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Biomedical subjects

L Cruz

Publications and source records attributed to L Cruz.

At least 19 recordsLinked to original sources
PubMed2003

Neuron recognition by parallel Potts segmentation.

Identifying neurons and their spatial coordinates in images of the cerebral cortex is a necessary step in the quantitative analysis of spatial organization in the brain. This is especially important in the study of Alzheimer's disease (AD), in which spatial neuronal organization and relationships are highly disrupted because of neuronal loss. To automate neuron recognition by using high-resolution confocal microscope images from human brain tissue, we propose a recognition method based on statistical physics that consists of image preprocessing, parallel image segmentation, and cluster selection on the basis of shape, optical density, and size. We segment a preprocessed digital image into clusters by applying Monte Carlo simulations of a q-state inhomogeneous Potts model. We then select the range of Potts segmentation parameters to yield an ideal recognition of simplified objects in the test image. We apply our parallel segmentation method to control individuals and to AD patients and achieve recognition of 98% (for a control) and 93% (for an AD patient), with at most 3% false clusters.

Alzheimer Disease↗
PubMed2002

Neurotoxic effects of thioflavin S-positive amyloid deposits in transgenic mice and Alzheimer's disease.

Despite extensive deposition of putatively neurotoxic amyloid-beta (Abeta) protein in the brain, it has not been possible to demonstrate an association of Abeta deposits with neuronal loss in Alzheimer's disease (AD), and neuronal loss is minimal in transgenic mouse models of AD. Using triple immunostaining confocal microscopy and analyzing the images with the cross-correlation density map method from statistical physics, we directly compared Abeta deposition, Abeta morphology, and neuronal architecture. We found dramatic, focal neuronal toxicity associated primarily with thioflavin S-positive fibrillar Abeta deposits in both AD and PSAPP mice. These results, along with computer simulations, suggest that Abeta develops neurotoxic properties in vivo when it adopts a fibrillar beta-pleated sheet conformation.

Alzheimer Disease↗
PubMed2001

Plaque-induced abnormalities in neurite geometry in transgenic models of Alzheimer disease: implications for neural system disruption.

Neurites that pass through amyloid-beta deposits in Alzheimer disease (AD) undergo 3 changes: they develop phosphorylated tau immunoreactivity; the density of SMI-32-positive dendrites diminishes; and they also develop a marked alteration in their geometric features, changing from being nearly straight to being quite curvy. The extent to which the latter 2 phenomena are related to phosphorylated tau is unknown. We have now examined whether amyloid-beta deposits in APP695Sw transgenic mice, which have only rare phosphorylated tau containing neurites. develop these changes. We found that dendritic density is diminished within the boundaries of amyloid-beta plaques, with the greatest loss (about 80%, p < 0.001) within the boundaries of thioflavine S cores. Remaining dendrites within plaques develop substantial morphological alterations quantitatively similar to those seen in AD. A statistically significant but smaller degree of change in geometry was seen in the immediate vicinity around plaques, suggesting a propagation of cytoskeletal disruption from the center of the plaque outward. We examined the possible physiological consequences of this change in dendritic geometry using a standard cable-theory model. We found a predicted delay of several milliseconds in about one quarter of the dendrites passing through a thioflavine S plaque. These results are consistent with previous observations in AD, and suggest that thioflavine S-positive amyloid-beta deposits have a marked effect on dendritic microarchitecture in the cortex, even in the relative absence of phosphorylated tau alterations.

Action Potentials↗
PubMed2000

Description of microcolumnar ensembles in association cortex and their disruption in Alzheimer and Lewy body dementias.

The cortex of the brain is organized into clear horizontal layers, laminae, which subserve much of the connectional anatomy of the brain. We hypothesize that there is also a vertical anatomical organization that might subserve local interactions of neuronal functional units, in accord with longstanding electrophysiological observations. We develop and apply a general quantitative method, inspired by analogous methods in condensed matter physics, to examine the anatomical organization of the cortex in human brain. We find, in addition to obvious laminae, anatomical evidence for tightly packed microcolumnar ensembles containing approximately 11 neurons, with a periodicity of about 80 microm. We examine the structural integrity of this new architectural feature in two common dementing illnesses, Alzheimer disease and dementia with Lewy bodies. In Alzheimer disease, there is a dramatic, nearly complete loss of microcolumnar ensemble organization. The relative degree of loss of microcolumnar ensembles is directly proportional to the number of neurofibrillary tangles, but not related to the amount of amyloid-beta deposition. In dementia with Lewy bodies, a similar disruption of microcolumnar ensemble architecture occurs despite minimal neuronal loss. These observations show that quantitative analysis of complex cortical architecture can be applied to analyze the anatomical basis of brain disorders.

Alzheimer Disease↗
PubMed1999

A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti-E-selectin monoclonal antibody to activated human endothelial cells.

Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5' to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the VH-Linker-VL 32-33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.

Amino Acid Sequence↗
PubMed1999

Plaque-induced neurite abnormalities: implications for disruption of neural networks in Alzheimer's disease.

The brains of Alzheimer's disease patients contain extracellular Abeta amyloid deposits (senile plaques). Although genetic evidence causally links Abeta deposition to the disease, the mechanism by which Abeta disrupts cortical function is unknown. Using triple immunofluorescent confocal microscopy and three-dimensional reconstructions, we found that neuronal processes that cross through an Abeta deposit are likely to have a radically changed morphology. We modeled the electrophysiological effect of this changed morphology and found a predicted delay of several milliseconds over an average plaque. We propose that this type of delay, played out among thousands of plaques throughout neocortical areas, disrupts the precise temporal firing patterns of action potentials, contributing directly to neural system failure and dementia.

Aged↗
PubMed1999

Dynamics of plaque formation in Alzheimer's disease.

Plaques that form in the brains of Alzheimer patients are made of deposits of the amyloid-beta peptide. We analyze the time evolution of amyloid-beta deposition in immunostained brain slices from transgenic mice. We find that amyloid-beta deposits appear in clusters whose characteristic size increases from 14 microm in 8-month-old mice to 22 microm in 12-month-old mice. We show that the clustering has implications for the biological growth of amyloid-beta by presenting a growth model that accounts for the experimentally observed structure of individual deposits and predicts the formation of clusters of deposits and their time evolution.

Alzheimer Disease↗
PubMed1999

Role of P-selectin expression in hepatic ischemia and reperfusion injury.

BACKGROUND: Researchers have shown that reperfusion of ischemic tissues initiates a complex series of reactions that paradoxically injure tissues. Although several mechanisms have been proposed to explain the pathobiology of ischemic/reperfusion (I/R) injury, much attention has focused on adhesion molecules. Our research is intended to show the kinetics of P-selectin in the liver in response to I/R injury. METHODS: Left-lobar hepatic ischemia was induced for 30 min in 35 C57BL-6 mice and 20 P-selectin-deficient (K-O) mice. P-selectin expression was measured in these mice at 20 min, 2, 5, 12 and 24 h reperfusion times, as well as in control and sham animals. The animals were injected with radio-labeled P-selectin monoclonal antibody and the organs were harvested for counts/g tissue, expressed as the percentage injected dose. Serum liver enzymes were measured and pathological sections of ischemic and control livers were performed. The unpaired t-test was used for statistical analysis. RESULTS: P-selectin expression showed two peaks in this animal model. The first peak was at 20 min and the second peak at 5 h of reperfusion (p < 0.001). We documented an 8-fold increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels 10 h following I/R injury. Pathological specimens showed periportal necrosis consistent with an ischemic event. P-selectin K-O mice showed no up-regulation as a separate control group, and the liver enzymes were significantly lower than the wild-type mice at 10 h (p < 0.001). CONCLUSION: P-selectin has a bimodal expression following hepatic I/R injury. The first peak is attributed to the Weibel-Palade bodies and the second to new translational P-selectin. We noted no difference in the up-regulation of P-selectin in the ischemic and non-ischemic liver lobes in the same animal.

Alanine Transaminase↗
PubMed1999

Dynamic feedback in an aggregation-disaggregation model.

We study an aggregation-disaggregation model which is relevant to biological processes such as the growth of senile plaques in Alzheimer disease. In this model, during the aggregation each deposited particle has a probability of producing a new particle in its vicinity, while during disaggregation the particles are anihilated randomly. The model is held in a dynamic equilibrium by a feedback mechanism which changes the disaggregation probability in proportion to the change in the total number of particles. We also include surface diffusion which influences the morphology of growing aggregates and colonies. A colony includes the descendents of a single particle. We investigate the statistical properties of the model in two dimensions. We find that unlike the colonies, individual aggregates are fractals with a fractal dimension of D(f)=1.92+/-0.06 in the absence of surface diffusion. We show that the surface diffusion changes the fractal dimension of aggregates: at a small aggregation-disaggregation rate, D(f) is independent of the strength of the surface diffusion, D(f)=1.73+/-0.03. At larger aggregation-disaggregation rates and different strengths of surface diffusion, aggregates with fractal dimensions between D(f)=1.73 and 1.92 form. The steady-state distribution of aggregate sizes is shown to be power law if the aggregation-disaggregation process dominates over the surface diffusion. In the limit of weak aggregation-disaggregation and strong surface diffusion the size distribution is log-normal.

Alzheimer Disease↗
PubMed1999

Ingestion of chilli pepper (Capsicum annuum) reduces salicylate bioavailability after oral asprin administration in the rat.

The bioavailabilities of aspirin (acetylsalicylic acid) and of salicylic acid were studied in male Wistar rats after acute and chronic administration of a Capsicum annuum extract, containing 100 mg of capsaicin per gram. With a single administration of 100 mg/kg of the extract, aspirin blood levels remained unchanged, but salicylic acid bioavailability was reduced in 44% compared with control animals. With a single administration of 300 mg/kg of the extract, aspirin blood levels were undetectable while salicylic acid bioavailability was reduced in 59%. Chronic administration once daily for 4 weeks of 100 and 300 mg/kg of the extract resulted in undetectable aspirin blood levels, while salicylic acid bioavailability was reduced in 63 and 76%, respectively, compared with controls. Results show that Capsicum ingestion reduces oral drug bioavailability, likely as a result of the gastrointestinal effects of capsaicin.

Administration, Oral↗
PubMed1998

Non-enzymatic production of nitric oxide (NO) from NO synthase inhibitors.

The gaseous signal molecule, nitric oxide (NO*), is generated enzymatically by NO synthase (NOS) from L-arginine. Overproduction of NO contributes to cell and tissue damage as sequelae of infection and stroke. Strategies to suppress NO synthesis rely heavily on guanidino-substituted L-arginine analogs (L-NAME, L-NA, L-NMMA, L-NIO) as competitive inhibitors of NOS, which are often used in high doses to compete with millimolar concentrations of intracellular arginine. We show that these analogs are also a source for non-enzymatically produced NO. Enzyme-independent NO release occurs in the presence of NADPH, glutathione, L-cysteine, dithiothreitol and ascorbate. This non-enzymatic synthesis of NO can produce potentially toxic, micromolar concentrations of NO and can oppose the effects of NOS inhibition. NO production driven by NOS inhibitors was demonstrated ex vivo in the central nervous and peripheral tissues of gastropod molluscs Aplysia and Pleurobranchaea using electron paramagnetic resonance and spin-trapping techniques. These results have important implications for therapeutic regulation of NO homeostasis.

Animals↗
PubMed1997

Aggregation and disaggregation of senile plaques in Alzheimer disease.

We quantitatively analyzed, using laser scanning confocal microscopy, the three-dimensional structure of individual senile plaques in Alzheimer disease. We carried out the quantitative analysis using statistical methods to gain insights about the processes that govern Abeta peptide deposition. Our results show that plaques are complex porous structures with characteristic pore sizes. We interpret plaque morphology in the context of a new dynamical model based on competing aggregation and disaggregation processes in kinetic steady-state equilibrium with an additional diffusion process allowing Abeta deposits to diffuse over the surface of plaques.

Alzheimer Disease↗
PubMed1997

Nitrite and nitrate levels in individual molluscan neurons: single-cell capillary electrophoresis analysis.

Cell and tissue concentrations of NO2- and NO3- are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO2- and NO3- levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-microm separation capillary and the NO2- and NO3- were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO2- and NO3- were <200 fmol (<4 microM in the neurons under study). The NO2- and NO3- levels in individual neurons varied from 2 mM (NO2-) and 12 mM (NO3-) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO2- was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3- averaged 1.8 +/- 0.2 x 10(-3) M. Hemolymph NO3- in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 +/- 0.2 x 10(-5) M) and for another opisthobranch, Aplysia californica (3.6 +/- 0.7 x 10(-4) M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2-/NO3- levels in single cells and small amounts of tissue.

Animals↗
PubMed1997

[Clinical characteristics of the peripheral form of epidemic neuropathy in the province of Cienfuegos].

INTRODUCTION: From October 1992 to September 1993 clinical observations of the civil population of Cienfuegos revealed the presence of epidemic neuropathy (EN) reaching about 2,000 patients. The clinical manifestations were not uniform. Although numerous studies have been carried out in our country, none have established the characteristics of EN 'the peripheral form'. MATERIAL AND METHODS: We studied the first 50 patients assisted by the neurological services of the clinical surgical hospital Dr. Gustavo Aldereguia Lima of Cienfuegos diagnosed of EN 'peripheral form' according to the procedure established by Ministry of Public Health of Cuba. A clinical history was made for each patient consisting of: General data, history of toxic, nutritional, malabsorption factors and chronic illnesses; symptoms and a neurological examinations. Laboratory test, in a group of patients, were done in sera, urine, cerebrospinal fluid (CSF), gastric juice and neurophysiological studies that included computerized evoked potentials and electroencephalograms registers. RESULTS: Upon analysis of the clinical history, nutritional factors was common to all the patients associated to toxic and malabsorption. The clinical forms were neuropathy (NP) 48%, myeloneuropathy (MNP) 42% and myelopathy (MP) 10%. Retrobulbar optic neuropathy was observed in 42% of the patients and auditory neuropathy in 22%. CONCLUSIONS: The clinical characteristics of NP, in our cases, indicated that this disease fundamentally affects the sensorial neurons and the sensitive peripheral nerves, bilaterally, symmetrically, distally and predominantly in lower limbs. The pathological process has been associated with a distal axonopathy. However, clinic signs of myelopathy can be found up to 40% frequently combined with neuropathy or in isolated form, and seems to affect the posterior and lateral columns of the spinal cord, mainly at thoraciclumbar level. Neuropathy of central nervous system is unknown since no patient has died of EN.

Adult↗
PubMed1996

Detection of radionuclides in capillary electrophoresis using a phosphor-imaging detector.

A capillary electrophoresis (CE) postcolumn radionuclide detector has been developed that uses a commercial phosphor-imaging detector and has been optimized for low-energy beta emitters. Eluant from the separation capillary is deposited on a membrane. Emission from radioactive analytes on the membrane is integrated using the phosphor-imaging system for 10-72 h. Results from the phosphor-imaging system are converted to conventional electropherograms. Modifications to a prior postcolumn CE deposition system have been accomplished by adding a buffer makeup capillary; this increases the electrolyte pH range and improves reproducibility. The limit of detection (LOD) for 35S-labeled analytes is 0.13 amol (8.7 pM or 0.007 Bq), while the LOD for 32P-labeled analytes is 4.9 zmol (0.33 pM or 0.002 Bq), with a linear range for 35S-Met from 1.5 amol to 1.5 fmol.

Animals↗
PubMed1996

Studies of the degradation products of nisin, a peptide antibiotic, using capillary electrophoresis with off-line mass spectrometry.

The utility of capillary electrophoresis (CE) for assessing the purity and stability of pharmaceutical peptides is investigated. The degradation of nisin, a pentacyclic peptide antibiotic, depends upon sample preparation and storage conditions and is followed by CE. With conventional UV detection, peaks are not identified and unresolved components are not detected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) of isolated CE fractions provides molecular mass information that aids in identification of the nisin degradants and determination of peak purity. The purity of pure and degraded nisin in the absence of any separation is also determined using electrospray ionization mass spectrometry (ESI-MS) and MALDI-TOFMS.

Amino Acid Sequence↗
PubMed1995

Structural and biosynthetic properties of peptides in cone snail venoms.

Venoms of the predatory cone snails Conus textile, Conus striatus, and Conus magus were subjected to comprehensive analysis of peptide content. With the fish-eating cone snails C. magus and C. striatus, the most abundant venom peptides were of > 30-50 residues, whereas the predominant peptides in the venom of the mollusc-eating snail, C. textile, were of 20-35 residues. Amino acid sequencing revealed an identical but unusual amino acid in a conserved position in four novel omega-type peptides from the C. textile venom. Two conserved amino acid sequences were obtained from the venoms of both C. magus and C. striatus. The amino acid compositions of the isolated C. textile peptides and the expected processing products of the propeptides (42) were compared. Despite the recovery in abundance of the carboxyl-terminal omega-type peptides, none of the isolated peptides had compositions expected from the propeptide amino-terminal fragments. We conclude that there are likely mechanisms for excluding the amino-terminal propeptide fragments from this venom, resulting in a venom with greater potency. Amounts of the different omega-type peptides in the venom vary widely, suggesting a distinct mechanism that results in the selective synthesis of different bioactive carboxyl-terminal propeptide fragments at elevated levels.

Amino Acid Sequence↗