Valores de referência para níveis séricos do Fator de crescimento semelhante à insulina tipo I (IGF-I) numa população adulta do Estado do Rio de Janeiro, Brasil

O nível sérico do Fator de crescimento semelhante à insulina tipo I (IGF-I) é fundamental para auxiliar no dignóstico e controle terapêutico dos transtornos relacionados à secreção do Hormônio de Crescimento (GH), bem como no diagnóstico e seguimento de outras doenças. Estabelecer valores de referência para as dosagens séricas de IGF-I por um ensaio imunoquimioluminométrico (ICMA), utilizando o sistema automatizado Immulite 2000/Diagnostic Products Corporation (DPC), e por um ensaio imunoradiométrico (IRMA), utilizando o kit comercial ACTIVE IGF-I/Diagnostic System Laboratories (DSL)-5600, numa população brasileira adulta da cidade do Rio de Janeiro. Este estudo, aprovado pelo Comitê de Ética do Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brasil, incluiu amostras de 484 indivíduos saudáveis (251 homens e 233 mulheres) com idades entre 18 e 70 anos. As amostras foram estudadas por ICMA- Immulite 2000/DPC and IRMA- ACTIVE IGF-I/DSL-5600. Para análise dos dados foram utilizados modelos específicos para idade e sexo, após transformação dos dados de IGF-I. Foi observada uma lenta diminuição dos níveis de IGF-I com a idade usando ambos os ensaios. Os níveis de IGF-I foram signicativamente (p=0,0181) mais elevados em mulheres do que em homens, quando as amostras foram analisadas usando ICMA. Não houve diferença significativa dos níveis de IGF-I entre homens e mulheres quando as amostras foram analisadas usando IRMA. Este estudo estabeleceu valores de referência de IGF-I específicos para idade e sexo, determinados com o sistema automatizado ICMA-Immulite 2000/DPC, e valores de referência de IGF-I específicos para idade, determinados com o kit comercial IRMA- ACTIVE IGF-I/DSL-5600, em uma população adulta brasileira, da cidade do Rio de Janeiro.

Marcadores séricos do metabolismo ósseo no hipertireoidismo felino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

PTH-related protein is released into the mother's bloodstream during location: evidence for beneficial effects on maternal calcium-phosphate metabolism

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradiometric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/l), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 +/- 10 vs. 13 +/- 9 micrograms/l; p = 0.018) and so was PTHrP (2.8 +/- 0.35 vs. 0.52 +/- 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 +/- 0.02 mmol/l) to B (1.22 +/- 0.02, mmol/l), whereas total Ca corrected for protein (2.18 +/- 0.02 mmol/l) or uncorrected (2.18 +/- 0.02 mmol/l) significantly rose during lactation (2.31 +/- 0.02 mmol/l, p = 0.003 and 2.37 +/- 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 +/- 0.38 vs. 2.11 +/- 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 +/- 2.3 vs. 24 +/- 1.9 ng/ml and 29.5 +/- 6.0 vs. 21.9 +/- 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 +/- 0.03 vs. 0.86 +/- 0.05 mmol/l GF, p = 0.003 and 1.25 +/- 0.03 vs. 0.96 +/- 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 +/- 7.1 vs. 52.6 +/- 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 +/- 0.003 vs. 0.017 +/- 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the Kd of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar Kd values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims: Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies. Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples. Conclusions: The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not. Significance and Impact of the Study: The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

The impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25-hydroxyvitamin D concentrations

Studies have examined the associations between cancers and circulating 25-hydroxyvitamin D [25(OH)D], but little is known about the impact of different laboratory practices on 25(OH)D concentrations. We examined the potential impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25(OH)D concentrations. Blood samples from 20 healthy volunteers underwent alternative laboratory procedures: four centrifuging times (2, 24, 72, and 96 h after blood draw); three types of collection tubes (red top serum tube, two different plasma anticoagulant tubes containing heparin or EDTA); and two types of assays (DiaSorin radioimmunoassay [RIA] and chemiluminescence immunoassay [CLIA/LIAISON®]). Log-transformed 25(OH)D concentrations were analyzed using the generalized estimating equations (GEE) linear regression models. We found no difference in 25(OH)D concentrations by centrifuging times or type of assay. There was some indication of a difference in 25(OH)D concentrations by tube type in CLIA/LIAISON®-assayed samples, with concentrations in heparinized plasma (geometric mean, 16.1 ng ml−1) higher than those in serum (geometric mean, 15.3 ng ml−1) (p = 0.01), but the difference was significant only after substantial centrifuging delays (96 h). Our study suggests no necessity for requiring immediate processing of blood samples after collection or for the choice of a tube type or assay.

Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh

This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh by using Quantitative PCR (qPCR) of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely, enterococci. The number of enterococci in lake water samples ranged from 1.1 x 104 to 1.9 x 105 CFU/100 ml of water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for the HF183 and CF128 markers, respectively. The numbers of the HF183 and CF128 markers in lake water samples were 3.9 x 104 to 6.3 × 107 and 9.3 x 103 to 6.3 x 105 genomic units (GU)/100 ml of water, respectively. The high numbers of enterococci and the HF183 markers indicate sewage pollution and potential health risks to those who use the lake water for non-potable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking (MST) markers in Dhaka, Bangladesh where diarrhoeal diseases is one of the major causes of childhood mortality. The molecular assay as used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimise potential health risks.

Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay

Reviewing the available literature, one could conclude that marrow-derived mesenchymal stem cells (BMSCs) are the ‘gold standard’ source for bone tissue engineering applications, due to their multilineage differentiation potential and easy accessibility. However, comprehensive studies comparing their osteogenic potential with bone-derived osteoblasts (OBs) to justify the preferred application of BMSCs based on performance are few. To address these shortfalls, in the present study, ovine BMSCs and OBs seeded onto scaffolds were characterized in vitro and transplanted subcutaneously into NOD/SCID mice in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7). It was hypothesized that cell origin, ossification type and degree of vascularization and ossification depends on the nature and commitment of transplanted cells and stimulating growth factors, such as rhBMP-7. After retrieval, specimens were analysed by biomechanical testing, µCT analysis, scanning electron microscopy/energy-dispersive X-ray spectroscopy and histo- and immunohistochemistry for osteocalcin, type II collagen and BrdU. The results showed a high degree of cell survival and proliferation ectopically, resulting in active contribution to endochondral osteogenesis. When compared to BMSCs, OBs showed a higher degree of bone deposition while OB-derived bone was of higher maturation. Stimulation with rhBMP-7 increased the rate of bone synthesis for both BMSCs and OBs, additionally promoting neovascularization and osteoclast activity. These results suggest that the origin and commitment of transplanted cells highly influence the type and degree of ossification, that rhBMP-7 represents a powerful adjuvant for bone tissue-engineering applications, and that mature bone is an adequate alternative cell source for bone tissue-engineering applications.

A mass spectrometric assay for the quantification of neuropeptide PYY in plasma

A multiple reaction monitoring mass spectrometric assay for the quantification of PYY in human plasma has been developed. A two stage sample preparation protocol was employed in which plasma containing the full length neuropeptide was first digested using trypsin, followed by solid-phase extraction to extract the digested peptide from the complex plasma matrix. The peptide extracts were analysed by LC-MS using multiple reaction monitoring to detect and quantify PYY. The method has been validated for plasma samples, yielding linear responses over the range 5–1,000 ng mL−1. The method is rapid, robust and specific for plasma PYY detection.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.