High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity,Stability, Milk Coagulation Profile and Gel Development

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.     

Preliminary analysis of reproductive success in a large mammal with alternative mating tactics,the Northern chamois,Rupicapra rupicapra

In polygynous mating systems, reproductive skew depends on the ability of males to monopolize females, which in turn may promote the development of contrasting traits in the two sexes. Although dominant individuals normally enjoy a higher reproductive success (RS) than subordinates, the use of genetic markers has shown that behavioural observations of male mating success may not provide reliable clues of RS. We report the preliminary results of the first DNA‐based paternity analysis on the Northern chamois (Rupicapra rupicapra), a scarcely dimorphic mountain ungulate described as highly polygynous, in relation to mating tactic and age. Because of sampling difficulties, the success in parentage assignment was low, and the interpretation of results requires caution. Territorial males had a greater RS than nonterritorial ones but they were unable to monopolize mating events. Age had a weak effect on paternity outcome but only males ≥ 6 years showed siring success. Although future studies are needed to assess the opportunity for sexual selection in male chamois, the concurrence of limited sexual size dimorphism, compensatory growth, unbiased sex‐specific survival, RS of alternative mating tactics and, possibly, long breeding tenure, may hint at the adoption of a conservative mating strategy in this species.     

On choice of substrate and habitat in bdelzoid rotifers

Information on the distributions of 77 bdelloid rotifers from diverse waters in south and central Sweden was analyzed to reveal their relationships to substrate and habitat. Only two species were reported from plankton, both in low frequency. An artificial substrate, white cotton, was utilized only by few bdelloids, mainly by species with eyes. Only one species occurred at higher frequency on the saprobic substrate Sphaerotilus. Most bdelloids prefer environments rich in oxygen, but some species may be found in soft-bottom sediments. Several species dwell preferentially in diverse habitats of bogs. Some bdelloids have an exceptionally broad ecological range, but even for these is it possible to distinguish a pattern of preference and avoidance.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

Long-Term Structural and Functional Myocardial Adaptations in Healthy Living Kidney Donors: A Pilot Study

Background and Aims

Compensatory renal hypertrophy following unilateral nephrectomy (UNX) occurs in the remaining kidney. However, the long-term cardiac adaptive process to UNX remains poorly defined in humans. Our goal was to characterize myocardial structure and function in living kidney donors (LKDs), approximately 12 years after UNX.

Methods and Results

Cardiac function and structure in 15 Italian LKDs, at least 5 years after UNX (median time from donation = 8.4 years) was investigated and compared to those of age and sex matched U.S. citizens healthy controls (n = 15). Standard and speckle tracking echocardiography (STE) was performed in both LKDs and controls. Plasma angiotensin II, aldosterone, atrial natriuretic peptide (ANP), N terminus pro B-type natriuretic peptide (NT-proBNP), cyclic guanylyl monophosphate (cGMP), and amino-terminal peptide of procollagen III (PIIINP) were also collected. Median follow-up was 11.9 years. In LKDs, LV geometry and function by STE were similar to controls, wall thickness and volumes were within normal limits also by CMR. In LKDs, CMR was negative for myocardial fibrosis, but apical rotation and LV torsion obtained by STE were impaired as compared to controls (21.4 ± 7.8 vs 32.7 ± 8.9 degrees, p = 0.04). Serum creatinine and PIIINP levels were increased [1.1 (0.9–1.3) mg/dL, and 5.8 (5.4–7.6)] μg/L, respectively), while urinary cGMP was reduced [270 (250–355) vs 581 (437–698) pmol/mL] in LKDs. No LKD developed cardiovascular or renal events during follow-up.

Conclusions

Long-term kidney donors have no apparent structural myocardial abnormalities as assessed by contrast enhanced CMR. However, myocardial deformation of the apical segments, as well as apical rotation, and LV torsion are reduced. The concomitant increase in circulating PIIINP level is suggestive of fibrosis. Further studies, focused on US and EU patients are warranted to evaluate whether these early functional modifications will progress to a more compromised cardiac function and structure at a later time.     

Infection with Soil-Transmitted Helminths Is Associated with Increased Insulin Sensitivity

Objective

Given that helminth infections have been shown to improve insulin sensitivity in animal studies, which may be explained by beneficial effects on energy balance or by a shift in the immune system to an anti-inflammatory profile, we investigated whether soil-transmitted helminth (STH)-infected subjects are more insulin sensitive than STH-uninfected subjects.

Design

We performed a cross-sectional study on Flores island, Indonesia, an area with high prevalence of STH infections.

Methods

From 646 adults, stool samples were screened for Trichuris trichiura by microscopy and for Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, and Strongyloides stercoralis by qPCR. No other helminth was found. We collected data on body mass index (BMI, kg/m²), waist-to-hip ratio (WHR), fasting blood glucose (FBG, mmol/L), insulin (pmol/L), high sensitive C-reactive protein (ng/ml) and Immunoglobulin E (IU/ml). The homeostatic model assessment for insulin resistance (HOMAIR) was calculated and regression models were used to assess the association between STH infection status and insulin resistance.

Results

424 (66%) participants had at least one STH infection. STH infected participants had lower BMI (23.2 vs 22.5 kg/m², p value = 0.03) and lower HOMAIR (0.97 vs 0.81, p value = 0.05). In an age-, sex- and BMI-adjusted model a significant association was seen between the number of infections and HOMAIR: for every additional infection with STH species, the HOMAIR decreased by 0.10 (p for linear trend 0.01). This effect was mainly accounted for by a decrease in insulin of 4.9 pmol/L for every infection (p for trend = 0.07).

Conclusion

STH infections are associated with a modest improvement of insulin sensitivity, which is not accounted for by STH effects on BMI alone.