Modeling the inhibitor activity and relative binding affinities in enzyme-inhibitor-protein systems: application to developmental regulation in a PG-PGIP system

Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Blending chitosan with polycaprolactone: effects on physicochemical and antibacterial properties

Chitosan is a well sought-after polysaccharide in biomedical applications and has been blended with various macromolecules to mitigate undesirable properties. However, the effects of blending on the unique antibacterial activity of chitosan as well as changes in fatigue and degradation properties are not well understood. The aim of this work was to evaluate the anti-bacterial properties and changes in physicochemical properties of chitosan upon blending with synthetic polyester poly(epsilon-caprolactone) (PCL). Chitosan and PCL were homogeneously dissolved in varying mass ratios in a unique 77% acetic acid in water mixture and processed into uniform membranes. When subjected to uniaxial cyclical loading in wet conditions, these membranes sustained 10 cycles of predetermined loads up to 1 MPa without break. Chitosan was anti-adhesive to Gram-positive Streptococcus mutans and Gram-negative Actinobacillus actinomycetemcomitans bacteria. Presence of PCL compromised the antibacterial property of chitosan. Four-week degradation studies in PBS/lysozyme at 37 degrees C showed initial weight loss due to chitosan after which no significant changes were observed. Molecular interactions between chitosan and PCL were investigated using Fourier transform infrared spectroscopy (FTIR) which showed no chemical bond formations in the prepared blends. Investigation by wide-angle X-ray diffraction (WAXD) indicated that the crystal structure of individual polymers was unchanged in the blends. Dynamic mechanical and thermal analysis (DMTA) indicated that the crystallinity of PCL was suppressed and its storage modulus increased with the addition of chitosan. Analysis of surface topography by atomic force microscopy (AFM) showed a significant increase in roughness of all blends relative to chitosan. Observed differences in biological and anti-bacterial properties of blends could be primarily attributed to surface topographical changes.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Dynamics of diffusivity and pressure drop in flow-through and parallel-flow bioreactors during tissue regeneration

In this study, transport characteristics in flow-through and parallel-flow bioreactors used in tissue engineering were simulated using computational fluid dynamics. To study nutrient distribution and consumption by smooth muscle cells colonizing the 100 mm diameter and 2-mm thick scaffold, effective diffusivity of glucose was experimentally determined using a two-chambered setup. Three different concentrations of chitosan-gelatin scaffolds were prepared by freezing at -80°C followed by lyophilization. Experiments were performed in both bioreactors to measure pressure drop at different flow rates. At low flow rates, experimental results were in agreement with the simulation results for both bioreactors. However, increase in flow rate beyond 5 mL/min in flow-through bioreactor showed channeling at the circumference resulting in lower pressure drop relative to simulation results. The Peclet number inside the scaffold indicated nutrient distribution within the flow-through bioreactor to be convection-dependent, whereas the parallel-flow bioreactor was diffusion-dependent. Three alternative design modifications to the parallel-flow were made by (i) introducing an additional inlet and an outlet, (ii) changing channel position, and (iii) changing the hold-up volume. Simulation studies were performed to assess the effect of scaffold thickness, cell densities, and permeability. These new designs improved nutrient distribution for 2 mm scaffolds; however, parallel-flow configuration was found to be unsuitable for scaffolds more than 4-mm thick, especially at low porosities as tissues regenerate. Furthermore, operable flow rate in flow-through bioreactors is constrained by the mechanical strength of the scaffold. In summary, this study showed limitations and differences between flow-through and parallel-flow bioreactors used in tissue engineering.     

Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in three-dimensional environment. The structural, biological and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.Key words: colonization, pore size, porosity, topography, mechanotransduction, degradation, matrix turnover     

Moderate alcohol consumption is associated with better endothelial function: a cross sectional study

Background

Natural heterologous valved conduits with a diameter greater than 22 mm that can be used for right ventricular outflow tract reconstruction in adults are not commercially available. The purpose of this study was to measure by ultrasonography the maximum diameter of the distended jugular veins of horses and cattle, respectively, to identify a population of animals that would be suitable for post-mortem collection of jugular veins at sizes greater than 22 mm.

Methods

The study population included 60 Warmblood horses, 25 Freiberger horses, 20 Brown Swiss cows, and 20 Holstein cows (including 10 Holstein and 10 Red Holstein). The maximum cross-sectional diameter of the distended jugular veins was measured at a location half-way between the mandibular angle and the thoracic inlet. The thoracic circumference (heart girth length) was used as a surrogate of body size. The jugular vein diameters of the different populations were compared by analysis of variance and the association between heart girth length and jugular vein diameter was determined in each of the four study populations by linear regression analysis.

Results

There was considerable individual variation of jugular vein diameters within each of the four study populations. There was no statistically significant relationship between thoracic circumference and jugular vein diameter in any of the populations. The jugular vein diameters of Brown Swiss cows were significantly larger than those of any of the other populations. Warmblood horses had significantly larger jugular vein diameters compared to Freiberger horses.

Conclusion

The results of this study suggest that the production of bovine or equine xenografts with diameters of greater than 22 mm would be feasible. Differences between species and breeds need to be considered. However, prediction of the jugular vein diameter based on breed and heart girth length in an individual animal is inaccurate.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae)

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.      

Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan-gelatin porous structures

Influence of mechanical characteristics and matrix architecture of substrates used in cell culture is an important issue to tissue engineering. Chitosan‐based materials have been processed into porous structures, injectable gels and membranes, and are investigated to regenerate various tissues. However, the effect of these structures on cell growth and matrix production in accordance with each of the differing scaffolds has not been examined. We investigated the influence of porous structures, hydrogels, and membranes on the growth of normal human fibroblasts and their matrix production in a serum‐free system. We used chitosan alone and in combination with gelatin. Injectable hydrogels were prepared using 2‐glycerol phosphate. From the same solution, porous scaffolds and membranes were formed using controlled rate freezing and lyophilization, and air‐drying, respectively. Fibroblast growth was evaluated on the 4th and 10th days using flow cytometry and CFDA‐SE pre‐staining. Cell morphology was assessed using actin and nucleus staining. Total protein content, collagen, tropoelastin, and MMP2/MMP‐9 activity in the media supernatant were assessed by BCA, Sircol?, Fastin Elastin, and fluorogeneic peptide assays. Collagen accumulated in the matrix was assessed by Sircol? assay after pepsin/acetic acid digestion and by Masson's Trichrome staining. These results showed increased viability of fibroblasts on chitosan–gelatin porous scaffold with decreased proliferation relative to tissue culture plastic (TCP) surface despite the cells showing spindle shape. The total protein, collagen, and tropoelastin contents were higher in the spent media from chitosan–gelatin porous scaffolds compared to other conditions. MMP2/MMP9 activity was comparable to TCP. An increase in collagen content was also observed in the matrix, suggesting increased matrix deposition. In summary, matrix production is influenced by the form of chitosan structures, which significantly affects the regenerative process. Biotechnol. Bioeng. 2012; 109:1314–1325. © 2011 Wiley Periodicals, Inc.