Biomedical subjects

I G Bravo

Publications and source records attributed to I G Bravo.

11 recordsLinked to original sources

PubMed2006

Bead-based multiplex genotyping of human papillomaviruses.

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

Cervix Uteri↗

PubMed2005

Effects of extracellular matrix on the morphology and behaviour of rabbit auricular chondrocytes in culture.

Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis.

Journal Article↗

PubMed2004

The E5 protein of the human papillomavirus type 16 modulates composition and dynamics of membrane lipids in keratinocytes.

The E5 protein of the human papillomavirus type 16 is a small protein found associated to membranes, mainly in the Golgi apparatus, and expressed in the early stages of viral infection. Its expression modifies the cell response towards growth factors and stress exposures, and also blocks the surface expression of MHC molecules. A global explanation for these multiple effects is hitherto not available. Here we present data showing that the expression of HPV16-E5 increases the amount of free cholesterol readily extractable from the plasma membrane, without altering the total cholesterol content. In addition, HPV16-E5 modifies the composition of the cell membranes, increasing the synthesis rate of phosphatidylcholine and phosphatidylserine, while diminishing that of phosphatidylglycerol. We propose that these changes in the lipid composition of the membrane are the central effect of HPV16-E5 on the cell. The multiple and apparently disconnected effects of HPV16-E5 on tyrosine-kinase receptors, induction of the apoptosis and impairment of MHC trafficking could follow the initial alteration on the membrane composition.

Animals↗

PubMed2001

N-Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system.

The N-acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37 degrees C in 50 mM Tris-HCl buffer, pH 8.0, containing 50 microg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K(m) for NeuAc was 0.1 microM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N'-dicyclohexyl-carbodiimide (50%), and 2,4-dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.

2,4-Dinitrophenol↗

PubMed2001

A normalized plot as a novel and time-saving tool in complex enzyme kinetic analysis.

A new data treatment is described for designing kinetic experiments and analysing kinetic results for multi-substrate enzymes. Normalized velocities are plotted against normalized substrate concentrations. Data are grouped into n + 1 families across the range of substrate or product tested, n being the number of substrates plus products assayed. It has the following advantages over traditional methods: (1) it reduces to less than a half the amount of data necessary for a proper description of the system; (2) it introduces a self-consistency checking parameter that ensures the 'scientific reliability' of the mathematical output; (3) it eliminates the need for a prior knowledge of Vmax; (4) the normalization of data allows the use of robust and fuzzy methods suitable for managing really 'noisy' data; (5) it is appropriate for analysing complex systems, as the complete general equation is used, and the actual influence of effectors can be typified; (6) it is amenable to being implemented as a software that incorporates testing and electing among rival kinetic models.

Enzymes↗

PubMed2001

Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2.

Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43 kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153 m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77 mM for CTP and 1.82 mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.

Amino Acid Sequence↗

PubMed2001

Chemical organization of the macaque monkey olfactory bulb: II. Calretinin, calbindin D-28k, parvalbumin, and neurocalcin immunoreactivity.

The distribution and morphologic features of calcium-binding protein- (calbindin D-28k, calretinin, neurocalcin, and parvalbumin) immunoreactive elements were studied in the macaque monkey olfactory bulb by using specific antibodies and the avidin-biotin-immunoperoxidase method. A characteristic laminar pattern of stained elements was observed for each marker. Scarce superficial short-axon cells and superficial stellate cells demonstrated calbindin D-28k immunoreactivity in the outer layers, whereas a moderate number of calbindin D-28k-immunoreactive granule cells and scarce deep short-axon cells were observed in the inner layers. Calretinin-staining demonstrated abundant periglomerular cells and granule cells and a scarce number of other interneuronal populations. Most neurocalcin-immunopositive elements were external and medial tufted cells and periglomerular cells, although other scarcer interneuronal populations were also immunostained. A few superficial and deep short-axon cells as well as small interneurons in the external plexiform layer were the only elements immunoreactive to parvalbumin. The distribution of the immunoreactive elements in the olfactory bulb of the macaque monkey showed a high similarity to that reported in the human, whereas it demonstrated a different and simpler pattern to what has been reported in the olfactory bulb of macrosmatic animals. It suggests more homogeneous calcium-mediated cell responses after stimulation that could be correlated to the lower capability to modulate olfactory signals in microsmatic animals. In addition, these results indicate that experimental models in rodents do not provide an accurate estimation of calcium-binding protein-immunoreactive neuronal populations in the primate olfactory system.

Animals↗

PubMed1999

Coexpression of neurocalcin with other calcium-binding proteins in the rat main olfactory bulb.

The distribution patterns of four calcium-binding proteins (CaBPs)-calbindin D-28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)-in the rat main olfactory bulb were compared, and the degrees ofcolocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques. All investigated CaBPs were detected in groups of periglomerular cells and Van Gehuchten cells, whereas other cell types expressed some of the investigated proteins but not all four. Double-labeling techniques demonstrated the colocalization of NC with CB, CR, or PV in periglomerular cells, whereas each neurochemical group constituted entirely segregated populations in the remaining neuronal types. This is evident in granule cells that demonstrated large but segregated populations immunoreactive to either NC or CR. This study provides a further biochemical characterization of interneuronal types in the rat main olfactory bulb. On the basis of the distinct calcium-binding affinities, each neurochemically defined population may have different responses to calcium influx that would result in the existence of distinct functional subgroups within morphologically defined neuronal types. The expression of the investigated CaBPs in periglomerular cells with both single and colocalized patterns suggests that the local circuits in the glomerular layer are constituted by a complex network of elements with particular calcium requirements.

Animals↗

PubMed1998

Neurocalcin immunoreactivity in the rat main olfactory bulb.

The morphological characteristics and distribution of neurocalcin (NC)-immunoreactive elements were studied in the rat main olfactory bulb (OB) using a polyclonal antibody and the avidin-biotin immunoperoxidase method. NC-positive elements were abundant in the glomerular layer (GL), where numerous immunostained external tufted cells and periglomerular cells were detected. Other less abundant NC-immunolabeled populations included middle and internal tufted cells, Van Gehuchten cells, horizontal cells, vertical cells of Cajal, deep short-axon cells and granule cells. This study demonstrates the presence of NC immunoreactivity in subsets of different neuronal types in the rat main OB. This calcium-binding protein has been found in interneurons, and no evidence of immunoreactivity to NC is detected in projecting neurons. Despite the large population of labeled external tufted cells, most of them belong according to morphological criteria to the local circuit group and some others to those with interbulbar and/or intrabulbar connections. The identification of neuronal subpopulations expressing NC provides a further characterization and shows the existence of biochemical differences within morphologically identical neurons. Thus, this marker may be a useful tool in unravelling the circuitries of the rodent OB in both normal and experimental conditions. The exact physiological function of NC in the olfactory system remains unknown. On the basis of similarities to recoverin, it could be involved in mechanisms responsible for sensory adaptation. Additionally, its calcium-binding abilities may contribute to improve the temporal precision of stimuli transmission, or be concerned with general calcium-related events occurring in specific interneuronal groups.

Animals↗

PubMed1995

Colocalization of NADPH-diaphorase and acetylcholinesterase in the rat olfactory bulb.

The colocalization of NADPH-diaphorase and acetylcholinesterase activities in the rat main and accessory olfactory bulbs has been studied by successive double histochemical staining of the same sections. In the main olfactory bulb, three patterns of glomerular labeling were found: typical/NADPH-diaphorase-positive, typical/NADPH-diaphorase-negative, and atypical/NADPH-diaphorase-negative glomeruli. Although both enzymatic activities were present in periglomerular cells and superficial short-axon cells, colocalization of NADPH-diaphorase and acetylcholinesterase was not observed in these neuronal types. By contrast, both enzymes were colocalized in a small subpopulation (less than 3% of NADPH-diaphorase- or acetylcholinesterase-positive cells) of short-axon cells located in the external plexiform layer, internal plexiform layer, granule cell layer, and white matter. In the accessory olfactory bulb, deep short-axon cells were the only neurons where both enzymes were present, and colocalization of both markers was observed in some of these cells located in the granule cell layer.

Acetylcholinesterase↗

PubMed1984

The pharmacokinetics of cotrimoxazole sulphonamide in malnourished (marasmic) infants.

The pharmacokinetics of trimethoprim-sulphamethoxazole was examined in seven malnourished (marasmic) infants receiving cotrimoxazole (CMZ) for treatment of urinary tract infection. Comparisons were made with the SMZ level of ten nutritionally normal infants, hospitalized for first and second degree burns, receiving CMZ for treatment of bronchitis. CMZ was administered as an oral suspension (20 mg TMP and 100 mg SMZ, 5 ml), patients receiving 22 mg SMZ/kg body weight. Capillary blood samples, 0.05 ml were taken at prescribed intervals. Elimination half-life of SMZ in the marasmic infants was prolonged, 9.6 vs 4.9 hr, in their eutrophic counterparts. In addition, greater area under the curve (AUC), 573 vs 328 micrograms/ml/h, was noted in the malnourished group. This disparity may be due to differences in body fluid distribution between the two groups.

Drug Combinations↗