Insulin-like growth factor 1 (IGF-1) concentrations in synovial fluid of sound and osteoarthritic horses, and its correlation with proinflammatory cytokines IL-6 and TNF

El factor de crecimiento similar a la insulina I (IGF-1) es el factor de crecimiento conocido más importante para la reparación del cartílago en caballos. Promueve la mitosis de los condrocitos, la expresión de colágeno II y la producción de matriz extracelular. La osteoartritis (OA) es la condición musculoesquelética más común que causa cojera y bajo rendimiento en caballos deportivos. Se evaluó clínica y radiográficamente un total de 11 caballos cojos, y se confirmó que todos sufrían una cojera metacarpofalángica frontal mediante una prueba de flexión positiva, un bloqueo nervioso en 4 puntos bajos y un bloqueo intraarticular. La proteína total, IGF-1, IL-6 y TNFα se determinaron por ELISA, lo que demostró cambios y diferentes correlaciones entre la condición clínica, los cambios radiográficos y el grado de inflamación. Todos los caballos con dolor asociado a las articulaciones y, por lo tanto, asociado a la cojera, mostraron un aumento significativo de la proteína total (P<0.0001) y la concentración de IGF-1 (P<0.05). Las concentraciones de IL-6 y TNFα entre los controles y los caballos cojos mostraron diferencias significativas (P<0.01 y P<0.001 respectivamente). Los caballos con menos cambios radiográficos mostraron la mayor expresión de IGF-1 en el líquido sinovial, y los caballos con condiciones de OA más crónicas tuvieron niveles de expresión de IGF-1 muy similares a los de las articulaciones de control. En todas las articulaciones cojas, se identificó por medio de Western blot una isoforma de IGF-1 más ligera (~ 7.5 kDa) que estaba relacionada con la inflamación y es el peso molecular del péptido maduro, y todas las articulaciones de control expresaron una isoforma más pesada (~ 12 kDa). Este hallazgo podría conducir a una nueva investigación para secuenciar y apuntar a la isoforma que no se expresa durante un proceso inflamatorio dentro de una articulación, y para tener una mejor comprensión de su papel en la articulación del caballo.Insulin-like growth factor I (IGF-1) is the most important known growth factor for cartilage repair in horses. It promotes mitosis of chondrocytes, collagen II expression, and extra cellular matrix production. Osteoarthritis (OA) is the most common musculoskeletal condition that causes lameness and poor performance in sport horses. A total of 11 lame horses were clinically and radiographically evaluated, and all were confirmed to suffer a front metacarpophalangeal lameness by a positive flexion test, a low-4-point nerve block and an intraarticular block. Total protein, IGF-1, IL-6 and TNFa were determined by ELISA, demonstrating changes and different correlations between clinical condition, radiographic changes and degree of inflammation. All horses with joint associated pain and therefore associated lameness, demonstrated a significant increase of total protein (P<0.0001) and IGF-1 concentration (P<0.05). Concentrations of IL-6 and TNFa between controls and lame horses demonstrated significant differences (P<0.01 and P<0.001 respectively). Horses with less radiographic changes, demonstrated the highest IGF-1 expression in synovial fluid, and horses with more chronic OA conditions had very similar IGF-1 expression levels than control joints. In all lame joints, it was identified by Western blot a lighter isoform of IGF-1 (~7.5 kDa) which was inflammation related and it is the molecular weight of the mature peptide, and all control joints expressed a heavier isoform (~12 kDa). This finding could lead to new research for sequencing and targeting the isoform which is not expressed during an inflammatory process within a joint, and to have a better understanding of its role in the horse’s joint

Purification, concentration and recovery of small fragments of DNA from Giardia lamblia and their use for other molecular techniques

Purification of nucleic acids is an essential procedure for most experiments in molecular biology. In this paper, the freeze-squeeze method with some modifications is proposed as an alternative methodology for the purification, concentration and recovery of small DNA fragments from agarose gels. The advantage of this alternative methodology is that it enables the recovery of fragments that are less than 100 bp in length and enables suspension of products in smaller volumes compared to several commercially available kits. In addition, the purified fragments were re-amplified by PCR and used for cloning and sequencing. Moreover, this protocol was used to perform the isolation and identification of microRNAs from Giardia lamblia, as previously reported. This protocol has the advantage of being inexpensive and easy and can be employed for various molecular applications. The advantages of this protocol include • A modified classical method was used for purification of small DNA fragments from G. lamblia. • The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits. • The modified method allows concentration and recovery of fragments up to 60 bp in length. • The modified freeze-squeeze method allows re-suspension of the products in volumes of up to 2.5 μL

Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions

Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion), followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP) binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine) ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in “sensu strictu” yeasts suggests this protein has a yet undiscovered physiological function

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug. Keywords: Giardiasis, Drug repurposing, Neglected disease, Recombinant protein, Enzyme inactivatio