Quantification of the bombesin/gastrin releasing peptide antagonist RC-3095 by liquid chromatography-tandem mass spectrometry

Bombesin (BN) and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. BN/GRP and their receptors were shown to be critical for the growth of various human malignancies, such as small-cell lung, prostate, ovary, stomach and breast cancers in the human tumor xenograft model. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of a BN/GRP receptor antagonist RC-3095 (D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leupsi(CH2NH)Leu-NH2), in human plasma by liquid chromatography coupled with tandem mass spectrometry. RC-3095 was extracted from 0.2 ml human plasma by protein precipitation using cold acetonitrile (0.4 ml). The method has a chromatographic run of 10 min using a C(8) analytical column (150 mm x 4.6 mm i.d.) and the linear calibration curve over the range was linear from 20 to 10000 ng ml(-1) (r(2)>0.994). The between-run precision, based on the relative standard deviation replicate quality controls, was 5.7% (60 ng ml(-1)), 7.1% (600 ng ml(-1)) and 6.8% (8000 ng ml(-1)). The between-run accuracy was +/-0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively. The developed procedure allows the quantitative determination of peptide RC-3095 for pharmacokinetics studies in human plasma.     

Measurements of CFTR-Mediated Cl? Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

Background

Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl⁻) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases.

Methodology/Principal Findings

To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl⁻ secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with “Classic CF”, presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl⁻ secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl⁻ secretion (10–57%) and non-CF controls show CFTR-mediated Cl⁻ secretion ≥30–35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in “CF suspicion” individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl⁻ secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups.

Conclusions/Significance

Determination of CFTR-mediated Cl⁻ secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.     

Cyclooxygenase pathway is involved in the vascular reactivity and inhibition of the Na+, K+-ATPase activity in the tail artery from L-NAME-treated rats

L-NAME (LN) induces hypertension by blocking nitric oxide (NO) synthesis. It produces vascular hyperreactivity to phenylephrine (PHE) associated with a reduced vascular Na+, K+-ATPase activity. The aim of this work was to investigate whether products of the cyclooxygenase pathway are involved in alterations of vascular reactivity and Na+-pump activity in the tail artery from LN-induced hypertension rats. Four groups of rats were used: Control (CT, normotensive), LN (50 mg/kg/day, hypertensive), indomethacin (Indo-4 mg/kg/day, normotensive), and LN plus Indo (LN + Indo, partially prevented hypertension). All drugs were administered in drinking water during 7 days. In isolated rat tail vascular beds; the reactivity to PHE, acetylcholine (ACh), sodium nitroprusside (SNP), the functional activity of the Na+, K+-ATPase (K+-induced relaxation) and the modulation of PHE-induced vasoconstriction by constitutively available NO were evaluated. LN increased vascular sensitivity (pD2) and reactivity (Emax) to PHE and Indo blocked the effect of LN on Emax without changing pD2. Emax and pD2 values for ACh were reduced by LN and partially reverted by Indo. SNP-induced vasodilatation was similar in all groups. LN reduced the activity of Na+, K+-ATPase and Indo prevented LN effects. LN also abolished NO ability to modulate PHE-induced contractions. This effect was partially prevented by Indo suggesting that products from the cyclooxygenase pathway might reduce NO actions. Indo itself did not affect vascular reactivity to PHE, ACh or SNP or the Na+,K+-ATPase activity. Results suggested that products from cyclooxygenase pathway are involved in the genesis or maintenance of LN-induced hypertension, playing a role in the increased vascular reactivity, in the reduction of the endothelium-dependent relaxation and in the inhibition of the functional activity of the Na+, K+-ATPase.     

A new species of Polystomoides ward, 1917 (Monogenea: polystomatidae) from freshwater chelonians (Testudines: chelidae) in Brazil

This report describes the first occurrence of Polystomoides brasiliensis n. sp. (Monogenea: Polystomatidae), a new monogenean species in the buccal and pharyngeal cavities of the freshwater turtles in Brazil. Live monogeneans were collected from Hydromedusa maximiliani and Phrynops geoffroanus at the Mariano Procópio Museum's lake, in Juiz de Fora, Minas Gerais, Brazil. Polystomoides brasiliensis differs from all other species of this genus in having 8-9 genital spines, except for Polystomoides uruguayensis, which has 8-10 genital spines. However, the new species differs morphometrically from P. uruguayensis in the greater size of the outer and inner hamuli, as well as having a testis that is proportionally greater than the pharynx and oral sucker. The current study is the first report of monogeneans in chelonians of Brazil, and the first record of helminths in H. maximiliani.     

Distribution and composition of particulate organic matter in the Ross Sea (Antarctica)

The biochemical composition and spatial distribution of particulate organic matter (POM) were studied in the Ross Sea (Antarctica) in summer 1989 to assess the quantitative role of organic carbon fractions in the cycling of organic matter in the water column. Large differences in chemical composition were observed between surface and deep layers. The results indicated that, despite large geographical differences, POM was quite homogeneous, of phytoplankton origin and mostly detrital. Different ratios were used to investigate the changes in biochemical composition of particulate organic matter in relation to the ice-melting: CN (organic carbonorganic nitrogen ratio) and C-POMPOC (sum of carbohydrate, protein and lipid carbontotal organic carbon ratio) were used to analyse the percentage of refractory organic material. PPRTPCHO (proteincarbohydrate ratio) were used to establish POM age and RNADNA ratios as a relative measure of particulate activity; POCChl a and N-PPRTChl a ratios were used to estimate the autotrophic contribution to the suspended particulate organic matter. Despite its low caloric value (5.3 Kcal g POM^–1), an high caloric content in the photic layer (1.6 Kcal m^–3 of POM and 2.5 Kcal m^–3 of POC) was found thus indicating that a large amount of food was available to higher trophic levels.     

Hypoxia–Ischemia Alters Nucleotide and Nucleoside Catabolism and Na+,K+-ATPase Activity in the Cerebral Cortex of Newborn Rats

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system––NTPDase, 5′-nucleotidase (5′-NT) and adenosine deaminase (ADA)––in cerebral cortex of rats immediately post insult. Furthermore, the Na⁺/K⁺-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5′-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na⁺/K⁺ ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5′-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na⁺/K⁺-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.     

Diet of marine fish larvae and juveniles that use rocky intertidal pools at the Portuguese coast

The use of tidal pools during early ontogeny is likely to enhance growth, condition and survival chances of the transient marine fish larvae and juveniles that use them. However, the diet of such individuals within tidal pools is poorly known; this knowledge is important to understand why such high numbers of individuals use these environments in spring and summer on the Portuguese coast. Transient marine fishes were sampled monthly over a two‐year period in four tidal pools of a rocky reef on the west Portuguese coast. The diet composition in the tidal pools of the most abundant marine fish larvae/juveniles, Diplodus sargus and Atherina spp., were investigated. Stomach and gut contents of 483 individuals (354 D. sargus ranging in total length from 9 to 87 mm and 134 Atherina spp. ranging in total length from 10 to 31 mm) were analyzed and dietary indices estimated for the different developmental stages. The diet of D. sargus composed mainly harpacticoid copepods, chironomid larvae, ostracods, chironomid adults and amphipods; whereas the diet of Atherina spp. was mainly harpacticoid copepods, ostracods and gastropods. Along the ontogenetic development, the proportions and diversity of food items in the D. sargus diet varied. Importance of the harpacticoid copepods decreased with increasing fish size, while that of amphipods and isopods increased. Larvae and juvenile D. sargus and Atherina spp. found in rocky reef tidal pools are opportunistic consumers of a wide range of prey and thus take advantage of the high prey availability in these habitats.     

Life history and description of larva and pupa of Platyphileurus felscheanus Ohaus, 1910, a scarabaeid feeding on bromeliad tissues in Brazil,to be excluded from Phileurini (Coleoptera,Scarabaeidae, Dynastinae)

The third instar larvae and the pupae of Platyphileurus felscheanus Ohaus, 1910 (Phileurini), recently synonymized with Surutu jelineki Endrődi, 1975 (Cyclocephalini), are described and illustrated, and some life history information is given. The larvae were collected and reared in bromeliads in rain forests of Santa Catarina state in southern Brazil. The systematic position of this monotypic genus is reassessed at the tribe level by considering larval and adult morphological characters. Both character sets, being described and illustrated, suggest the placement of Platyphileurus in the tribe Oryctini.     

An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Length-weight relationships of fish species from oxbow lakes on the floodplain of the middle Purus River in western Brazilian Amazon

The length-weight relationship (LWRs) was estimated for eight fish species collected from oxbow lakes on the floodplain of the middle Purus River in western Brazilian Amazonia in January, May, and September 2012. The specimens were collected using 12 gillnets (80 m in length × 4 m in height, meshes of between 1.5 cm and 12.0 cm), which were set during both diurnal and nocturnal periods. The species had allometric coefficients (b) of between 2.92 and 3.37, and correlation coefficients (r²) ranging from 0.954 to 0.993 and we highlight that the work presents new data for literature.