Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K _m than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

Targeted Deletion of Fibrinogen Like Protein 1 Reveals a Novel Role in Energy Substrate Utilization

Fibrinogen like protein 1(Fgl1) is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.     

Absorptive root area and stem resistivity in whole trees of contrasting structure and size – improvement of methods

Aims

The study was focused on comparing the results of the three instrumental methods applied simultaneously for root studies in several tree species representing contrasting situations: root systems of different structure and stems of a wide range of diameters (especially when considering their resistivity). We want to learn properties of the methods, make some improvements and test their validity, before they will be applied to a large series of trees at the stand level.

Material and methods

Douglas fir (Pseudotsuga menziessii (Mirbel) Franco) with very asymmetric root system and Blue spruce (Picea pungens Engelm.) with homogeneous root system growing in the Mendel University Training Forest Enterprise in K?tiny, were selected as the main sample trees. Three variants of stem impedance measurements needed for absorptive root area estimates were applied to an additional series of over 20 trees. In order to characterize vertical and circumferential (around stem) root distribution we applied (1) the sap flow radial patterns measured by the multi-point sensors based on the heat field deformation (HFD) method, and (2) a modified earth impedance (MEI) method from the group of thermodynamic and electric measuring methods and finally we (3) almost harmlessly excavated the whole root system by supersonic air stream. Three steps of absorptive root area measurements were improved: (a) Impact of stem impedance was almost eliminated, (b) Excessive variation of stem impedance values measured too close to stems (in a place with the most heterogeneous materials) was compensated by extrapolation of several close points, (c) Impact of high curvature of small stems was determined and eliminated by an equation.

Results

All the methods gave similar results when considering differences between individual trees as well as between stem sides. Sap flow density was interesting when expressed per measured absorptive root area and leaf area. Experimental data of main and additional sample trees confirmed validity of relationship, which can be applied to improve stem resistivity especially in small trees.

Conclusions

Results indicated, that all the instrumental methods are field applicable and suitable for quantitative measurements, when specific properties of the methods and stem macrostructure are taken into account. Soil electric parameters characterize the important properties related to presence of cracks, water content, and ion concentration, which are being analyzed now.     

Characterization of the intestinal microbiome of Hirschsprung’s disease with and without enterocolitis

Hirschsprung’s disease (HD) is a congenital malformation of the gastrointestinal tract characterized by the absence of the distal enteric nervous system. Hirschsprung-associated enterocolitis (HAEC) is severe life threatening complication of HD. The disease pathogenesis is still unclear, but evidences suggest that the intestinal microbiota may play important role in the development of HD and HAEC. Because microbial abundance and diversity might differ in HD patients with enterocolitis, we sought to generate comparative metagenomic signatures to characterize the structure of the microbiome in HD patients with and without enterocolitis. Our experimental design is to enroll four HD patients (two with enterocolitis and two without enterocolitis). The microbiome was characterized by 16S rRNA gene, and the data obtained will be used to taxonomically classify and compare community structure among different samples. We found that the structure of the microbiome within HAEC patients are differ from those without enterocolitis. This study helps us to understand microbial contributions to the etiology of Hirschsprung-associated enterocolitis.     

Relationship between Auditory and Cognitive Abilities in Older Adults

Objective

The objective was to evaluate the association of peripheral and central hearing abilities with cognitive function in older adults.

Methods

Recruited from epidemiological studies of aging and cognition at the Rush Alzheimer’s Disease Center, participants were a community-dwelling cohort of older adults (range 63–98 years) without diagnosis of dementia. The cohort contained roughly equal numbers of Black (n=61) and White (n=63) subjects with groups similar in terms of age, gender, and years of education. Auditory abilities were measured with pure-tone audiometry, speech-in-noise perception, and discrimination thresholds for both static and dynamic spectral patterns. Cognitive performance was evaluated with a 12-test battery assessing episodic, semantic, and working memory, perceptual speed, and visuospatial abilities.

Results

Among the auditory measures, only the static and dynamic spectral-pattern discrimination thresholds were associated with cognitive performance in a regression model that included the demographic covariates race, age, gender, and years of education. Subsequent analysis indicated substantial shared variance among the covariates race and both measures of spectral-pattern discrimination in accounting for cognitive performance. Among cognitive measures, working memory and visuospatial abilities showed the strongest interrelationship to spectral-pattern discrimination performance.

Conclusions

For a cohort of older adults without diagnosis of dementia, neither hearing thresholds nor speech-in-noise ability showed significant association with a summary measure of global cognition. In contrast, the two auditory metrics of spectral-pattern discrimination ability significantly contributed to a regression model prediction of cognitive performance, demonstrating association of central auditory ability to cognitive status using auditory metrics that avoided the confounding effect of speech materials.     

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes

Chromosoma - In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered...     

mTOR�� Splicing Isoform Promotes Cell Proliferation and Tumorigenesis

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TORβ, which, in contrast to the full-length protein (mTORα), has the potential to regulate the G₁ phase of the cell cycle and to stimulate cell proliferation. mTORβ is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTORβ transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.