Screening for enzyme activity in turbid suspensions with scattered light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.     

Dynamic fibroblast cytoskeletal response to subcutaneous tissue stretch ex vivo and in vivo

Cytoskeleton-dependent changes in cell shape are well-established factors regulating a wide range of cellular functions including signal transduction, gene expression, and matrix adhesion. Although the importance of mechanical forces on cell shape and function is well established in cultured cells, very little is known about these effects in whole tissues or in vivo. In this study we used ex vivo and in vivo models to investigate the effect of tissue stretch on mouse subcutaneous tissue fibroblast morphology. Tissue stretch ex vivo (average 25% tissue elongation from 10 min to 2 h) caused a significant time-dependent increase in fibroblast cell body perimeter and cross-sectional area (ANOVA, P < 0.01). At 2 h, mean fibroblast cell body cross-sectional area was 201% greater in stretched than in unstretched tissue. Fibroblasts in stretched tissue had larger, "sheetlike" cell bodies with shorter processes. In contrast, fibroblasts in unstretched tissue had a "dendritic" morphology with smaller, more globular cell bodies and longer processes. Tissue stretch in vivo for 30 min had effects that paralleled those ex vivo. Stretch-induced cell body expansion ex vivo was inhibited by colchicine and cytochalasin D. The dynamic, cytoskeleton-dependent responses of fibroblasts to changes in tissue length demonstrated in this study have important implications for our understanding of normal movement and posture, as well as therapies using mechanical stimulation of connective tissue including physical therapy, massage, and acupuncture. mechanotransduction; connective tissue; tensegrity; musculoskeletal manipulations; acupuncture     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Ontogeny of the Neutral Amino Acid Transporter SNAT1 in the Developing Rat

Summary System A is a highly regulated, Na⁺-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.     

Selective foraging of fungi by collembolans in soil

Soils contain highly diverse communities of microorganisms and invertebrates. The trophic interactions between these species are largely unknown. Collembolans form an abundant part of the invertebrate community in soils. A prevailing view is that soil collembolans are generalist feeders on fungi, lichens, fragmented litter and bacteria. However, in laboratory food choice experiments, it has been shown that collembolans preferentially select certain taxa of fungi. To examine this apparent contradiction, we developed a molecular technique based on the analysis of 18S ribosomal DNA (rDNA) sequences to explore the diversity of fungi in soils and in the guts of collembolans. We report that the diversity of fungi found in the natural soil was 33 times higher than that in the guts of the collembolan Protaphorura armata. The data support the view that collembolan species can be highly selective when foraging on fungi in soils.     

Variogram analysis of the spatial genetic structure of continuous populations using multilocus microsatellite data

A geostatistical perspective on spatial genetic structure may explain methodological issues of quantifying spatial genetic structure and suggest new approaches to addressing them. We use a variogram approach to (i) derive a spatial partitioning of molecular variance, gene diversity, and genotypic diversity for microsatellite data under the infinite allele model (IAM) and the stepwise mutation model (SMM), (ii) develop a weighting of sampling units to reflect ploidy levels or multiple sampling of genets, and (iii) show how variograms summarize the spatial genetic structure within a population under isolation-by-distance. The methods are illustrated with data from a population of the epiphytic lichen Lobaria pulmonaria, using six microsatellite markers. Variogram-based analysis not only avoids bias due to the underestimation of population variance in the presence of spatial autocorrelation, but also provides estimates of population genetic diversity and the degree and extent of spatial genetic structure accounting for autocorrelation.     

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Mutation of the binding site for Cbl (Tyr1045) in the EGF receptor (EGFR) results in impaired ubiquitination but does not affect EGFR internalization. However, the Y1045F mutation resulted in strongly decreased degradation of the EGFR, as well as efficient recycling of EGFR to the plasma membrane. Significantly, more wild-type EGFR than Y1045F EGFR was found localizing to multivesicular late endosomes. Ubiquitination of the EGFR was in HeLa cells inhibited both upon overexpressing the N-terminal part of Cbl and upon overexpressing a double mutant Grb2 incapable of interacting with Cbl and thereby being incapable of indirectly recruiting Cbl to the EGFR. Collectively, these data suggest that the ubiquitination resulting from direct binding of Cbl to pTyr1045 of the EGFR is critical for lysosomal sorting of the EGFR in contrast to ubiquitination resulting from Grb2-mediated binding of Cbl to the EGFR.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.