Requirement of mouse BCCIP for neural development and progenitor proliferation

Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR) pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors.     

The non-peptide kinin receptor antagonists FR 173657 and SSR 240612: preclinical evidence for the treatment of skin inflammation

Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases.     

Evaluation of 59Fe-lignosulfonates complexes as Fe-sources for plants

Iron chlorosis is a wide-spread limiting factor of production in agriculture. To cope with this problem, synthetic chelates (like EDTA or EDDHA) of Fe are used in foliar-spray or in soil treatments; however, these products are very expensive. Therefore paper-production byproducts, like Lignosulfonates (LS), with varying content of carboxylate and sulfonate groups, were tested with respect to their ability to maintain Fe in the solution of soils and to feed plants grown in hydroponics with Fe through foliar sprays or application to the nutrient solution. Results show that LS had a low capability to solubilize ⁵⁹Fe-hydroxide and that preformed ⁵⁹Fe(III)-LS complexes had poor mobility through a soil column (pH 7.5) and scarce stability when interacting with soils compared to ⁵⁹Fe(III)-EDDHA. However when ⁵⁹Fe(III)-LS were supplied to roots in a hydroponic system, they demonstrated an even higher capability to fed Fe-deficient tomato plants than ⁵⁹Fe(III)-EDDHA. Hence, data here presented indicate that the low Fe use efficiency from Fe-LS observed in soil-applications is due to interactions of these Fe-sources with soil colloids rather than to the low capability of roots to use them. Foliar application experiments of ⁵⁹Fe(III)-LS or ⁵⁹Fe(III)-EDTA to Fe-deficient cucumber plants show that uptake and reduction rates of Fe were similar between all these complexes; on the other hand, when ⁵⁹Fe(III)-LS were sprayed on Fe-deficient tomato leaves, they showed a lower uptake rate, but a similar reduction rate, than ⁵⁹Fe(III)-EDTA did. In conclusion, Fe-LS may be a valid, eco-compatible and cheap alternative to synthetic chelates in dealing with Fe chlorosis when applied foliarly or in the nutrient solution of hydroponically grown plants.     

Heavy metal sorption by released polysaccharides and whole cultures of two exopolysaccharide-producing cyanobacteria

The metal removal capacity of cultures of two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested using copper (II) as the model metal. C. capsulata cultures removed the greatest amount of copper, with a maximum per unit of biomass (q _max) of 115.0±5.1 mg copper g⁻¹ of protein, compared with 85.0±3.2 removed with Nostoc PCC7936 cultures. Water solutions of pure polysaccharides (RPSs) released into the culture medium by C. capsulata and Nostoc PCC7936 achieved q _max values of 20.2±0.8 mg g⁻¹ copper per polysaccharide dry weight with C. capsulata RPS and 11.0±1.5 mg g⁻¹ with Nostoc PCC7936 RPS. Cultures of the two cyanobacteria also removed Zn (II) and Ni (II), in both single-metal systems and in multimetal systems with Cu; in the various single-metal systems more copper was removed than Zn or Ni, while in the multimetal systems a smaller amount of each individual metal was removed but the overall amount of all metal ions sorbed or the amount of copper sorbed in the copper-only system was almost the same with C. capsulata, and slightly higher with Nostoc PCC7936.     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

PERK inhibition attenuates the abnormalities of the secretory pathway and the increased apoptotic rate induced by SIL1 knockdown in HeLa cells

Loss-of-function mutations in the SIL1 gene are linked to Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of infancy characterized by cerebellar and skeletal muscle degeneration. SIL1 is a ubiquitous adenine nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone BiP. The complexity of mechanisms by which loss of SIL1 causes MSS is not yet fully understood. We used HeLa cells to test the hypothesis that impaired protein folding in the ER due to loss of SIL1 could affect secretory trafficking, impairing the transport of cargoes essential for the function of MSS vulnerable cells. Immunofluorescence and ultrastructural analysis of SIL1-knocked-down cells detected ER chaperone aggregation, enlargement of the Golgi complex, increased autophagic vacuoles, and mitochondrial swelling. SIL1-interefered cells also had delayed ER-to-plasma membrane transport with retention of Na⁺/K⁺-ATPase and procollagen-I in the ER and Golgi, and increased apoptosis. The PERK pathway of the unfolded protein response was activated in SIL1-interfered cells, and the PERK inhibitor GSK2606414 attenuated the morphological and functional alterations of the secretory pathway, and significantly reduced cell death. These results indicate that loss of SIL1 is associated with alterations of secretory transport, and suggest that inhibiting PERK signalling may alleviate the cellular pathology of SIL1-related MSS.     

Oral clonidine provocative test in the diagnosis of growth hormone deficiency in childhood: should we make the timing uniform?

INTRODUCTION: Oral clonidine is one of the most frequent drugs used for the diagnosis of growth hormone deficiency (GHD), but the duration of the test, depending on which European centres use it, is not uniform and can vary from 120 to 150 min or even 180 min. SUBJECTS AND METHODS: To standardize this test, evaluating the possibility to shorten it to 90 min, we investigated the response of GH to the oral clonidine test in 291 children evaluated for short stature (height <-2 SD). Of these, 164 were diagnosed as idiopathic short stature (ISS) and 127 as GHD. In these patients, we calculated: (1) the frequency distribution of the GH peaks to clonidine in GHD and in ISS at various times; (2) the percentage of GH peaks to clonidine before and after 90 min in all and in ISS children; (3) the percentage of the first GH value >or=10 ng/ml before 90 min and after 90 min in ISS. RESULTS: GH peak distribution varied between 30 and 180 min, even though the vast majority of peaks occurred between 30 and 60 min. There was no significant difference (p > 0.05) in the peak distribution between ISS and GHD children. The percentages of GH peaks within 90 min were 92.1% in all children and 95.7% in ISS. If considering the first value of GH >or=10 ng/ml this last percentage reaches 96.3%. CONCLUSION: Our study suggests that the oral clonidine test can be administered for only 90 min without significantly changing its validity. This test should be standardized at 90 min in European protocols just as in those currently used in the USA in order to reduce the discomfort of patients and the cost of this diagnostic procedure.     

Transfer factor in chronic mucocutaneous candidiasis

Fifteen patients suffering from chronic mucocutaneous candidiasis were treated with an in vitro produced TF specific for Candida albicans antigens and/or with TF extracted from pooled buffy coats of blood donors. CMI of the patients was assessed using the LMT and the LST in presence of candidine. The aim of the study was the clinical evaluation of TF treatment and the incidence of positive tests before, during, and after therapy. Immunological data were matched using the Chi square test. 87 LMT were performed for each antigen dose and at the dilution of 1/50, 58.9% (33/56) tests were positive during non-treatment or non-specific TF treatment. On the contrary 83.9% (26/31) were positive during specific TF treatment (P<0.05). In the LST, a significant decrease of thymidine uptake in the control cultures in presence of autologous or AB serum was observed when patients were matched according to non-treatment, and both non specific (P<0.05) and specific TF treatment (P<0.01). Only during specific TF treatment was a significant increase of reactivity against the Candida antigen at the highest concentration noticed, when compared with the period of non specific treatment (P<0.01). Clinical observations were encouraging: all but one patient experienced significant improvement during treatment with specific TF. These data confirm that orally administered specific TF, extracted from induced lymphoblastoid cell-lines, increases the incidence of reactivity against Candida antigens in the LMT. LST reactivity appeared not significantly increased with respect to the periods of non treatment, but was significantly increased when it was compared to the non-specific TF treatment periods. At the same time, a clinical improvement was noticed.     

Selenium,zinc, and thyroid hormones in healthy subjects

Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T₃ production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T₄ to T₃ with a lower T₃/T₄ ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T₃/T₄ ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T₃/T₄ ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T₄ conversion is related to impaired Se status in the elderly.