Effects of 915 nm laser irradiation on human osteoblasts: a preliminary in vitro study.

Photobiomodulation (PBM) is a non-invasive treatment that uses laser or led devices making its effects a response to light and not to heat. The possibility of accelerating dental implant osteointegration and orthodontic movements and the need to treat refractory bone lesions, such as bisphosphonate related osteonecrosis of the jaws, has led researchers to consider the effects of PBM on bone for dentistry purposes. The aim of our study was to investigate the effects of 915 nm light supplied with a GaAs diode laser on human osteoblasts in vitro. Osteoblasts were isolated from mandibular cortical bone of a young healthy donor. The irradiation parameters were as follows: doses = 5, 15 and 45 J/cm2; power densities = 0.12 and 1.25 W/cm2; and irradiation times = 41.7, 125 and 375 s. We performed one irradiation per day for 3 and 6 days to study proliferation and differentiation, respectively. Microscopic analysis showed a greater amount of bone nodules in samples treated with 5 J/cm2 and 0.12 W/cm2 compared to controls (56.00 ± 10.44 vs 19.67 ± 7.64, P = 0.0075). Cell growth and quantification of calcium deposition did not show any differences when comparing irradiated and non-irradiated samples. Photobiomodulation, with the parameters investigated in the present study, positively modulated the mineralization process in human osteoblasts, inducing the formation of a greater amount of bone nodules, but did not increase cell proliferation.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea.

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (Papp = 0.75 ± 0.56 × 10-6 cm/s), about 2.6-fold higher than 5-FAM (Papp = 0.29 ± 0.08 × 10-6 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (Papp = 6.18 ± 1.46 × 10-6 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens.

Glomerular planted antigens (histones, DNA, and C1q) are potential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patients with LN. Prevalent antibody isotypes were defined, levels were determined, and glomerular colocalization was investigated. Renal and circulating antibodies were matched, and serum levels were compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (anti-α-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti-α-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and anti-α-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria>3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis.

Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells.

The dynamics of microtubule networks are known to have an impact on replication of influenza A virus in some cellular models. Here we present evidence suggesting that at late stages of LLC-MK2 cell infection by influenza A (H1N1) virus the ubiquitin-proteasome protein degradation system participates in destabilization of microtubules, and favours virus replication. Chemical inhibition of proteasome activity partially suppresses influenza A virus replication, while stimulation of proteasome activity favours influenza A virus replication. Conversely, in another cellular model, A549 cells, inhibitors and activators of proteasomes have a small effect on influenza A virus replication. These data suggest that influenza A virus might take selective advantage of proteasome functions in order to set up a favourable cytoskeletal "environment" for its replication and spread. Furthermore, the relationship between influenza virus and the host cell is likely to depend on both the cellular model and the virus strain.

Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential (Δψm). The aim of our study is to evaluate "in vitro" the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on Δψm. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and Δψm following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of Δψm in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of Δψm, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.

Polyphenon E(R), a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis.

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 µg/ml] than PC3 (IC50 = 145 µg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Structural and functional studies on a proline-rich peptide isolated from swine saliva endowed with antifungal activity towards Cryptococcus neoformans.

A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC(50) of 2.2µM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, ß-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage.

ß2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

The thrombogenic effect of ß2-glycoprotein I (ß2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since ß2GPI behaves as LPS scavenger, LPS/ß2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between ß2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among ß2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of ß2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of ß2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and ß2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-ß2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-ß2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that ß2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. ß2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between ß2GPI and TLR4 is confirmed by the reduction of anti-ß2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. ß2GPI binding is not affected by LPS at concentrations comparable to those found in both ß2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that ß2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to ß2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-ß2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of ß2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals.

Conserved alternative splicing of Arabidopsis transthyretin-like determines protein localization and S-allantoin synthesis in peroxisomes.

S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL(1-)) and the absence (TTL(2-)) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL(1-) localizes in peroxisomes, whereas TTL(2-) localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.

Study of 1,4-naphthoquinone as a new useful derivatization reagent for LC analysis of aliphatic thiols in dietary supplements and pharmaceuticals.

The use of 1,4-naphthoquinone as an advantageous pre-column reagent for liquid chromatography analysis of aliphatic thiol compounds is proposed. The compound reacts selectively in mild conditions (5 min at room temperature; pH 7.5) with thiol function. The resulting adducts were separated under isocratic conditions by using a reversed-phase column (C-12n) with a mobile phase corresponding to methanol/triethylammonium phosphate buffer (pH 3; 0.05 mol L(-1)) 65:35, v/v, at a flow rate of 0.4 mL min(-1) in presence of quercetin as internal standard. Detection was set at a wavelength of 420 nm. The effect of the derivatization reaction conditions on the N-acetylcysteine (NAC) reaction yield was investigated by a series of experiments. The yield of NAC derivative was found to be quantitative at a reagent thiol molar ratio of about 3 by comparison with an authentic specimen of synthesized NAC adduct, which was characterized by (1) H NMR, IR, and UV. Similar linear responses were observed by standard and placebo solutions (determination coefficient, 0.9998). The within- and between-day standard deviations (RSD) were ≤0.47 %. Recovery studies showed good results (100.03 %) with RSD 0.76 %. The limit of detection was about 20 pmol. The utility of the validated method for the determination of NAC in a new dietary supplement and commercial formulations is demonstrated.

Impaired phagocytosis in macrophages from patients affected by lysinuric protein intolerance.

Lysinuric Protein Intolerance (LPI, MIM 222700) is a recessive aminoaciduria caused by defective cationic amino acid transport in epithelial cells of intestine and kidney. SLC7A7, the gene mutated in LPI, codifies for the y+LAT1 subunit of system y(+)L amino acid transporter. LPI patients frequently display severe complications, such as pulmonary disease, haematological abnormalities and disorders of the immune response. The transport defect may explain only a part of the clinical aspects of the disease, while the mechanisms linking the genetic defect to the clinical features of the patients remain thus far obscure. The aim of the study is to investigate the consequences of SLC7A7 mutations on specific macrophage functions, so as to evaluate if a macrophage dysfunction may have a role in the development of pulmonary and immunological complications of LPI. The results presented 1) confirm previous data obtained in one LPI patient, demonstrating that arginine influx through system y(+)L is markedly compromised in LPI macrophages; 2) demonstrate that also system y(+)L-mediated arginine efflux is significantly lower in LPI macrophages than in normal cells and 3) demonstrate that the phagocytic activity of LPI macrophages is severely impaired. In conclusion, SLC7A7/y+LAT1 mutations lead to a defective phenotype of macrophages, supporting the pathogenetic role of these cells in the development of LPI-associated complications.

Serum thyroid-stimulating hormone as a predictor of cognitive impairment in an elderly cohort.

BACKGROUND: It is unclear whether in late life serum thyroid-stimulating hormone (TSH) predicts risk of developing cognitive impairment. OBJECTIVE: This study investigated the prospective relationship of serum TSH with the risk of developing mild cognitive impairment (MCI), Alzheimer's disease (AD) and vascular dementia (VaD) in an elderly cohort with a 4-year follow-up. METHODS: Data are for 660 subjects aged 65 years and older from an Italian population-based cohort who were cognitively normal at an extensive assessment in 1999/2000 and underwent follow-up assessment in 2003/2004. Serum TSH was measured at baseline. Multinomial logistic models adjusted for sociodemographic and cardiovascular risk factors were used to investigate the association of serum TSH (both as a tertile and continuous log-transformed variable) with risk of incident MCI, AD and VaD diagnosed according to international criteria. RESULTS: Over 3.8 ± 0.7 years of follow-up, there were 149 incident MCI cases (77 with impairment of memory and 72 with impairment of nonmemory domains) and 86 incident dementia cases (53 with AD, 28 with VaD). No association between baseline TSH and risk of developing any MCI subtype or AD was found. The highest TSH tertile had a threefold higher increased risk of VaD (OR: 3.25, 95% CI: 1.01-10.77, p = 0.048) compared to the lowest tertile. Risk of VaD increased about 60% for each 1 SD increase in log-transformed TSH (OR: 1.61, 95% CI: 1.06-2.44, p = 0.025). CONCLUSIONS: In this elderly cohort, baseline TSH was not related to the risk of developing MCI or AD, but high TSH was associated with an increased risk of VaD. These results suggest further need for research using larger samples to examine the role of TSH as a predictor of VaD and the role of thyroid autoimmunity in vascular cognitive impairment.

Autoimmunity in membranous nephropathy targets aldose reductase and SOD2.

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Synergistic activity of letrozole and sorafenib on breast cancer cells.

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 µM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Modulatory effect of rRNA synthesis and ppUL83 nucleolar compartmentalization on human cytomegalovirus gene expression in vitro.

The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. Here we have investigated the possible effects of rRNA synthesis inhibition upon the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate-early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolar matrix and the normal development of HCMV lytic infection.

Quantitative amino acids profile of monofloral bee pollens by microwave hydrolysis and fluorimetric high performance liquid chromatography.

Bee pollen is an attractive resource in the field of alternative remedies and thanks to the content of carbohydrates, crude fibers, proteins and lipids must be considered as a supplementary food of high potential rate. In characterization of bee pollen with the aim to define its value in human nutrition, the amino acids profile is one of the most important attributes. In the present study, the determination of amino acids composition of different monofloral bee pollen samples was obtained by an approach combining microwave acidic hydrolysis (60 min at 150 °C instead of 22 h at 120 °C in conventional oven) followed by derivatization using 9-fluorenylmethylchloroformate (FMOC-Cl) and separation of amino acids derivatives using a Phenomenex Kinetex core-shell 5 µm C18 (150 x 4.6 mm i.d.) column under a ternary gradient elution. Separation of 19 amino acids was achieved in about 40 min and fluorimetric detection (λexc = 265 nm λem = 315 nm) allowed selective and sensitive quantitation with LOQ values ranging within 0.14-3.00 µg/mL. Interestingly, the present approach allowed determination of some amino acids e.g., tryptophan and trans-4-hydroxyproline that are often lost by other methods of analysis. Significant differences in the composition of the considered samples were found confirming the impact of botanical origin of the product on its nutritional value. Principal Component Analysis was applied to treat the obtained data, highlighting the importance for discrimination, of detecting low abundance amino acids. The proposed method can be used as an advantageous alternative to the existing ones for characterization of bee pollen as an important source of dietary proteins.

Combinatorial co-expression of pheromone receptors, V2Rs.

Basal neurons of the vomeronasal organ of the mouse express a superfamily of about 120 pheromone receptors, named V2Rs, that are grouped in four families, A, B, C, and D, according to sequence homology. Family-A, -B, and -D V2Rs are expressed as one receptor gene per cell, but we previously reported their co-expression with family-C V2Rs. Here, we show that basal neurons can be further grouped according to the combinatorial expression of different V2Rs. Altogether, these findings suggest that in each basal neuron a transcriptional program is active for expressing a combination of two compatible receptors and for excluding, at the same time, the expression of all other V2Rs. Further analyses revealed non-random combinations of co-expression between family-C V2Rs and genes of the class Ib major histocompatibility complex. Thus, each basal neuron of the vomeronasal organ represents a highly qualified sensory unit for detecting very specific combinations of pheromonal cues.