Sunflower Seed Aminopeptidase-Catalyzed Hydrolysis of Phe-Xaa Dipeptides — Exploring the S1′ Specificity of the Enzyme

The S₁′ substrate specificity of the sunflower seed major aminopeptidase was studied with a series of dipeptide substrates with phenylalanine at P₁ and a hydrophobic amino acid at P₁′ position. The kinetic parameters of hydrolysis are significantly affected by the structure, side chain hydrophobicity and configuration of the P₁′ moiety. Its binding during enzyme-substrate complex formation takes place at a hydrophobic site of limited size following an extraction mechanism as seen from the applied structure-activity correlation. Attempts to establish such dependencies for the catalytic step of the reaction reveal the presence of additional S₁′-P₁′ enzyme-substrate interactions of greater complexity.     

Impaired esophageal function in patients with irritable bowel syndrome

The aim of the study was to determine prevalence of the signs and symptoms related to esophageal dysfunction in irritable bowel syndrome (IBS) patients, and to investigate sensorimotor function impairment based on the esophageal manometry study, thus to determine the correlation between them. The study included 30 patients with IBS, 14 of them with diarrhea (IBSd) and 16 with constipation (IBSc) as a predominant discomfort. Control group consisted of 30 healthy subjects. The patients were included in the study on the basis of the Rome criteria for IBS. In addition to thorough history and physical examination patient underwent esophagogastroduodenoscopy and esophageal manometry. The values of esophageal manometry obtained in healthy subjects served as controls in manometry studies. The patients with IBS suffered a great number of both colonic and extracolonic signs and symptoms, however, there was no statistically significant difference in the prevalence of particular symptoms between the two patient subgroups. In comparison with healthy subjects, the patients suffering from IBS showed pathologically altered values in the majority of parameters of esophageal motility. Comparison of the two subgroups of IBS patients according to esophageal motility characteristics yielded differences in only few of them. The results obtained in the study could explain why the patients with IBS quite commonly complain of the symptoms related to upper gastrointestinal tract, such as heartburn and chest pain of noncardiac genesis. The results also suggest that the IBS might be associated with considerably more extensive smooth muscle or innervation changes than presumed before.     

Combined metabolic activity within an atrazine-mineralizing community enriched from agrochemical factory soil

The main objective of this work was to characterize an atrazine-mineralizing community originating from agrochemical factory soil, especially to elucidate the catabolic pathway and individual metabolic and genetic potentials of culturable members. A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO₂ and NH₃. Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization.Scientific relevance: Despite numerous reports on atrazine degradation by pure bacterial cultures, the pathways and the atrazine-degrading gene combinations harboured by bacterial communities are only poorly described. In this work, we characterized a four-member atrazine-mineralizing community enriched from an agrochemical factory soil, which was capable of rapidly metabolizing atrazine to CO₂. This study will contribute towards better understanding of the genetic potential and metabolic activities of atrazine-degrading communities, which are generally considered to be responsible for atrazine mineralization in the natural environment.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.     

CHANGES IN ABUNDANCE OF HARBOR SEALS IN MAINE, 1981–2001

Aerial counts of harbor seals ( Pboca vitulina concolor ) on ledges along the Maine coast were conducted during the pupping season in 1981, 1986, 1993, 1997, and 2001. Between 1981 and 2001, the uncorrected counts of seals increased from 10,543 to 38,014, an annual rate of 6.6 percent. In 2001 30 harbor seals were captured and radio-tagged prior to aerial counts. Of these, 19 harbor seals (six adult males, two adult females, seven juvenile males, and four juvenile females) were available during the survey to develop a correction factor for the fraction of seals not observed. The corrected 2001 abundance estimate was 99,340 harbor seals. Productivity in this population has increased since 1981 from 6.4% pups to 24.4% pups. The number of gray seals ( Halichoerus grypus ) counted during the harbor seal surveys increased from zero in both 1981 and 1986 to 1,731 animals in 2001.     

NC4 Domain of cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.     

Molecular dynamics shows complex interplay and long-range effects of post-translational modifications in yeast protein interactions

Post-translational modifications (PTMs) play a vital, yet often overlooked role in the living cells through modulation of protein properties, such as localization and affinity towards their interactors, thereby enabling quick adaptation to changing environmental conditions. We have previously benchmarked a computational framework for the prediction of PTMs’ effects on the stability of protein-protein interactions, which has molecular dynamics simulations followed by free energy calculations at its core. In the present work, we apply this framework to publicly available data on Saccharomyces cerevisiae protein structures and PTM sites, identified in both normal and stress conditions. We predict proteome-wide effects of acetylations and phosphorylations on protein-protein interactions and find that acetylations more frequently have locally stabilizing roles in protein interactions, while the opposite is true for phosphorylations. However, the overall impact of PTMs on protein-protein interactions is more complex than a simple sum of local changes caused by the introduction of PTMs and adds to our understanding of PTM cross-talk. We further use the obtained data to calculate the conformational changes brought about by PTMs. Finally, conservation of the analyzed PTM residues in orthologues shows that some predictions for yeast proteins will be mirrored to other organisms, including human. This work, therefore, contributes to our overall understanding of the modulation of the cellular protein interaction networks in yeast and beyond.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.