Local polynomial regression modeling of human plasma melatonin levels

Accurate characterization of features of the melatonin production curve is important for research in chronobiology. Local polynomial regression is used to model the plasma melatonin concentration curve and obtain consistent and asymptotically normally distributed estimates of synthesis onset and offset times, duration, peak concentration, and area under the curve. A simple production-clearance model for melatonin kinetics provides the connection between plasma concentrations and the melatonin production curve. This model identifies onset and offset times with critical points of derivatives of the concentration curve. The method proposed has the advantage of flexibility and ease of implementation. Local polynomial regression can be shown to produce consistent estimates of the regression curve and parameters of interest. Furthermore, the method can be implemented in any software package with weighted least squares routines.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Stereoselectivity in the placental transfer and kinetic disposition of racemic bupivacaine administered to parturients with or without a vasoconstrictor

The aim of the present study was to investigate the stereoselectivity in the kinetic disposition and the transplacental distribution of bupivacaine in term parturients during labor. Maternal age ranged from 18-37 years and fetal gestational age from 37.6-41.5 weeks. Healthy parturients (n = 23) received epidural 0.5% racemic bupivacaine alone (group A) or combined with epinephrine (group B). Maternal venous blood was sampled at regular intervals until 8 h after drug administration and umbilical venous blood was obtained at delivery. Bupivacaine enantiomers were determined in plasma samples by HPLC using a Chiralcel(R) OD-R column and a UV detector. One- or two-compartment models were fitted to data and differences between the (+)-(R) and (-)-(S) enantiomers were compared with the paired Wilcoxon test (P< 0.05). The influence of epinephrine was evaluated using the unpaired Mann-Whitney test (P< 0.05). The disposition of bupivacaine in maternal plasma was stereoselective, with higher V(d/f) (140.60 vs. 132.81 L for group A and 197.86 vs. 169.46 L for group B) and C(l/f) (29.00 vs. 25.43 L/h for group A and 33.15 vs. 26.39 L/h for group B) and lower t(1/2)beta (3.24 vs. 3.30 h for group A and 4.36 vs. 4.45 h for group B) being observed for (+)-(R)-bupivacaine. The combined administration of epinephrine resulted in higher V(d/f) (197.86 vs. 140.60 L for (+)-(R) and 169.46 vs. 132.81 L for (-)-(S)) and t(1/2)beta values (4.36 vs. 3.24 h for (+)-(R) and 4.45 vs. 3.30 h for (-)-(S)). The transplacental distribution of bupivacaine was stereoselective only when bupivacaine was administered without epinephrine (group B), with a higher cord blood/maternal blood ratio being observed for (-)-(S)-bupivacaine (0.40 vs. 0.35). Chirality 16:65-71, 2004.     

Tumor-induced endothelial cell activation: role of vascular endothelial growth factor

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl₂ significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl₂, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects. vascular endothelial growth factor receptor 2; MG63-conditioned medium     

The Electron Transfer Flavoprotein <Emphasis Type="Italic">fixABCX</Emphasis> Gene Products from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Show a NifA-Dependent Promoter Regulation

The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric pfixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.     

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.