Modulation of proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal mice by granulocyte-macrophage colony-stimulating factor and IL-4: role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.     

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

Background  

The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins.     

Proteasome protease mediated regulation of cytokine induction and inflammation

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin-proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effects of acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin-induced diabetic rats

We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca²⁺ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca²⁺ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35–36°C. At 8–12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca²⁺ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca²⁺ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca²⁺ and altered mechanisms of sarcoplasmic reticulum Ca²⁺ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat. (Mol Cell Biochem 261: 227–233, 2004)     

Detection of Antibody against Fungal Glucosylceramide in Immunocompromised Patients: A Potential New Diagnostic Approach for Cryptococcosis

We have developed an ELISA to determine the value of anti-glucosylceramide antibody for the prediction of disseminated cryptococcosis in immunocompromised subjects and performed a clinical prospective study at the Medical University of South Carolina. The study enrolled a total of 53 patients who were free of active fungal diseases at the time of enrollment but at risk of developing one because they were all immunocompromised, e.g., (1) patients positive for HIV and (2) patients post- or awaiting solid organ transplantation. Among 53 patients enrolled, two patients developed invasive cryptococcosis, and in both patients, IgM anti-GlcCer was detected in sera using the ELISA at least 6 weeks prior to the clinical presentation of the brain disease. These results were corroborated by a cryptococcal antigen lateral flow assay, which was also positive in serum prior to the development of meningoencephalitis. However, a high number of positive results were also detected in patients with no evidence of cryptococcosis. This study highlights the potential utility of this new assay in early diagnostic testing algorithms for patients at risk for cryptococcosis, but further investigations are needed to validate the sensitivity and specificity of the glucosylceramide ELISA as a predictor of cryptococcosis.     

The effects of neuropeptide Y on skeletal muscle contractile properties in streptozotocin diabetic rats

Diabetes induces changes in the structural, biochemical, electrical, and contractile properties of skeletal muscles. Neuropeptide Y (NPY) administered locally can induce angiogenesis in a rat ischemic limb model and restore the contractile function of the ischemic muscle. The effects of NPY on the contractile characteristics of limb skeletal muscles were examined in streptozotocin-induced diabetic rats. Rats were treated with sham pellets (control groups) or NPY-containing pellets (1 mg of NPY/pellet, 14 days releasing time) administered locally to the rat hind limb 2 months after induction of diabetes. Contractile properties and fatigability of the slow-twitch soleus and fast-twitch gastrocnemius medials muscle were compared in control (sham), control NPY, diabetic (sham), and diabetic NPY groups. In order to induce fatigue trains of repetitive tetanic stimulation were used (600 ms/1 s simulation-rest cycle per train, 112 trains at an 85-Hz fusion frequency). Two months of untreated diabetes significantly prolonged soleus contraction and slowed its relaxation, but had minimal effects on soleus tension. NPY ameliorated the diabetic effects on soleus speed-related contractile properties, restoring its contraction and relaxation times. Diabetes significantly reduced gastrocnemius medials tetanic tension, leaving its contractile characteristics mostly unaffected. NPY partially restored gastrocnemius tetanic tension production capacity. Diabetes significantly increased fatigability of both muscles, which was partially restored by NPY, as evidenced by restored endurance of soleus muscle. The results suggest that NPY administered locally tends to normalize muscle performance and improve fatigue resistance of skeletal muscles in streptozotocin diabetes. Further examination is needed to establish the mechanisms of local NPY action on muscle contractile properties in streptozotocin-induced diabetes.     

On the fixation of genes of large effects due to continued truncation selection in small populations of polygenic systems with linkage

Summary The proportion of fixed loci for desirable genes and the time required for fixation is studied in simulated diploid populations, which have initially aHardy-Weinberg structure. A symmetric ten-locus system of additive or dominant genes is simulated with linkages between adjacent loci varying as .005, .05, or .5. A constant degree of upper truncation selection within a population is considered over the generations. In different populations the intensity of truncation is varied asN/N,N/N+2,N/N+4, ..., whereN is the parental population size, specified as 2,4,8 or 16. The selection differential in initial generation, , thereby varies from zero to more than two standard deviations in some cases. The initial mean gene frequency,p, simulated in an initial population is .1 or .5.It is pointed out that when selective advantage of a gene is large and is changing with gene frequency, diffusion approximations assuming constant selective advantage, gives higher values for proportion of fixed genes in the case ofp equal to .1 and lower values forp equal to .5. With parental population size of 16 or less, a relation withN alone does not give the proportion of fixed genes. Higher order terms ofN appear to be involved in the relation. For the sameN , the proportion is much higher for lowN.The depressing effect of low recombinations between loci is of different magnitude for differentN andp for a givenN . The increase in the proportion of fixed genes due to increasingN is not as large when is low. High intensity of selection offsets considerably the effects of population size and linkage when gene effects are large. It appears that with increased inbreeding and selection intensity, almost all the genes of large effects and at intermediate frequencies can be rapidly fixed regardless of linkage.Linkage has been shown to cause faster fixation of genes in the absence of selection. With selection, linkage tends to delay fixation. But in the case of very low recombinations, there appears to be a level of population size and selection intensity, below which there is more rapid fixation because of linkage. Selection for dominant genes in the case of very close linkage, delays fixation for a number of generations and this delay results in reducing the depressing effect of linkage.

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Zusammenfassung Der Anteil fixierter Loci für erwünschte Gene und die für die Fixierung erforderliche Zeit werden in einer simulierten diploiden Population untersucht, wobei eine ursprünglicheHardy-Weinberg-Struktur angenommen wird. Es wird ein symmetrisches 10-Locus-System von additiven oder dominanten Genen mit Koppelung zwischen benachbarten Loci, die von 0,005 über 0,05 bis zu 0,5 variiert wird, simuliert. Hierbei wird ein konstantes Ausmaß von trunkierender (stutzender) Selektion für die Obergrenze der Verteilung in der Population betrachtet. In verschiedenen Populationen wird die Intensität der Verteilungsstutzung variiert in der folgenden FormN/N,N/N+2,N/N+4, ..., wobeiN die elterliche Populationsgröße ist, die mit 2,4,8 oder 16 spezifiziert wird. Das Selektionsdifferential der Ursprungsgeneration,i, variiert hierbei in einigen Fällen von 0 bis auf mehr als 2 Standardabweichungen. Die ursprüngliche mittlege Genfrequenz,p, die in einer Ausgangspopulation simuliert wird, ist 0,1 oder 0,5.Es wird gezeigt, daß, im Vergleich zu großem selektivem Vorteil eines Gens und frequenzabhängiger Änderung des Selektionskoeffizienten, Diffusionsnäherungen, die konstante selektive Vorteile voraussetzen, höhere Werte für den Anteil fixierter Gene im Fallp=0,1 und niedrigere Werte fürp=0,5 ergeben. Mit einer elterlichen Population der Größe 16 oder kleiner ergibt die BeziehungNi allein nicht den Anteil fixierter Gene, da Termini höherer Ordnung vonNi in die Bezichung einbezogen sind. Bei gleichemNi ist der Anteil bei kleinemN viel höher. Der reduzierende Effekt einer niederen Rekombinationsrate zwischen den Loci ist von unterschiedlicher Größenordnung bei verschiedenemN und bei einem gegebenenNi. Der Zuwachs im Anteil fixierter Gene infolge eines wachsendenN ist nicht so groß, wennp niedrig ist. Eine hohe Intensität der Selektion gleicht die Wirkungen der Populationsgröße und Koppelung erheblich aus, wenn die Genwirkungen groß sind. Es zeigt sich, daß praktisch alle Gene mit großer Wirkung und intermediärer Frequenz unabhängig von der Koppelung schnell fixiert werden können, wenn eine zunehmende Inzucht und Selektionsintensität vorliegt.Koppelung hat sich als eine Ursache für eine schnellere Fixierung von Genen in der Abwesenheit von Selektion erwiesen. Mit Selektion tendiert Kopplung dazu, die Fixierung zu verzögern. Es zeigt sich jedoch im Falle einer sehr niederen Rekombinationsrate, daß es für die Populationsgröße und Selektionsintensität einen Schwellenwert zu geben scheint, unterhalb dessen eine schnellere Fixierung als Folge der Koppelung auftritt. Eine Selektion auf dominante Gene verzögert im Fall der sehr engen Koppelung die Fixierung für eine Anzahl von Generationen und diese Verzögerung führt dazu, daß der verlangsamende Effekt der Koppelung reduziert wird.

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On leave from West Pakistan Agricultural University, Lyallpur.

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Journal paper No. 5870, Iowa Agriculture and Home Economics Experiment Station, Ames, supported by National Institute of Health Grant No. GM 13827.     

Prioritizing Tiger Conservation through Landscape Genetics and Habitat Linkages

Even with global support for tiger (Panthera tigris) conservation their survival is threatened by poaching, habitat loss and isolation. Currently about 3,000 wild tigers persist in small fragmented populations within seven percent of their historic range. Identifying and securing habitat linkages that connect source populations for maintaining landscape-level gene flow is an important long-term conservation strategy for endangered carnivores. However, habitat corridors that link regional tiger populations are often lost to development projects due to lack of objective evidence on their importance. Here, we use individual based genetic analysis in combination with landscape permeability models to identify and prioritize movement corridors across seven tiger populations within the Central Indian Landscape. By using a panel of 11 microsatellites we identified 169 individual tigers from 587 scat and 17 tissue samples. We detected four genetic clusters within Central India with limited gene flow among three of them. Bayesian and likelihood analyses identified 17 tigers as having recent immigrant ancestry. Spatially explicit tiger occupancy obtained from extensive landscape-scale surveys across 76,913 km² of forest habitat was found to be only 21,290 km². After accounting for detection bias, the covariates that best explained tiger occupancy were large, remote, dense forest patches; large ungulate abundance, and low human footprint. We used tiger occupancy probability to parameterize habitat permeability for modeling habitat linkages using least-cost and circuit theory pathway analyses. Pairwise genetic differences (F_ST) between populations were better explained by modeled linkage costs (r>0.5, p<0.05) compared to Euclidean distances, which was in consonance with observed habitat fragmentation. The results of our study highlight that many corridors may still be functional as there is evidence of contemporary migration. Conservation efforts should provide legal status to corridors, use smart green infrastructure to mitigate development impacts, and restore habitats where connectivity has been lost.