Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

Growth of Pleurotus ostreatus on wheat straw and wheat-grain-based media: biochemical aspects and preparation of mushroom inoculum

Mycelial growth, intracellular activity of proteases, laccases and β-1,3-glucanases, and cytoplasmic protein were evaluated in the vegetative phase of Pleurotus ostreatus grown on wheat straw and in wheat-grain-based media in Petri dishes and in bottles. The productivity of the wheat straw and wheat-grain-based spawn in cylindrical polyethylene bags containing 5 kg of chopped straw was also determined. We observed high activity of proteases and high content of intracellular protein in cultures grown on wheat straw. This suggests that the proteases are not secreted into the medium and that the protein is an important cellular reserve. On the contrary, cultures grown on wheat straw secreted laccases into the medium, which could be induced by this substrate. P. ostreatus grown on media prepared with a combination of wheat straw and wheat grain showed a high radial growth rate in Petri dishes and a high level of mycelial growth in bottles. The productivities of wheat straw and wheat-grain-based spawn were similar. Our results show that cheaper and more productive mushroom spawn can be prepared by developing the mycelium on wheat straw and wheat-grain-based substrates.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Expression of the catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.     

Medicinal use of fauna by a traditional community in the Brazilian Amazonia

Background

Zootherapy inventories are important as they contribute to the world documentation of the prevalence, importance and diversity of the medicinal use of animals in traditional human communities. The present study aims to contribute with a more valuable example of the zootherapy practices of a traditional community in the Brazilian Amazonia ?C the ??Riozinho do Anfrísio?? Extractive Reserve, in Northern Brazil.

Methods

We used the methods of participant observation and semi-structured interviews, applied to 25 informants. We employed the combined properties of two indices to measure the medicinal importance of each cited species to the studied community, as well as their versatility in the treatment of diseases: the well known Use Value (UV) and the Medicinal Applications Value (MAV) that we developed.

Results

We recorded 31 species of medicinal animals from six taxonomic categories, seven of which are new to science. The species are used for the treatment of 28 diseases and one species is used as an amulet against snakebites. The five species with the highest UV indices are the most popular and valued by the studied community. Their contrasting MAV indices indicate that they have different therapeutic properties: specific (used for the treatment of few diseases; low versatility) and all-purpose (several diseases; high versatility). Similarly, the most cited diseases were also those that could be treated with a larger number of animal species. Ten species are listed in the CITES appendices and 21 are present in the IUCN Red List. The knowledge about the medicinal use of the local fauna is distributed evenly among the different age groups of the informants.

Conclusions

This study shows that the local fauna represents an important medicinal resource for the inhabitants of the protected area. The combined use of the UV and MAV indices allowed identifying the species with the highest therapeutic potential. This type of information about a species may be of interest to pharmacological research, and is crucial to its conservation, since it helps signaling the species that may undergo higher hunting pressures. Data on zootherapy can also be of interesting to ecologists by contributing to indicators of local biodiversity richness.     

Plasma neurotensin release and gastric emptying in the dumping syndrome

Seventy-three patients were studied after ingesting a liquid glucose meal, tagged with ¹¹³Indium. Nineteen of these patients were awaiting surgery for their duodenal ulcer, while 54 were studied postoperatively, 25 of whom experienced troublesome postprandial (dumping) symptoms in their daily lives. The radioactive marker emptied significantly faster in the symptomatic patients than in the symptomfree, pre and post-operative groups (initial emptying rate 3.45 ± 0.23, compared with 1.16 ± 0.19 and 1.27 ± 0.15% fall in counts/min respectively; p < 0.01). Initial (20 min) rises in the plasma concentrations of neurotensin-like immunoreactivity measured during the test correlated significantly with the rate of gastric emptying in all patients, being greatest in patients with dumping symptoms. Physiological concentrations of neurotensin have been shown to delay gastric emptying. The excessive rise in plasma neurotensin-like immunoreactivity in patients with dumping symptoms, presumably occuring as a result of the rapid passage of nutrients to the neurotensin-rich ileum, may possibly have a compensatory role in slowing further emptying from the stomach.     

On the relationship between an Asian haplotype on chromosome 6 that reduces androstenone levels in boars and the differential expression of SULT2A1 in the testis

Background

Androstenone is one of the major compounds responsible for boar taint, a pronounced urine-like odor produced when cooking boar meat. Several studies have identified quantitative trait loci (QTL) for androstenone level on Sus scrofa chromosome (SSC) 6. For one of the candidate genes in the region SULT2A1, a difference in expression levels in the testis has been shown at the protein and RNA level.

Results

Haplotypes were predicted for the QTL region and their effects were estimated showing that haplotype 1 was consistently related with a lower level, and haplotype 2 with a higher level of androstenone. A recombinant haplotype allowed us to narrow down the QTL region from 3.75 Mbp to 1.94 Mbp. An RNA-seq analysis of the liver and testis revealed six genes that were differentially expressed between homozygotes of haplotypes 1 and 2. Genomic sequences of these differentially expressed genes were checked for variations within potential regulatory regions. We identified one variant located within a CpG island that could affect expression of SULT2A1 gene. An allele-specific expression analysis in the testis did not show differential expression between the alleles of SULT2A1 located on the different haplotypes in heterozygous animals. However a synonymous mutation C166T (SSC6: 49,117,861 bp in Sscrofa 10.2; C/T) was identified within the exon 2 of SULT2A1 for which the haplotype 2 only had the C allele which was higher expressed than the T allele, indicating haplotype-independent allelic-imbalanced expression between the two alleles. A phylogenetic analysis for the 1.94 Mbp region revealed that haplotype 1, associated with low androstenone level, originated from Asia.

Conclusions

Differential expression could be observed for six genes by RNA-seq analysis. No difference in the ratio of C:T expression of SULT2A1 for the haplotypes was found by the allele-specific expression analysis, however, a difference in expression between the C over T allele was found for a variation within SULT2A1, showing that the difference in androstenone levels between the haplotypes is not caused by the SNP in exon 2.     

Heritability of cortisol response to confinement stress in European sea bass dicentrarchus labrax

Background

In fish, the most studied production traits in terms of heritability are body weight or growth, stress or disease resistance, while heritability of cortisol levels, widely used as a measure of response to stress, is less studied. In this study, we have estimated heritabilities of two growth traits (body weight and length) and of cortisol response to confinement stress in the European sea bass.

Findings

The F1 progeny analysed (n = 922) belonged to a small effective breeding population with contributions from an unbalanced family structure of just 10 males and 2 females. Heritability values ranged from 0.54 (±0.21) for body weight to 0.65 (±0.22) for standard body length and were low for cortisol response i.e. 0.08 (±0.06). Genetic correlations were positive (0.94) between standard body length and body weight and negative between cortisol and body weight and between cortisol and standard body length (−0.60 and −0.55, respectively).

Conclusion

This study confirms that in European sea bass, heritability of growth-related traits is high and that selection on such traits has potential. However, heritability of cortisol response to stress is low in European sea bass and since it is known to vary greatly among species, further studies are necessary to understand the reasons for these differences.     

Iterleukin 1 alpha is a marker of endothelial cellular senescent

Background  

The functional changes associated with endothelial senescence may be involved in human aging and age-related vascular disorders. Since the inflammatory cytokine interleukin (IL-)1 inhibits endothelial growth, we evaluated the expression of IL-1α, IL-1β and their antagonist, the IL-1 receptor antagonist (IL-1ra), in endothelial in vitro senescence and quiescence. We also examined the expression of IL-1α in human senescent and progeric fibroblasts.