Measurement of endogenous and TNFα-mediated H2O2 production in supernatants of SK-N-SH neuroblastoma cells with an enhanced chemiluminescence assay

A sensitive peroxidase-dependent luminol-enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H₂O₂) generation by tumour cells was established. This test system allows determination of H₂O₂ in concentrations as low as 25 pmol (50nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin flurorescence assay. After 3h incubation time 10⁴SK-N-SH neuroblastoma cells released 60° 5 pmol H₂O₂ in the supernatant and this level was significantly (p<0.025) increased by about 70% in the presence of 5pmol (100 ng/mL) recombinant tumour necrosis factor α (TNFα). In contrast, H₂O₂ production was slightly reduced by TNFα at a very low concentration of 0.5 fmol (0.01 ng/mL).     

Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis

An opioid receptor agonist, [D-Ala2,Me-Phe4,Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the mu-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The mu-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly mu-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.     

Repurposing the estrogen receptor modulator raloxifene to treat SARS-CoV-2 infection

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates strategies to identify prophylactic and therapeutic drug candidates to enter rapid clinical development. This is particularly true, given the uncertainty about the endurance of the immune memory induced by both previous infections or vaccines, and given the fact that the eradication of SARS-CoV-2 might be challenging to reach, given the attack rate of the virus, which would require unusually high protection by a vaccine. Here, we show how raloxifene, a selective estrogen receptor modulator with anti-inflammatory and antiviral properties, emerges as an attractive candidate entering clinical trials to test its efficacy in early-stage treatment COVID-19 patients.Subject terms: Infectious diseases, Drug development     

Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection

BACKGROUND: A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. MATERIALS AND METHODS: To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. RESULTS: No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). CONCLUSIONS: Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.     

The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.     

A crucial role for tryptophan catabolism at the host/Candida albicans interface

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-gamma- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-gamma may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.     

Production of a monoclonal antibody directed against the recombinant SEL1L protein

SEL1L, highly similar to the C elegans sel-1 gene, is a recently cloned human gene whose function is under investigation. SEL1L is differentially expressed in tumors and normal tissues and seems to play a role in tumor growth and aggressiveness. We used the recombinant N-terminus of the SEL1L protein to immunize a Balb/c mouse and produce a monoclonal antibody. A hybridoma secreting an antibody specifically reacting on the SEL1L recombinant fragment was selected. This monoclonal antibody, named MSel1, recognizes the SEL1L protein by Western blotting, immunofluorescence and immunohistochemistry on normal and tumor cells. MSel1 is able to recognize SEL1L even on archival tumor specimens and is therefore particularly appropriate to study SEL1L involvement in tumor progression.     

Effect of an implant containing the GnRH agonist deslorelin on secretion of LH, ovarian activity and milk yield of postpartum dairy cows

Prevention of high plasma progesterone concentrations in the early postpartum period may improve fertility. Our objective was to determine whether a Deslorelin implant (DESL; 2100 microg, s.c.) would reduce secretion of LH and alter follicle dynamics, plasma concentrations of progesterone, estradiol and PGF2alpha metabolite (PGFM) in postpartum dairy cows. Cows received DESL on Day 7 postpartum (Day 7, n=8) or were untreated (Control, n=9). All cows were injected with GnRH (100 microg, i.m.) on Day 14 to assess LH response. A protocol for synchronization of ovulation with timed AI was initiated on Day 60 (GnRH [Day 60], CIDR [Day 60 to Day 67], PGF2alpha [Day 67, 25 mg and Day 68, 15 mg], GnRH [Day 69] , AI [Day 70]). The LH response to injection of GnRH on Day 14 was blocked in animals treated with DESL. Numbers of Class 1 (<6 mm) follicles were unaffected (P > 0.05) whereas numbers of Class 2 (6 to 9 mm) (P < 0.01) and Class 3 (>9 mm) follicles were less (P < 0.01) in DESL cows between Day 7 and Day 21. From Day 22 to Day 60, DESL-treated cows had more of Class 1 follicles and less Class 2 (P < 0.01) and Class 3 (P < 0.01) follicles, and lower plasma concentrations of progesterone and estradiol (P < 0.01). Concentrations of PGFM between Day 7 and Day 42 were not affected by treatment (P > 0.05). All cows ovulated in response to GnRH on Day 69. Subsequent luteal phase increases in plasma progesterone concentrations (Day 70 to Day 84) did not differ. The use of the DESL implant associated with PGF2alpha given 14 days later suppressed ovarian activity and caused plasma progesterone concentrations to remain < 1 ng/mL between Day 22 and Day 51. The DESL implant did not affect milk production.     

Relationship between pollen emission and fruit production in olive (Olea europaea L.)

Aerobiological data of pollen emission concentrations is used to predict fruit production. The principal aim of this work was to study the relationship between pollen emission patterns, emission homogeneity and fruit production in olive (Olea europaea L.). Data of daily pollen concentrations in the atmosphere during the flowering period, collected over a 20‐year period in Perugia (Central Italy), and the corresponding fruit production data were analysed. Correlation and regression analyses on the partial pollen amounts (subdivisions of the whole flowering period), their statistical variability (expressed as coefficients of variation of the daily pollen concentration), and the production values in the different years demonstrate that pollen emission, during the seven to ten day period immediately preceding the maximum pollen emission day, appears to be most closely related to fruit production. Moreover, the pollen emission homogeneity (minimal variability in daily pollen concentrations) during the “critical” flowering period is very important for fertilization.     

Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction

BackgroundImpaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.ConclusionPLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.