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The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies.

Urinalysis could be helpful to investigate the health status of giraffes held in captivity using noninvasive methods to avoid animal handling or anesthesia. We collected 52 voided urine samples from 20 giraffes of different ages, sexes, and subspecies from the ground. To evaluate potential interference by soil contaminants, a pilot study was performed using 20 urine samples obtained from 10 cows. All bovine and 29 giraffe samples were subjected to routine urinalysis including urine specific gravity (USG). All samples were analyzed for urine total protein (uTP), urine creatinine (uCrea) concentration, and urine protein-to-urine creatinine ratio (UPC). Urinary proteins were separated by SDS-PAGE electrophoresis. No significant differences were determined between free-catch and urine sampled from the ground in cows. Giraffe urine was pale-yellow, with alkaline pH (>8.0) and a mean USG of 1.035 ± 0.013. The uTP, uCrea, and UPC expressed as median (range) were 0.20 (0.08–0.47) g/L, 2.36 (0.62–5.2) g/L, and 0.08 (0.05–0.15), respectively. SDS-PAGE allowed the separation of protein bands with different molecular masses, including putative uromodulin at 90 kD, putative albumin at 64 kD, and putative immunoglobulin heavy and light chains at 49 kD and 25 kD, respectively. Urine collection from the ground appears to be a reliable technique for urinalysis and urine electrophoresis in giraffes.

Urinalysis is widely recognized to be a useful tool in routine health investigations, since it can diagnose numerous pathologies. Considering the paucity of knowledge concerning giraffes, urine from 44 giraffes (Giraffa camelopardalis) (18 males and 26 females, from 3 months of age to 21 years of age) underwent routine urinalysis, 1D-electrophoresis, and protein identification using mass spectrometry, with the aim of identifying the urinary reference values and the urine proteome. The urine specific gravity (USG), urine total proteins (uTP), urine creatinine (uCr), and urine protein:creatinine ratio (UPC) reference values, reported as the median, and lower limit (LL) and upper limit (UL), were 1.030 (1006&ndash, 1.049), 17.58 (4.54&ndash, 35.31) mg/dL, 154.62 (39.59&ndash, 357.95) mg/dL, and 0.11 (0.07&ndash, 0.16), respectively. Mass spectrometry, together with electrophoresis, revealed a pattern of common urinary proteins, albumin, lysozyme C, and ubiquitin were the most represented proteins in the giraffe urine. It has been hypothesized that these proteins could act as a defense against microbes. Moreover, in giraffes, urinalysis could be a valid tool for gauging renal function and physiological status changes.

Generation of 3′ single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5′ strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3′-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.

Molecular food traceability requires continuous updates to identify more robust, efficient and affordable methodologies to guarantee food quality and safety and especially consumers’ health. Available commercial kits are often unsatisfactory and require modifications to successfully detect single components on complex and transformed food matrices. Here we report a simple method for molecular traceability of cold-pressed hazelnut oil based on microsatellite DNA markers. Different genomic extraction methodologies were tested and a total genome pre-amplification step was applied on PCR-negative samples. PCR-capillary electrophoresis using nine microsatellites demonstrates the accuracy of the fingerprint analysis even for filtered oil.

Multiwalled carbon nanotube (MWCNT)-based gas sensors were decorated by an electrophoretic deposition of electrochemically preformed gold nanoparticles (Au NPs) with controlled size and loading, for the detection of gaseous pollutants at sub-ppm concentrations and operating temperature in the range of 100-200 °C. The effects of the tailored Au content on the sensitivity and selectivity of MWCNT-based gas sensors were evaluated towards the NO2 monitoring, and also towards some interfering reducing gases, such as NH3 and H2S. Gas sensing measurement revealed the highest NO2 response up to sub-ppm level by using MWCNTs functionalized by the lowest Au content; instead, the worse NO2 response was obtained by modified MWNTs containing the highest Au loading. Moreover, the control of the deposited gold loading has allowed to control the MWCNT sensing response; specifically the increase of gold content on MWCNT has reduced the selectivity and sensitivity towards NO2 gas, and, on the contrary, at the same time it has improved those towards H2S and NH3 interfering gases. Finally, binary gas mixtures (NO2/H2S and NO2/NH3) were performed to evaluate the detection of the targeted NO2 gas, simulating possible real-world conditions. © 2015 Elsevier B.V. All rights reserved.

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96±1.12g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed.

Doping hematite with different elements is a common strategy to improve the electrocatalytic activity towards the water oxidation reaction, although the exact effect of these external agents is not yet clearly understood. Using a feasible electrophoretic procedure, we prepared modified hematite films by introducing in the deposition solution Ti(IV) butoxide. Photoelectrochemical performances of all the modified electrodes were superior to the unmodified one, with a 4-fold increase in the photocurrent at 0.65 V vs. SCE in 0.1 M NaOH (pH 13.3) for the 5% Ti-modified electrode, which was the best performing electrode. Subsequent functionalization with an iron-based catalyst led, at the same potential, to a photocurrent of ca. 1.5 mA?cm-2 , one of the highest achieved with materials based on solution processing in the absence of precious elements. AFM, XPS, TEM and XANES analyses revealed the formation of different Ti(IV) oxide phases on the hematite surface, that can reduce surface state recombination and enhance hole injection through local surface field effects, as confirmed by electrochemical impedance analysis.

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-d-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12–39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.

Uterine leiomyoma is the most common smooth benign neoplasm. In the present study, we analyzed the global interstitial fluid (IF) profile of leiomyoma vs. normal myometrium to identify protein dysregulation involved in leiomyoma pathogenesis. Two-dimensional gel electrophoresis and mass spectrometry were used to generate and compare the global interstitial fluid profiles of the leiomyoma and of the normal tissue. Two proteins were validated by immunohistochemistry. By comparing the interstitial fluid profile of the leiomyoma with that of the normal myometrium, the levels of seven proteins were found to be significantly different: four structural organization proteins (desmin, prelamin-A/C, transgelin and α-actinin-1), an inflammatory response (α1-antitrypsin), a response to oxidative stress (peroxiredoxin-2), and a folding protein (heat shock 70 kDa protein 1A/1B). Desmin, α1-antitrypsin and peroxiredoxin-2 were upregulated in the leiomyoma, whereas heat shock 70 kDa protein 1A/1B, α-actinin-1, prelamin-A/C and transgelin were downregulated. Desmin and α1-antitrypsin were further validated by immunohistochemistry. By identifying proteins with altered expression levels compared to the myometrium from several pathways of the leiomyoma pathogenesis, we found the leiomyoma interstitial fluid to have a characteristic proteomic profile. A better appreciation of the pathophysiology of the disease can be useful in the development of conservative treatments that serve as viable alternatives to hysterectomy.

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited. The aims of this study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. Urinalysis was performed and urine protein to creatinine ratio (UPC) was calculated. Urine proteins were separated by SDS-PAGE on 4-12% gels, 20 different protein bands were cut, reduced, alkylated and digested by trypsin before ESIQ-TOF mass spectrometry analysis; protein identification was performed using MASCOT science search engine. CKD cats had significantly higher UPC (median 0.9, P < .01) and significantly lower urine specific gravity (USG, median 1.014, P < .01) than healthy cats (UPC 0.1; USG 1.070). SDS-PAGE allowed visualization of qualitative and quantitative differences in protein profiles. CKD cats had significantly less bands (P < .01), particular at molecular weight (MW) > 100 kDa (P < .01). Eleven different proteins were identified by mass spectrometry in cat urine. In CKD a2-macroglobulin (168 kDa) and uromodulin (90 kDa) disappeared, while albumin (64 kDa), retinol-binding protein (RBP, 22 kDa) and cystatin (16 kDa) were enhanced. Cauxin (carboxylesterase 5A, 61 kDa), transferrin (80 kDa), angiotensin-converting enzyme 2 (93 kDa), inter-a-trypsin inhibitor heavy chain H4 (103 kDa), albumin- (55 kDa) and cauxin- (40 kDa) fragments, haptoglobin (45 kDa) and immunoglobulin light chains (24 kDa) were also identified. In conclusion, proteomic techniques were successfully used to investigate urine proteins, revealing different patterns between healthy and CKD cats; some of these proteins could be considered as promising biomarkers of chronic renal damage in feline patients.

Data on urine proteome in big cats are lacking, therefore the aim of our research is to study urine proteome with sensitive analytical methods to monitor the health status in Panthera leo, Panthera tigris and Panthera pardus. Urine samples were collected 4 times from May to December 2012, by noninvasive methods from 2 tigers, 2 lions and 3 leopards from Rome’s bioparco. Urinary total proteins (UTP), creatinine and the urinary protein to creatinine ratio (UP/C) were measured by automated methods (Olympus AU400) routinely used for dogs and cats. Urine samples were analyzed by sodium-dodecyl-sulphate poliacrilamide gel electrophoresis (SDS-PAGE, Invitrogen®) to separate urinary proteins. Mean UTP values were not significantly different in lions, tigers and leopards (40, 43, 46 mg/dl respectively), while mean UP/C values were significantly lower in lions (0.11) than in tigers (0.28) but not than leopards (0.22). In absence of reference ranges, these values could be considered as representative of healthy animals. Regarding qualitative proteinuria, with the highly sensitive SDS-PAGE, 29 different protein bands were visualized in lions, 26 in tigers and 30 in leopards, including the 2 most abundant ones, which could be identified as albumin and putative cauxin, a specific feline’s protein. Noninvasive sampling was easy to apply without causing stress to animals and allowed to collect valuable samples for routine and/or more sensitive analysis.

Serum/plasma protein electrophoresis is the current standard technique in human and veterinary medicine to investigate dysproteinemias and to identify and monitor specific pathologies. Serum/plasma protein electrophoresis application in reptile medicine is limited because of lack of reference values. Aims of this study were to provide reference intervals for plasma protein fractions of 30 clinically healthy Hermann’s tortoises (Testudo hermanni) and compare the results with plasma samples collected from 10 diseased animals. 0.5-0.7 mL of blood were collected from the dorsal cervical sinus and flushed into 1.3 mL lithium heparin tubes. In the experimental conditions, at least 4 fractions were always present in all specimens: albumin, α globulins (frequently resolved in two peaks) β globulins (sometimes separated in two peaks) and γ globulins. In healthy young specimens (1-2 years old), plasma proteins were represented by 42% of albumin, 18,5 % of α globulins, 29,5 % of β globulins and 10% of γ globulins. In healthy adult specimens lower values of β (20 – 22 %) and γ (4 – 5 %) fractions were found, while values of α fractions were increased (28 – 32 %). The high amount of α globulins in adult tortoises could be related to the abundance of two specific protein components of α zone: α2 macroglobulin (α2M) and α lipoproteins (HDL). In unhealthy specimens (traumatized or subjected to surgical operations) a huge increase of β fraction was evident (40-42%) associated to a decrease of albumin fraction (21%). Changes in this fraction are also dramatic in birds with acute disease.

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited (1,2,3). The aims of our study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. For all samples urinalysis and urine protein to creatinine ratio (UPC) were performed. Urine proteins were further separated by SDS-PAGE and the bands were reduced, alkylated and then digested by trypsin before ESI-Q-TOF mass spectrometry analysis. Protein identification was performed using MASCOT science search engine. Healthy cats had significantly (p

In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited. The aims of our study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. For all samples urinalysis and urine protein to creatinine ratio (UPC) were performed. Urine proteins were further separated by SDS-PAGE and the bands were reduced, alkylated and then digested by trypsin before ESI-Q-TOF mass spectrometry analysis. Protein identification was performed using MASCOT science search engine. Healthy cats had significantly (p

A significant proportion (∼15%) of colorectal cancer (CRC), either sporadic or arising in the setting of the hereditary non-polyposis colorectal carcinoma syndrome, features microsatellite instability (MSI). Five MSI loci, either mononucleotide or dinucleotide repeats (Bat25, Bat26, D2S123, D5S346 and D17S250), are included in the Bethesda panel and capillary electrophoresis represents the usual gold standard technique.1 Samples are classified as MSI-low (L) if only one locus is abnormal or as MSI-high (H) if alterations extend to two or more loci. In a study published in 2009 in the Journal of Clinical Pathology , Odenthal et al 2 validated, on the 2100 Bioanalyzer (Agilent …

Urine markers are advocated to early detect kidney damage in the clinical practice, nevertheless histology remains the gold standard. The aim of this study was to evaluate quali-quantitative proteinuria and possible renal damage using different non-invasive tests in dogs affected by leishmaniasis.Based on clinical signs and serology/cytology, 26 affected dogs (Leish) were included. Fifteen healthy, non-proteinuric dogs were selected as Control. Upon admission, all dogs underwent to physical examination, Systolic Blood Pressure (SBP) measurement, clinicopathological evaluation (CBC, Urea, Creatinine, ALT, ALP, Glucose, Calcium, Phosphorous, Sodium, Potassium, Cholesterol, Triglyceride, Albumin, Total Protein), serology, urinalysis, ultrasound examination and Renal Resistive Index (RRI) determination. Urine Total Protein and Urine Albumin to Creatinine ratios (UPC; UAC), urine High Resolution agarose and Silver Staining Sodium-Dodecyl-Sulphate-PolyAcrylamide gel electrophoresis (HRE; SDS-PAGE) were performed. A cut-off of 66 kDa was selected to classify bands in High or Low Molecular Weight (HMW; LMW). Data were analyzed with non-parametric statistics and ROC curve analysis (ROC). A difference was considered significant for p0.5) was detected in 16/26 dogs (P), 5/26 were borderline proteinuric (BLP; UPC 0.2-0.5) and 5/26 were nonproteinuric (NP, UPC 0.17, 89% Sensitivity and 88% Specificity and for UAC >0.013, 94% Sensitivity and 82% Specificity. The Areas under the Curve for UPC and UAC were 0.89 and 0.92, respectively. Non-invasive methods, particularly urinary SDS-PAGE, could be useful to detect an early renal damage in canine leishmaniasis. Further studies are required to correlate these findings to renal histology

Urine samples were collected from 66 feline hospital admissions by cystocentesis or free catch, and analyzed (UPC, HRE). Within-assay imprecision for high (H), medium (M) and low (L) concentrations, between-assay imprecision, linearity/accuracy and percentage recovery were determined for albumin. Albumin was assessed by urine to creatinine ratio (UAC), percentage (ALB%) and mg/dl (ALB mg/dl). Cases were categorized: a) as chronic kidney disease with (CKDC) and without complications (CKD), feline lower urinary tract disease (FLUTD) or other, and b) as proteinuric (P), borderline proteinuric (BP), or not proteinuric (NP), and c) by type of sediment active (A) or not active (NA). Results: HRE within-assay CV was 2.58%(H), 5.31%(M), 3.42%(L), between-assay CV was 3.52%, recovery percentage was 97%, 93% and 109% for high, medium and low concentration, respectively, and linearity was high (r=0.99). Compared with UPC at different cut-off values, HRE showed higher specificity and PPV but lower sensitivity and NPV than dipstick to measure proteinuria. Dipsticks showed frequent false positives compared with HRE. Based on UPC, measures of albuminuria were higher in proteinurics than non-proteinurics, and ALB% correlated to UPC (r=0.35, R2=0.12, p

A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the β-Amyloid peptide (Aβ(1-42)), a biomarker for Alzheimer's Disease (AD), at concentrations close to physiological conditions. The CE-LIF method allowed the interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles (NPs) and the soluble Aβ(1-42) peptide monomers to be highlighted. These results were confirmed by surface plasmon resonance (SPR) and confocal laser scanning microscopy (CLSM). Whereas SPR showed an interaction between the NPs and the Aβ(1-42) peptide, CLSM allowed the formation of large aggregates/assemblies at high NP and peptide concentrations to be visualized. All these results suggested that these nanoparticles could bind the Aβ(1-42) peptide and influence its aggregation kinetics. Interestingly, the non-PEGylated poly(alkyl cyanoacrylate) NPs did not alter the aggregation kinetics of the Aβ(1-42) peptide, thus emphasizing the high level of discrimination of the CE-LIF method with respect to NPs.

Microtubules play important roles during growth and morphogenesis of plant cells. Multiple isoforms of alpha- and beta-tubulin accumulate in higher plant cells and originate either by transcription of different genes or by post-translational modifications. The use of different tubulin isoforms involves the binding of microtubules to different associated proteins and therefore generates microtubules with different organizations and functions. Tubulin isoforms are differentially expressed in vegetative and reproductive structures according to the developmental program of plants. In grapevine (Vitis vinifera L.), vegetative and reproductive structures appear on the same stem, making this plant species an excellent model to study the accumulation of tubulin isoforms. Proteins were extracted from grapevine samples (buds, leaves, flowers and tendrils) using an optimized extraction protocol, separated by two-dimensional electrophoresis and analyzed by immunoblot with anti-tubulin antibodies. We identified eight alpha-tubulin and seven beta-tubulin isoforms with pI around 4.8-5 that group into separate clusters. More acidic alpha-tubulin isoforms were detected in buds, while more basic alpha-isoforms were prevalently found in tendrils and flowers. Similarly, more acidic beta-tubulin isoforms were used in the bud stage while a basic beta-tubulin isoform was essentially used in leaves and two central beta-tubulin isoforms were characteristically used in tendrils and flowers. Acetylated alpha-tubulin was not detected in any sample while tyrosinated alpha-tubulin was essentially found in large latent buds and in bursting buds in association with a distinct subset of tubulin isoforms. The implication of these data on the use of different tubulin isoforms during grapevine development is discussed.

An integrated readout circuit for Lab-on-a-Chip applications is presented. The overall system consist of a 640x480 array of capacitor sensors and actuators. Sensors detect dielectric permittivity variation thanks to dielectrophoresis (DEP) process. Usually for this kind of applications an off-chip analog-to-digital converter is used. As a consequence, the noise floor increases and high signal-to-noise ratios are difficulty achieved. On the other hand, these applications requires a stringent noise floor specification (>10b) and a relaxed linearity (approximate to 8b). In this design, the noise coupled to the signal at the chip pad is reduced by using an on-chip analog-to-digital converter. The complete sensor readout channel is composed by two main blocks: a pre-amplifier with programmable gain and an algorithmic analog-to-digital converter with a 1.5-bit/stage architecture. Each one is realized with fully differential switched capacitor technique. In order to save chip area and power consumption a time sharing technique has been taken into account using a single operational amplifier for the pre-amplification stage and the conversion stage. The proposed A/D converter has 11b resolution, a sampling rate of about 100ksample/s and an input full-scale range of 1.2Vp-p differential. Simulation results show a SNR=65.7 dB and an ENOB value of 10.6b. Its power consumption is about 150 mu W. Readout chain is implemented in 0.35 mu m CMOS technology with a 3.3V supply voltage

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1–2 h) of cells with 0.1–5 mMN-acetyl-l-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiolN-acetyl-d-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin α-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and β-actin. NAC pretreatment augmented integrin α-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, α-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H2O2 with ferrous ions, spermine–NO, KO2, and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.

Human seminal plasma proteins from fertile and infertile men were fractionated by electrophoresis. The amounts of three Coomassie-stained protein bands were measured by densitometry. Their relative ratios were constant in normospermic men but varied in the infertile patients. Laboratory manipulation of the semen was shown not to affect the protein ratios as observed after liquefaction, incubation at various temperatures, and storage at -20 degrees C. The three proteins were purified by chromatographic techniques and identified as lactoferrin, albumin, and acid phosphatase by electrophoresis, high-pressure liquid chromatography, and enzyme assays. The use of these proteins to evaluate the contributions of different fluids to seminal plasma is discussed.

Pig leucocytes contain inhibitors of neutral and thiol proteinases. These proteins could be isolated from post-granule supernatant fraction as well as from nuclear extract using ion exchange chromatography, gel chromatography and affinity chromatography. Inhibitors differ in molecular weight, isoelectric point, immunologically and their inhibition ability against tested enzymes.

Alloalbumin Tagliacozzo (313 Lys------Asn) was detected in the cerebrospinal fluid of a 27-yr-old woman. We studied this rare finding by means of high-resolution electrophoresis, isoelectric focusing, and immunofixation. We herein discuss the importance of molecular mass versus superficial electrical charge in the transfer of plasma proteins through the blood-brain barrier.

BACKGROUND: Urinary tract infection is the most common health problem affecting millions of people each year, mainly caused by a large genetically heterogeneous group of Escherichia coli called uropathogenic E. coli This study investigates the genotypic analysis of E. coli strains isolated from patients with cystitis and pyelonephritis. METHODS: During 2008-2009, 90 E. coli strains were analyzed, consisting of 48 isolates causing pyelonephritis in children and 42 isolates causing cystitis. Having identified the strains by standard methods, they were subtyped by pulsed field gel electrophoresis (PFGE) and their corresponding patterns were compared using dendrogram. RESULTS: Sixty five PFGE profiles were obtained from the genome of E. coli strains by this genotyping method. Thirty six and thirty three patterns were obtained for pyelonephritis and cystitis, respectively. Most strains exhi-bited twelve and thirteen bands and the patterns with eight or nineteen bands had the lowest rate. Genome sizes of the strains were between 1610-4170 kbp. CONCLUSION: With due attention to these results, genetic patterns showed that the strains had different clonalities and it could be suggested in some cases that the strains causing pyelonephritis or cystitis have common patterns and different diseases could be explained by different gene factors.