The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

The +838 C/G MT2A polymorphism, metals, and the inflammatory/immune response in carotid artery stenosis in elderly people

Carotid artery stenosis (CS) is a well-established risk factor for stroke. Increased proinflammatory chemokines, enhanced metallothionein (MT), and altered metal homeostasis may play roles in atherosclerosis progression and plaque destabilization. MT may sequester zinc during chronic inflammation, provoke zinc deficiency, and modulate NK cell cytotoxicity. A recent investigation of older patients with diabetes and atherosclerosis showed an association between the -209 A/G MT2A polymorphism, CS, and zinc status. In this study, we evaluated the relationship between two MT2A polymorphisms (-209 and + 838 locus), metal status, and inflammatory/immune response in older patients with CS only (the CS1 group) or with CS and previous cerebrovascular episodes (transient ischemic attack or stroke) (the CS2 group). A total of 506 individuals (188 CS1, 100 CS2, and 218 healthy controls) were studied. Atherosclerotic patients (CS1 and CS2) showed increased levels of MT, MCP-1, and RANTES, reduced NK cell cytotoxicity, and altered trace element concentrations (zinc, copper, magnesium, iron). The +838 C/G MT2A polymorphism was differently distributed in CS1 and CS2 patients, who displayed the GG genotype (C-) with significantly higher frequency than elderly controls. C- carriers showed increased MCP-1 and decreased NK cell cytotoxicity, CD56+ cells, and intracellular zinc availability along with decreased zinc, copper, and magnesium content in erythrocytes and increased iron in plasma. C- carriers also showed a major incidence of soft carotid plaques. In conclusion, the +838 C/G MT2A polymorphism seems to influence inflammatory markers, zinc availability, NK cell cytotoxicity, and trace element status, all of which may promote CS development.     

Genomic instability within centromeres of interspecific marsupial hybrids

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus x Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.     

An on-pathway intermediate in the folding of a PDZ domain

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.     

High Serum β-Lactams Specific/Total IgE Ratio Is Associated with Immediate Reactions to β-Lactams Antibiotics

Total serum IgE result from the combination of specific and non-specific pools. High specific/total IgE ratio may reflect high level of allergen-specific IgE on mast cells. No data regarding its applications to drug allergies is available. One hundred seventy-one patients with a history of immediate reactions to β-lactams, confirmed by positive skin testing, and 122 control subjects tolerant to β-lactams, were studied. CAP System was used for the detection of serum total and specific IgE antibodies. The specific/total IgE ratio was tested for diagnostic accuracy compared with conventional criteria. We finally performed a simulation study to expand our investigation of the performance of the specific/total IgE ratio index in a scenario in which the new CAP detection threshold is lowered further. Specific/total IgE ratio values ≥0.002 were observed more frequently in reactive than in controls. Seventy-four of 80 subjects with values ≥0.002 were allergic to β-lactams, yielding a positive predictive value of 92.5%. The application of specific/total IgE ratio significantly improves the positive likelihood ratio and the overall diagnostic performance. In addition, we showed the capability of this new criterion to identify true reactive patients even among subjects with high levels of total IgE (>200 kU/L). Significant increase in both receiver operator characteristic (ROC) curve and sensitivity were observed in imputed case of the simulation study. The β-lactams-specific/total IgE ratio may be an additional index compared to the common criterion of positivity to a single hapten in the allergological work-up of patients with β-lactams immediate adverse reactions.     

Generation and Validation of miR-142 Knock Out Mice

microRNA-142 (miR-142) is an important regulator of many biological processes and associated signaling pathways during embryonic development, homeostasis and disease. The miR-142 hairpin gives rise to the “guide strand” miR-142-3p and the sister "passenger" strand miR-142-5p. miR-142-3p has been shown to play critical, non-redundant functions in the development of the hematopoietic lineage. We have recently reported that miR-142-3p is critical for the control of Wnt signaling in the mesenchyme of the developing lung. miR-142-5p has been proposed to control adaptive growth in cardiomyocytes postnatally and its increase is associated with extensive apoptosis and cardiac dysfunction in a murine heart failure model. Using homologous recombination, we now report the generation and validation of miR-142-null mice. miR-142-null mice show a significant decrease in th expression levels of both the 3p and 5p isoforms. The expression of Bzrap1, a gene immediately flanking miR-142 is not altered while the expression of a long non-coding RNA embedded within the miR-142 gene is decreased. miR-142-null newborn pups appear normal and are normally represented indicating absence of embryonic lethality. At embryonic day 18.5, miR-142-null lungs display increased Wnt signaling associated with the up-regulation of Apc and p300, two previously reported targets of miR-142-3p and -5p, respectively. Adult miR-142-null animals display impaired hematopoietic lineage formation identical to previously reported miR-142 gene trap knockdown mice. We report, for the first time, the homologous recombination-based miR-142-null mice that will be useful for the scientific community working on the diverse biological functions of miR-142.     

Differences in Muscle Strength in Parkinsonian Patients Affected on the Right and Left Side

Background

Muscular weakness is a frequent cause of instability that contributes to falls in Parkinson’s disease (PD). Isokinetic dynamometry is a method of muscle assessment useful to measure the muscular strength giving a quantification of the weakness, but only few studies about isokinetic assessment were performed in PD. The aims of the study were to evaluate the muscle strength in PD and to investigate the differences in patients affected on the right and left side.

Methods

Knee flexor and extensor muscles strength was assessed using an isokinetic dynamometer in 25 patients in stage 3 H&Y and in 15 healthy controls. Subjects were tested in both legs at three fixed angular velocities: 90°/s, 120°/s, 180°/s.

Results

Considering the whole population of Parkinsonians, no difference in strength was observed with respect to controls. Considering the side, patients affected on the right side showed a clear tendency to be weaker than patients affected on the left side and controls.

Conclusions

PD patients affected on the right side, but not those affected on the left side, had a reduction in muscle strength as compared to controls. We postulate a central origin deficit in muscle strength in PD. It is known that dopamine transporter binding is more severely reduced in the left posterior putamen and our results suggest that the control of the muscle strength in PD is linked to the right–left hemispheric asymmetry of the functional organization of basal ganglia and with their connections to cortical motor and pre-motor areas.     

Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy

Objective

Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.

Methods

A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m² as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.

Results

Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027).

Conclusions

We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.     

Single and three-color flow cytometry assay for intracellular zinc ion availability in human lymphocytes with Zinpyr-1 and double immunofluorescence: relationship with metallothioneins.

BACKGROUND:: The amount of available intracellular zinc is pivotal to regulate many cellular processes, including oxidative stress response and apoptotic mechanisms. Therefore it is not surprising that zinc homeostasis and dyshomeostasis is involved in many physiological and pathological states, respectively. Cell permeable zinc probes allow intracellular applications with microscopy technology, but flow cytometry (FC) applications have been scarcely explored, albeit they can be suited to study zinc homeostasis in different cell types, including rare cells. METHODS:: We describe a FC method able to estimate intracellular zinc ion availability and the intracellular capability to activate a zinc signal after treatment with an NO-donor (AcOM-DEA/NO) in human PBMCs, using the fluorescent zinc-specific probe, Zinpyr-1 (ZP1), alone or in association with CD4-PE and CD8-Cychrome mAb. RESULTS:: This method was able to detect an increase/decrease of intracellular zinc available in human fresh cultured PBMC and in immune subsets using AcOM-DEA/NO or TPEN, respectively. ZP1 mean fluorescence on gated histograms was sensitive to the amount of zinc added in the culture medium and significantly correlated to metallothioneins and total intracellular zinc. CONCLUSIONS:: FC applications using ZP1 may be a fast and useful tool to study zinc homeostasis in immune cells.