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101.
Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D(1) receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D(1) receptor-stimulated cAMP formation, but failed to affect either adenosine A(2A) or DA D(2) receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D(1)-induced phosphorylation of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D(1) receptors, N/OFQ curtailed DA D(1) receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D(1) receptor signaling. 相似文献
102.
Pietro Dri Barbara Bisiacchi Rita Cramer Paolo Bellavite Giuseppina de Nicola Pierluigi Patriarca 《Molecular and cellular biochemistry》1978,22(2-3):159-166
Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O2
– and H2O2, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O2
– and H2O2 by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H2O2 is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H2O2 degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H2O2 degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs. 相似文献
103.
104.
Pilla P Sandomenico A Malachovská V Borriello A Giordano M Cutolo A Ruvo M Cusano A 《Biosensors & bioelectronics》2012,31(1):486-491
We present a fast and effective method for anchoring bioreceptors to optical waveguides exhibiting a poorly reactive polymer interface and that have to be minimally perturbed with respect to their design. The study originated from the need to biofunctionalize a fiber optic Long Period Grating (LPG) that is tuned in a highly sensitive working point, the so-called transition mode, through the deposition of a high refractive index overlay. In particular, a thin film of atactic polystyrene (PS) was dip-coated onto the LPG with a thickness suitable to optimize the LPG sensitivity to refractive index changes of the surrounding medium. Bovine serum albumin was selected as sacrificial layer for its well-known adhesion capabilities to PS surfaces, then glutaraldehyde was used to conjugate IgGs, serving as prototypical bioreceptor, on the device surface. The effectiveness of the immobilization method was assessed by studying the interaction between the immobilized IgG with a suitable anti-IgG. In a preliminary study performed by means of ELISA and surface plasmon resonance, optimal conditions for the biomolecular testing with the LPG were assessed. Four distinct interactions were thus monitored in real time following the shift of the LPG attenuation band. These experiments suggest a novel and interesting biofunctionalization approach of unreactive polymers with applications in immunosensing and basic life science research. 相似文献
105.
Federica Laudisi Manolo Sambucci Francesca Nasta Rosanna Pinto Rossella Lodato Pierluigi Altavista Giorgio Alfonso Lovisolo Carmela Marino Claudio Pioli 《Bioelectromagnetics》2012,33(8):652-661
Wireless local area networks are an increasing alternative to wired data networks in workplaces, homes, and public areas. Concerns about possible health effects of this type of signal, especially when exposure occurs early in life, have been raised. We examined the effects of prenatal (in utero) exposure to wireless fidelity (WiFi) signal‐associated electromagnetic fields (2450 MHz center‐frequency band) on T cell development and function. Pregnant mice were exposed whole body to a specific absorption rate of 4 W/kg, 2 h per day, starting 5 days after mating and ending 1 day before the expected delivery. Sham‐exposed and cage control groups were used as controls. No effects on cell count, phenotype, and proliferation of thymocytes were observed. Also, spleen cell count, CD4/CD8 cell frequencies, T cell proliferation, and cytokine production were not affected by the exposure. These findings were consistently observed in the male and female offspring at early (5 weeks of age) and late (26 weeks of age) time points. Nevertheless, the expected differences associated with aging and/or gender were confirmed. In conclusion, our results do not support the hypothesis that the exposure to WiFi signals during prenatal life results in detrimental effects on the immune T cell compartment. Bioelectromagnetics 33:652–661, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
106.
Nathan M. Kleczewski Daniel A. Herms Pierluigi Bonello 《Trees - Structure and Function》2012,26(2):525-533
In managed settings, seedlings are often fertilized with the objective of enhancing establishment, growth, and survival. However,
responses of seedlings to fertilization can increase their susceptibility to abiotic stresses such as drought. Seedlings acclimate
to variation in soil resources by reallocating carbon among different physiological processes and compartments, such as above
versus belowground growth, secondary metabolism, and support of ectomycorrhizal fungi (EMF). We examined the effects of nutrient
and water availability on carbon allocation to above and belowground growth of river birch (Betula nigra), as well as partitioning among root sugars, starch, phenolics, lignin, and EMF abundance. As nutrient availability increased,
total plant biomass and total leaf area increased, while percent root biomass decreased. Root sugars, total root phenolics
and EMF abundance responded quadratically to nutrient availability, being lowest at intermediate fertility levels. Decreased
water availability reduced total leaf area and root phenolics relative to well-watered controls. No interactions between nutrient
and water availability treatments were detected, which may have been due to the moderate degree of drought stress imposed
in the low water treatment. Our results indicate that nutrient and water availability significantly alter patterns of carbon
allocation and partitioning in roots of Betula nigra seedlings. The potential effects of these responses on stress tolerance are discussed. 相似文献
107.
Casadei R Piovesan A Vitale L Facchin F Pelleri MC Canaider S Bianconi E Frabetti F Strippoli P 《Genomics》2012,100(2):125-130
The "5' end mRNA artifact" issue refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5' end sequence. We performed a systematic identification of coding regions at the 5' end of all human known mRNAs, using an automated expressed sequence tag (EST)-based approach. Following parsing of more than 7 million BLAT alignments, we found 477 human loci, out of 18,665 analyzed, in which an extension of the mRNA 5' coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 cDNAs, and the consequences for the functional studies of these loci are discussed. We also generated a list of 20,775 human mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5' in the current form. 相似文献
108.
Pierluigi Maestrini Andrea Cavallini Milena Rizzo Tommaso Giordani Rodolfo Bernardi Mauro Durante Lucia Natali 《Journal of plant physiology》2009,166(14):391
Poplar is an important crop and a model system to understand molecular processes of growth, development and responses to environmental stimuli in trees. In this study, we analyzed gene expression in white poplar (Populus alba) plants subjected to chilling. Two forward suppression-subtractive-hybridization libraries were constructed from P. alba plants exposed to low non-freezing temperature for 6 or 48 h. Hundred and sixty-two cDNAs, 54 from the 6-h library and 108 from the 48-h library, were obtained. Isolated genes belonged to six categories of genes, specifically those that: (i) encode stress and defense proteins; (ii) are involved in signal transduction; (iii) are related to regulation of gene expression; (iv) encode proteins involved in cell cycle and DNA processing; (v) encode proteins involved in metabolism and energetic processes; and (vi) are involved in protein fate.Different expression patterns at 3, 6, 12, 24, 48 h at 4 °C and after a recovery of 24 h at 20 °C were observed for isolated genes, as expected according to the class in which the gene putatively belongs. Forty-four of 162 genes contained DRE/LTRE cis-elements in the 5′ proximal promoter of their orthologs in Populus trichocarpa, suggesting that they putatively belong to the CBF regulon. The results contribute new data to the list of possible candidate genes involved in cold response in poplar. 相似文献
109.
Flavia Pichiorri Hiroshi Okumura Tatsuya Nakamura Preston N. Garrison Pierluigi Gasparini Sung-Suk Suh Teresa Druck Kelly A. McCorkell Larry D. Barnes Carlo M. Croce Kay Huebner 《The Journal of biological chemistry》2009,284(2):1040-1049
We have previously shown that Fhit tumor suppressor protein interacts with
Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein.
Fhit-effector interactions are associated with a Fhit-dependent increase in
Fdxr stability, followed by generation of reactive oxygen species and
apoptosis induction under conditions of oxidative stress. To define Fhit
structural features that affect interactions, downstream signaling, and
biological outcomes, we used cancer cells expressing Fhit mutants with amino
acid substitutions that alter enzymatic activity, enzyme substrate binding, or
phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing
mutants that do not bind substrate or cannot be phosphorylated showed
decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization.
Expression of Fhit or mutants that bind interactor proteins results in
oxidative damage and accumulation of cells in G2/M or
sub-G1 fractions after peroxide treatment; noninteracting mutants
are defective in these biological effects. Gastric cancer clones expressing
noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity,
confirming that substrate binding, interaction with heat shock proteins,
mitochondrial localization, and interaction with Fdxr are important for Fhit
tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or
reduced in cancer cells because of rearrangement of the exquisitely DNA
damage-sensitive fragile FHIT gene. Restoration of Fhit expression
suppresses tumorigenicity of cancer cells of various types, and the ability to
induce apoptosis in cancer cells in vitro is reduced by specific Fhit
mutations (1,
2).Through studies of signal pathways affected by Fhit expression, by searches
for Fhit protein effectors, and by in vitro analyses of Fhit
activity, we and others have defined Fhit enzymatic activity in vitro
(3), apoptotic activity in
cells and tumors
(4–6),
and most recently identification of a Fhit protein complex that affects Fhit
stability, mitochondrial localization, and interaction with ferredoxin
reductase (Fdxr)5
(7). The complex includes Hsp60
and Hsp10 that mediate Fhit stability and may affect import into mitochondria,
where Fhit interacts with Fdxr, which is responsible for transferring
electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit
restoration in Fhit-deficient cancer cells increases production of
intracellular reactive oxygen species (ROS), followed by increased apoptosis
of cancer cells under oxidative stress conditions; conversely, Fhit-negative
cells escape apoptosis, likely carrying oxidative DNA damage that contributes
to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad
(HIT) family of proteins, suggested that the protein product would hydrolyze
diadenosine tetraphosphate or diadenosine triphosphate (Ap3A)
(8), and in vitro
studies showed that Ap3A was cleaved into ADP and AMP by Fhit. The
catalytic histidine triad within Fhit was essential for catalytic activity
(3), and a Fhit mutant that
substituted Asn for His at the central histidine (H96N mutant) was
catalytically inactive, although it bound substrate well
(3). Early tumor suppression
studies showed that cancer cells stably transfected with wild type (WT) or
H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested
the hypothesis that the Fhit-substrate complex sends the tumor suppression
signal (9,
10). To test this hypothesis,
a series of FHIT alleles was designed to reduce substrate-binding
and/or hydrolytic rates and was characterized by quantitative cell-death
assays on cancer cells virally infected with each allele. The allele series
covered defects as great as 100,000-fold in kcat and
increases as large as 30-fold in Km. Mutants with
2–7-fold increases in Km had significantly reduced
apoptotic indices and the mutant with a 30-fold increase in
Km retained little apoptotic function. Thus, the
proapoptotic function of Fhit, which is likely associated with tumor
suppressor function, is limited by substrate binding and is unrelated to
substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine
phosphorylation by the Src family protein kinases, which can phosphorylate
Tyr-114 of Fhit in vitro and in vivo
(12). After co-expression of
Fhit with the Elk tyrosine kinase in Escherichia coli to generate
phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated
Fhit were purified, and enzyme kinetics studies showed that monophosphorylated
Fhit exhibited monophasic kinetics with Km and
kcat values ∼2- and ∼7-fold lower, respectively,
than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic
kinetics; one site had Km and kcat
values ∼2- and ∼140-fold lower, respectively, than for
unphosphorylated Fhit; the second site had a Km
∼60-fold higher and a kcat ∼6-fold lower than for
unphosphorylated Fhit (13).
Thus, it was possible that the alterations in Km and
kcat values for phosphorylated forms of Fhit might favor
formation and lifetime of the Fhit-Ap3A complex and enhance tumor
suppressor activity (see Fhit forms
Kinetic parameters
% Sub-G1
Direct binding
Subcellular location
Co-IP in vivo
8-OHdG
Apoptosis
Tumor suppressor
Km (mm) kcat (s–1) A549 MKN74 Hsp60 Fdxr Hsp60 Fdxr
Fhit WT
1.6 +/– 0.19
2.7 +/– 0.95
43
24
Yes
Yes
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Catalyt mutants H96D
Up 2-fold
Down >2 × 104 29
NT
NT
NT
Cyt & mito
Yes
Yes
NT
Yes
NT
H96N
Up 2-fold
Down >5 × 105
31
14.4
NT
NT
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Loop mutants Y114A
Up 23-fold
Down 2-fold
3.7
NT
NT
NT
Cyt
+/–
+/–
+/–
No
No
Y114D
NT
NT
2.9
6
NT
NT
Cyt
+/–
+/–
–
No
–/+
Y114E
NT
NT
NT
NT
NT
NT
Cyt & mito
–/+
–/+
–
No
NT
Y114F
Up 5-fold
Up 1.1-fold
11.5
3
NT
NT
Cyt & mito
–/+
–/+
–
No
No
Y114W
Up 5-fold
Up 1.4-fold
NT
NT
NT
NT
Cyt & mito
–/+
–
–
NT
NT
del113–117
Up 10-fold
Down 38-fold
5
NT
NT
NT
NT
NT
NT
–
No
NT
Other mutants L25W
Up 7-fold
Down 4-fold
15
NT
NT
NT
Cyt
–
–
–
NT
–/+
I10W,L25W
Up 32-fold
Down 6-fold
11
NT
NT
NT
NT
NT
NT
NT
NT
NT
F5W
Up 3.3 fold
NT
NT
5
NT
NT
NT
NT
NT
+/–
No
NT
Purified pFhit pFhit
Down 0.4-fold
Down 7-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
ppFhit
Down 0.4-fold
Down > 100-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
Up 60-fold
Down 6-fold