Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Oligomerization of L-asparaginase from Erwinia carotovora

Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the oligomerization process of this enzyme, which is active only as the tetramer because its active sites are located in the contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunit interfaces, and the development of a new experimental approach for studies of enzyme oligomerization. L-Asparaginase was immobilized on a surface of CM5 optical chip of biosensor Biacore 3000, which is based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered.     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16).     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Photoaptameric heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinks with a target protein

Using DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nucleotides (nt) in length) to produce aptamer heterodimeric constructs results into affinity enhancement. At the linker lengths ranged from 35 to 55 nt the apparent dissociation constants (K _D^app) measured using the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (K _D^app) = 0.2–0.4 nM), which were approximately 30-fold less than for the complexes with the initial aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer could bind to the exosite 1. The (K _D^app value for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure of the equimolar mixtures of thrombin with the photoaptamer construct to the UV radiation at 308 nm the equal yield of the crosslinked complexes was observed at concentrations, which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.     

Isatin‐binding proteins of rat and mouse brain: Proteomic identification and optical biosensor validation

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X‐100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin‐binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5‐aminocarproyl‐isatin, and 5‐aminoisatin as the affinity ligands. The K_d values (of 0.1–20 μM) obtained during the optical biosensor experiments were consistent with the range of K_d values recently reported for [³H]isatin binding to brain sections. Although the number of isatin‐binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one‐third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.