Evidence of an insulin generated pyruvate dehydrogenase stimulating factor in rat brain plasma membranes

1. The results of this study indicates that the binding of insulin to brain plasma membranes activates a membrane protease which, by a trypsin like mechanism, produces a soluble factor that modulates the PDH behaviour when added to brain mitochondria. 2. The supernatant from brain plasma membranes incubated with 0.5 mg/ml trypsin added to mitochondria increases PDH activity levels and cancels PDH inhibition by NaF, as has already been seen when the plasma membranes are incubated with 25 microU/ml insulin. No such effects are obtained when the incubation is run out with 0.5 mg/ml chymotrypsin. 3. The supernatants from insulin or trypsin treated plasma membranes retain their activating properties on mitochondrial PDH also after dansylation; from these preparations a dansylated active on PDH material was separated by monodimensional chromatography on HPTLC silica Gel plates, using chloroform/1-butanol (93:7 v/v) as a solvent. 4. Insulin incubation of plasma membranes pretreated with protease inhibitors (leupeptin, phenylmethylsulfonylfluoride) or with exogenous trypsin, but not chymotrypsin substrates (esters of arginine and tyrosine) yields an inactive supernatant on PDH. 5. Insulin treated plasma membrane supernatants lose all stimulating properties on PDH after incubation for 1 hr with 2 mg/ml trypsin or chymotrypsin.     

Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis

Altered estrogen receptor α (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process.     

Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.     

Ragweed (Ambrosia) pollen presence in Livorno,Central Italy: Aerobiological and sensitization data

We have studied Ragweed pollen concentration in the atmosphere of Leghorn (Livorno). where Ragweed is not widespread. Our data, from 1991 to 1995, confirm the presence of Ragweed pollen in the atmosphere of Leghorn in low concentrations (highest value of 11 pollen grains/m³) from August to the end of September. Even if the sensitization level in our area is 6.76%, the clinical significance of Ragweed pollinosis is still very low.     

Contribution to the study of fungistatic action of N,N'-bis-n-butylamid of 2,2' dicarboxydiphenyldisulfide (OD 507)

Summary The authors have studied the fungistatic action in vitro of N,N'-bis-n-butylamide of 2,2' dicarboxydiphenyldisulfide (OD 507) on some strains ofC. albicans, C. neoformans, M. apiospermum, H. duboisii, P. pedrosoi andE. coronata isolated from human fungal infections (both superficial and deep) in Congo.Considering that this substance has an important fungistatic action on the strains studied and that its inhibitory action maintains itself satisfactorily even in presence of human blood (though to somewhat lesser extent), the authors think it usefull to report the results obtained and suggest further research on animals with a view to investigate the possibility of using this substance parenterally on deep human fungal infections.

---

Résumé Les Auteurs ont étudié l'action fungistatique in vitro du N,N'-bis-n.butylamide du 2,2' dicarboxydiphenyldisulfure (OD 507) sur quelques souches deC. albicans, C. neoformans, M. apiospermum H. duboisii, P. pedrosoi etE. coronata isolées au Congo à partir de mycoses humaines superficielles et profondes.Ayant constaté que cette substance présente une action fungistatique importante sur les souches étudiées et que l'action inhibitrice, bien que moins forte, se maintient satisfaisante même en présence de sang humain, les Auteurs estiment utile de signaler les résultats obtenus et de suggérer des espériences ultérieures sur l'animal afin d'évaluer les possibilités éventuelles d'un usage parenteral de l'OD 507 dans les cas de mycose humaine profonde.

---

Riassunto Gli A.A. hanno studiato l'azione fungistatica in vitro del N,N'-bis-n.butilamide del 2,2'dicarbossidifenildisolfuro (OD 507) su alcuni ceppi diC. albicans, C. neoformans, M. apiospermum, H. duboisii, P.pedrosoi edE. coronata isolati in Congo a partire da micosi umane supericiali e profonde.Avendo constatato che tale sostanza presenta una notevole azione fungistatica sui ceppi studiati e che l'azione inibitrice, benchè in minor grado, si mantiene soddisfacente anche in presenza di sangue umano, ritengono utile di segnalare i risultati da loro ottenuti e di suggerire delle ulteriori esperienze sull'animale onde indagare le eventuali possibilità di impiego parenterale dell'OD 507 nelle micosi umane profonde.

     

Micropolyspora faeni antigens of three commercial extracts

The protein composition of three commercial extracts of Micropolyspora faeni, produced in U.S.A., England and Italy has been evaluated by agarose gel electrophoresis.By crossed immunoelectrophoresis, tandem-crossed immunoelectrophoresis and by a modification of this last technique, the antigenic composition and the common antigens of the extracts have been investigated. Hyperimmune rabbit serum and a pool of five human sera with precipitins to Micropolyspora faeni have been used as source of antibody.Different quantity and quality of protein content was observed in the available batches. Different antigenic composition was also observed, not directly related to the different proteins contained therein; three antigens were definitely common to all extracts and two of them represented the major antigens of each extract.Despite the total protein content, the major and common antigens were found in similar concentrations in all three products examined. Therefore, the discrepancies observed in the precipitin reactions using the three commercial Micropolyspora faeni extracts are due to differences in the minor antigen composition of the extracts.     

Recent ostracods (Crustacea,Ostracoda) found in lowland springs of the provinces of Piacenza and Parma (Northern Italy)

The “fontanili” are artificial aquatic ecosystems, typical of the lowland plains of Northern Italy, exploiting natural resurgences of deep groundwater. These habitats are characterized by low variation in hydrologic, hydrochemical and thermal conditions throughout the year. Proper management is required to prevent the spring clogging by biomass accumulation. In spite of their importance as refugia for endangered species, many springs were completely abandoned in the last years and several of them will disappear. We report the results of a study carried out in 2001 on 31 springs of the provinces of Piacenza and Parma, distributed in seven areas defined on hydrological and geological considerations. Physical and chemical variables and parameters of waters were measured and ostracod samples were collected. Most of the springs showed high nitrate concentration, due to a diffuse pollution of agricultural origin. Twelve ostracod species in four families were identified. Ostracod valves were analysed by scanning electron microscopy. Cypria ophtalmica was found in all the springs; other relatively common species were Cyclocypris laevis,Notodromas persica, and Prionocypris zenkeri.The maximum number of species per site was four. Cypridopsis vidua,P. zenkeri, and N. persicashowed a very localized distribution in the study area. The ostracod fauna of the “fontanili” was compared to other species assemblages found in spring habitats and to the available information on recent freshwater ostracods reported for Italy.     

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (K_m) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.