Modulation of class III peroxidase pathways and phenylpropanoids in Arundo donax under salt and phosphorus stress.

Arundo donax L. is an invasive species that has been recently employed for biomass production due to its well-known ability to colonize harsh environment. Based on previous observations, the present study investigated the potential role of phenylpropanoids and class III peroxidases to confer adaptation through biochemical and transcriptomic analysis in A. donax after Na+ and P excess supply, both in single stress and in combination, and after growth at low P level. The levels of hydrogen peroxide, flavonoids (i.e., quercetin, apigenin and kaempferol derivatives) and the activity of class III peroxidases, as well as the expression of several genes encoding for their enzymes involved in their biosynthesis, increased when Na+ was supplied in combination with P. These results suggest that those biomolecules are involved in the response of A. donax, to the presence of +Na and P in the soil. Moreover, even though at the sampling time no significant accumulation of lignin has been determined, the trend of accumulation of such metabolite and most of all the increase of several transcripts involved in its synthesis was found. This work for the first time indicates the need for further investigation devoted to elucidating whether the strengthening of cell walls via lignin synthesis is one of the mechanisms used by A. donax to adapt to harsh environments.

Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Efficient Non-Invasive Plasmid-DNA Administration into Tibialis Cranialis Muscle of "Little" Mice.

BACKGROUND: An alternative treatment for growth hormone deficiency based on hGH-DNA administration, followed by electro gene transfer, was investigated by injecting the plasmid into surgically exposed or non-exposed quadriceps or tibialis muscle of immunodeficient "little" mice. METHODS: An optimization of electrotransfer conditions via a new combination of high/low voltage pulses is presented. After 3 days, serum hGH was determined and in a 28-day assay, the relative growth parameters were compared. RESULTS: Both groups exhibited similar results: 5.0 ± 2.2 (SD) and 3.5 ± 0.9 ng hGH/ml (P>0.05; n=7) for the exposed quadriceps and non-exposed tibialis treatments, respectively. The final body weight increases were 16.1% for the quadriceps and 18.9% for the tibialis group. The tail and nose-to-tail length increases were 4.5% and 7.1% for the quadriceps and 4.8 and 4.6% for the tibialis group. The right and left femur length increases, obtained from radiographic measurements, were 16.9% and 12.7% for the quadriceps and 19.4% and 12.3% for the tibialis, respectively. A non-significant difference between exposed quadriceps and non-exposed tibialis treatments (P=0.48) was confirmed via a completely integrated statistical analysis. Circulating mIGF-1 levels were 126 ± 47, 106 ± 93 (P>0.05) and 38 ± 15 ng/ml for the quadriceps, tibialis and saline treatments, respectively. CONCLUSION: These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment, much more suitable for pre-clinical testing than administration into exposed quadriceps.

Thin layered drawing media probed by THz time-domain spectroscopy.

Dry and wet drawing materials were investigated by THz time-domain spectroscopy in transmission mode. Carbon-based and iron-gall inks have been studied, some prepared following ancient recipes and others using current synthetic materials; a commercial ink was studied as well. We measured the THz signals on the thin films of liquid inks deposited on polyethylene pellicles, comparing the results with the thick pellets of dried inks blended with polyethylene powder. This study required the implementation of an accurate experimental method and data analysis procedure able to provide a reliable extraction of the material transmission parameters from a structured sample composed of thin layers, down to a thickness of a few tens of micrometers. THz measurements on thin ink layers enabled the determination of both the absorption and the refractive index in an absolute scale in the 0.1-3 THz range, as well as the layer thickness. THz spectroscopic features of a paper sheet dyed by using one of the iron-gall inks were also investigated. Our results showed that THz time-domain spectroscopy enables the discrimination of various inks on different supports, including the application on paper, together with the proper determination of the absorption coefficients and indices of refraction.

Supercooling and freezing processes in nanoconfined water by time-resolved optical Kerr effect spectroscopy.

Using heterodyne-detected optical Kerr effect (HD-OKE) measurements, we investigate the vibrational dynamics and the structural relaxation of water nanoconfined in Vycor porous silica samples (pore size ≃ 4 nm) at different levels of hydration and temperatures. At low levels of hydration corresponding to two complete superficial water layers, no freezing occurs and the water remains mobile at all the investigated temperatures with dynamic features similar, but not equal to, the bulk water. The fully hydrated sample shows the formation of ice at about 248 K. This process does not involve all the contained water; a part of it remains in a supercooled phase. The structural relaxation times measured from the decay of the time-dependent HD-OKE signal shows the temperature dependence largely affected by the hydration level; the low frequency (ν < 500 cm(-1)) vibrational spectra obtained by the Fourier transforms of the HD-OKE signal appear less affected by confinement.

Optical Kerr effect of liquid and supercooled water: the experimental and data analysis perspective.

The time-resolved optical Kerr effect spectroscopy (OKE) is a powerful experimental tool enabling accurate investigations of the dynamic phenomena in molecular liquids. We introduced innovative experimental and fitting procedures, that enable a safe deconvolution of sample response function from the instrumental function. This is a critical issue in order to measure the dynamics of liquid water. We report OKE data on water measuring intermolecular vibrations and the structural relaxation processes in an extended temperature range, inclusive of the supercooled states. The unpreceded data quality makes possible a solid comparison with few theoretical models: the multi-mode Brownian oscillator model, the Kubo's discrete random jump model, and the schematic mode-coupling model. All these models produce reasonable good fits of the OKE data of stable liquid water, i.e., over the freezing point. The features of water dynamics in the OKE data becomes unambiguous only at lower temperatures, i.e., for water in the metastable supercooled phase. We found that the schematic mode-coupling model provides the more rigorous and complete model for water dynamics, even if its intrinsic hydrodynamic approach does not give a direct access to the molecular information.

Human adipose tissue derived pericytes increase life span in Utrn (tm1Ked) Dmd (mdx) /J mice.

Duchenne muscular dystrophy (DMD) is still an untreatable lethal X-linked disorder, which affects 1 in 3500 male births. It is caused by the absence of muscle dystrophin due to mutations in the dystrophin gene. The potential regenerative capacity as well as immune privileged properties of mesenchymal Stem Cells (MSC) has been under investigation for many years in an attempt to treat DMD. One of the questions to be addressed is whether stem cells from distinct sources have comparable clinical effects when injected in murine or canine muscular dystrophy animal models. Many studies comparing different stem cells from various sources were reported but these cells were obtained from different donors and thus with different genetic backgrounds. Here we investigated whether human pericytes obtained from 4 different tissues (muscle, adipose tissue, fallopian tube and endometrium) from the same donor have a similar clinical impact when injected in double mutant Utrn (tm1Ked) Dmd (mdx) /J mice, a clinically relevant model for DMD. After a weekly regimen of intraperitoneal injections of 10(6) cells per 8 weeks we evaluated the motor ability as well as the life span of the treated mice as compared to controls. Our experiment showed that only adipose tissue derived pericytes are able to increase significantly (39 days on average) the life span of affected mice. Microarray analysis showed an inhibition of the interferon pathway by adipose derived pericytes. Our results suggest that the clinical benefit associated with intraperitoneal injections of these adult stem cells is related to immune modulation rather than tissue regeneration.

An optically coupled system for quantitative monitoring of MRI gradient currents induced into endocardial leads.

The time-varying gradient fields generated during Magnetic Resonance Imaging (MRI) procedures have the potential to induce electrical current on implanted endocardial leads. Whether this current can result in undesired cardiac stimulation is unknown. This paper presents an optically coupled system with the potential to quantitatively measure the currents induced by the gradient fields into endocardial leads during MRI procedures. Our system is based on a microcontroller that works as analog-to-digital (A/D) converter and sends the current signal acquired from the lead to an optical high-speed light-emitting-diode transmitter. Plastic fiber guides the light outside the MRI chamber, to a photodiode receiver and then to an acquisition board connected to a PC. The preliminary characterization of the performances of the system is also presented.

Evidence of two distinct local structures of water from ambient to supercooled conditions.

The liquid and supercooled states of water show a series of anomalies whose nature is debated. A key role is attributed to the formation of structural aggregates induced by critical phenomena occurring deep in the supercooled region; the nature of the water anomalies and of the hidden critical processes remains elusive. Here we report a time-resolved optical Kerr effect investigation of the vibrational dynamics and relaxation processes in supercooled bulk water. The experiment measures the water intermolecular vibrations and the structural relaxation process in an extended temperature range, and with unprecedented data quality. A mode-coupling analysis of the experimental data enables to characterize the intermolecular vibrational modes and their interplay with the structural relaxation process. The results bring evidence of the coexistence of two local configurations, which are interpreted as high-density and low-density water forms, with an increasing weight of the latter at low temperatures.

Isolation of the pituitary gonadotrophic α-subunit hormone of the giant amazonian fish: pirarucu (Arapaima gigas).

The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

Phase-locking to a free-space terahertz comb for metrological-grade terahertz lasers.

Optical frequency comb synthesizers have represented a revolutionary approach to frequency metrology, providing a grid of frequency references for any laser emitting within their spectral coverage. Extending the metrological features of optical frequency comb synthesizers to the terahertz domain would be a major breakthrough, due to the widespread range of accessible strategic applications and the availability of stable, high-power and widely tunable sources such as quantum cascade lasers. Here we demonstrate phase-locking of a 2.5 THz quantum cascade laser to a free-space comb, generated in a LiNbO(3) waveguide and covering the 0.1-6 THz frequency range. We show that even a small fraction (<100 nW) of the radiation emitted from the quantum cascade laser is sufficient to generate a beat note suitable for phase-locking to the comb, paving the way to novel metrological-grade terahertz applications, including high-resolution spectroscopy, manipulation of cold molecules, astronomy and telecommunications.

Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.

The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.

Electromagnetic compatibility of WLAN adapters with life-supporting medical devices.

This paper investigates the electromagnetic compatibility of 45 critical care medical devices (infusion pumps, defibrillators, monitors, lung ventilators, anesthesia machines and external pacemakers) with various types of wireless local area network (WLAN, IEEE 802.11 b/g, 2.45 GHz, 100 mW) adapters. Interference is evaluated by performing ad-hoc tests according to the ANSI C63.18 recommended practice. The behavior of the devices during the tests was monitored using patient simulators/device testers specific for each device class. Electromagnetic interference cases were observed in three of 45 devices at a maximum distance of 5 cm. In two cases the interference caused malfunctions that may have clinical consequences for the patient. The authors' findings show that the use of these wireless local area network adapters can be considered reasonably safe, although interference may occur if they are operated at very close distance (<10 cm) to the medical devices.

A pilot study on potency determination of human follicle-stimulating hormone: a comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.

Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice.

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography.

Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCG

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

Lymphocytic prolactin does not contribute to systemic lupus erythematosus hyperprolactinemia.

OBJECTIVES: Lymphocytic prolactin (PRL) gene expression is detected in the majority of the immune cells and it is not known if this source contributes to hyperprolactinemia in systemic lupus erythematosus (SLE). We have therefore evaluated lymphocytic PRL secretion and gene expression in SLE and healthy controls. METHODS: Thirty SLE patients (ACR criteria) and 10 controls were selected for the study. Serum levels of PRL and macroprolactin were detected by immunofluorometric assay and gel filtration chromatography, respectively. The lymphocytic biological activity was determined by Nb2 cells bioassays. Lymphocytic PRL gene expression was evaluated by RT-PCR assay. RESULTS: The median serum PRL levels of the 30 SLE patients was higher than the control group (9.65 (1.9-38.9) vs. 6.40 (2.4-10.3) ng/mL, p=0.03). A significant difference was detected between median serum PRL levels of active SLE, inactive SLE and controls (10.85 (5-38.9) vs. 7.65 (1.9-15.5) vs. 6.40 (2.4-10.3) ng/mL), p=0.01). The higher frequency of mild hyperprolactinemia was detected among active SLE in comparison with inactive SLE and controls (7 (38.9%) vs. 1 (8.3%) vs. 0 (0%)), with statistical significance (p=0.02). Nb2 cells assay revealed uniformly low levels of lymphocytic PRL in active, inactive and control groups without statistical significance among them (24.2 (8-63) vs. 27 (13.6-82) vs. 29.5 (8-72) ng/mL), p=0.84). Furthermore, median lymphocytic PRL gene expression evaluated by RT-PCR assay was comparable in both active and inactive SLE groups (p=0.12). CONCLUSIONS: This is the first study to exclude a lymphocytic source of PRL, pointing out a pituitary etiology for hyperprolactinemia in SLE. However, other sources from the immune system cannot be ruled out.

Structure and acoustic properties of hydrated nafion membranes.

The propagation of acoustic waves in water-hydrated Nafion membrane has been monitored using heterodyne-detected transient grating spectroscopy. At room temperature, upon increasing the water content, the speed of sound drops to a value lower than the respective velocities of sound in pure Nafion and pure water. This counterintuitive effect can be explained by a simple calculation of the sound velocity in an effective medium made of water and Nafion polymer. Upon cooling, a phase separation occurs in the sample, and the formation of ice is observed (M. Pineri et al. J. Power Sources 2007, 172, 587-596). This phase transition is characterized via a second acoustic wave observed in the signal. Sound propagation and X-ray diffraction confirm the formation of crystalline ice on the membrane surface, that reversibly melts upon heating. The amount of ice that forms in the sample is monitored as a function of temperature and represents an order parameter for the transition. This parameter follows a power law with an exponent of 0.5, indicating the critical nature of the observed process.