ochre codon results in expression of cat-86 in the absence of inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino-acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat-86 leader pentapeptide can alter the function of its translating ribosome.

A comparison of serum retinol concentration between human and different species of normo and hypercholesterolemic nonhuman primates fed semi-purified diets with defined amounts of vitamin A.

1. Serum retinol and total cholesterol concentrations were determined in several species of nonhuman primates fed semipurified diets. Two species of Old World and three species of New World nonhuman primates were examined. 2. Retinol levels were significantly lower (up to four-fold) in the serum of the smaller New World than the larger Old World animals and the difference could not be explained by differences in dietary make-up. 3. Cholesterol levels were not different between the groups but differed within a species when type of dietary fat was altered. 4. Differences in circulating levels of retinol may reflect differences in levels of retinol binding protein between the groups.

Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene.

The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer.

Parallel induction strategies for cat-86: separating chloramphenicol induction from protein synthesis inhibition.

Induction of cat-86 translation results from the stalling of a ribosome at a discrete location in the leader region of the transcript. Stalling destabilizes an adjacent region of secondary structure that sequesters the cat-86 ribosome binding site, thereby activating cat-86 translation. Two well characterized antibiotics, chloramphenicol and erythromycin, induce cat-86 by stalling a ribosome at the appropriate leader site. Here we demonstrate differences between the two antibiotics with respect to induction. First, induction by chloramphenicol is dependent on nucleotides in the leader sequence that are different from those necessary for erythromycin induction. Second, variants of Bacillus subtilis that are chloramphenicol resistant because of chromosome mutations permit cat-86 induction by chloramphenicol, whereas erythromycin-resistance host mutations block or greatly reduce cat-86 induction by erythromycin. Third, selected strains of B. subtilis bearing alterations in proteins of the 50S ribosomal subunit interfere with cat-86 induction by chloramphenicol, yet these strains are chloramphenicol sensitive. Lastly, induction by chloramphenicol is not reversed by removal of the antibiotic whereas erythromycin induction is reversible. The data indicate that chloramphenicol induction results from an effect of the drug that is not identical to its role as a general inhibitor of ribosome elongation. Induction by erythromycin, on the other hand, could not be distinguished from its antibiotic activity.

Evaluation of seven Cholestech L.D.X analyzers for total cholesterol determinations.

We assessed the performance of seven Cholestech L.D.X lipid analyzers under tightly controlled laboratory conditions for accuracy and precision in accordance with analytical guidelines of the National Cholesterol Education Program (NCEP). Venous heparinized whole blood (VB) and plasma (VP), venous serum (VS), and capillary fingerstick whole blood (FB) were collected from 18 individuals. Total cholesterol (TC) concentration was measured in VB, VP, and VS on all seven instruments. Three instruments were used for TC measurements of FB. Reference cholesterol values for each individual were generated in the same laboratory with a standardized method. The within-run coefficients of variation (CVs) for all instruments with a Level I pool (1560 mg/L, n = 10) ranged from 1.3% to 1.8% (mean = 1.59%). The between-run CVs with the same pool ranged from 2.2% to 3.4% (mean = 2.84%, n = 10). Correlation coefficients derived from comparison of total cholesterol values generated by the instruments for each specimen type vs the reference cholesterol values were all > 0.97. The average bias for all instruments for each sample type was 1.9% (FB), 4.3% (VB), 6.6% (VP), and 7.0% (VS). Predicted cholesterol concentration for each sample type from regression curves for total cholesterol at the suggested NCEP clinical decision cutoff values of 2000 and 2400 mg/L, respectively, were 2049 and 2431 mg/L for FB, 2081 and 2469 mg/L for VB, 2122 and 2522 mg/L for VP, and 2121 and 2521 mg/L for VS.

Mosquitoes (Diptera: Culicidae) captured in the Iquitos area of Peru.

A mosquito capture program was initiated to study mosquito species and their potential for arboviral transmission in the Peruvian Amazon. More than 35,000 mosquitoes of 13 different genera and at least 25 species were captured in urban and sylvan sites in the Iquitos area. These findings represent the first published list of Peruvian mosquitoes since 1971 and the first such list from the Peruvian Amazon.