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PubMed2004

A new method to measure intestinal activity of P-glycoprotein in avian and mammalian species.

Permeability-glycoprotein (Pgp) actively exports numerous potentially toxic compounds once they diffuse into the cell membrane of intestinal epithelial cells. We adapted the everted sleeve technique to make the first measures of intestinal Pgp function in an avian species (chicken) and in wild mammalian species, and compared them to laboratory rats. Tissues maintained both structural and functional integrity, and our method offers advantages over other in vitro techniques by using smaller intestinal sections (1 cm), and shorter incubation times (8-12 min). To determine Pgp function, we compared accumulation of [(3)H]-digoxin in sleeves incubated in Ringer solution with and without a transport-saturating concentration of a competitive inhibitor, cyclosporin A. We demonstrated significant variation in Pgp activity within individuals along the intestine, between populations fed different diets, and between species (laboratory rats had one-third to one-fifth the Pgp activity of wild rodents). In chicken, we also tested the effect of natural metabolites on digoxin accumulation. We found that among flavonoids, genistein (200 microM), found in soy and other legumes, but not quercetin (10, 30, 100, 330 microM) or the 3-beta-glycoside isoquercetrin (100 microM), significantly increased digoxin accumulation. Among fungal metabolites, sterigmatocystin (5 microM), but not aflatoxin B1 (5 microM), significantly increased digoxin accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

An fMRI study of anterior cingulate function in posttraumatic stress disorder.

BACKGROUND: Several recent neuroimaging studies have provided data consistent with functional abnormalities in anterior cingulate cortex in posttraumatic stress disorder (PTSD). In our study, we implemented a cognitive activation paradigm to test the functional integrity of anterior cingulate cortex in PTSD. METHODS: Eight Vietnam combat veterans with PTSD (PTSD Group) and eight Vietnam combat veterans without PTSD (non-PTSD Group) underwent functional magnetic resonance imaging (fMRI) while performing the Emotional Counting Stroop. In separate conditions, subjects counted the number of combat-related (Combat), generally negative (General Negative), and neutral (Neutral) words presented on a screen and pressed a button indicating their response. RESULTS: In the Combat versus General Negative comparison, the non-PTSD group exhibited significant fMRI blood oxygenation level-dependent signal increases in rostral anterior cingulate cortex, but the PTSD group did not. CONCLUSIONS: These findings suggest a diminished response in rostral anterior cingulate cortex in the presence of emotionally relevant stimuli in PTSD. We speculate that diminished recruitment of this region in PTSD may, in part, mediate symptoms such as distress and arousal upon exposure to reminders of trauma.

Case-Control Studies↗

PubMed2001

Neurons in amygdala mediate ear pinna vasoconstriction elicited by unconditioned salient stimuli in conscious rabbits.

We determined whether functional integrity of neurons in the amygdala is necessary for sudden episodes of cutaneous vasoconstriction that occur when the conscious animal detects a salient alerting stimulus. To inhibit neuronal function, muscimol (5 nmol in 300 nl), a long acting and potent GABA-A receptor agonist that hyperpolarizes neurons, was injected bilaterally into the amygdala or into a more dorsal control site in conscious rabbits. Cutaneous blood flow was measured in the ear pinna flow using an ultrasonic Doppler probe chronically implanted around the central ear artery. Ear flow responses to salient unconditioned alerting stimuli (fur touch, slight cage movement. removal of drape covering cage) were examined before and after injection of the muscimol, and the effects compared with effects of muscimol on the ear flow response to more nociceptive stimuli, including ear pinch. Muscimol injections into the dorsal control site did not significantly alter alerting-related episodes of ear pinna vasoconstriction. Muscimol injections into the amygdala almost completely abolished ear vasoconstriction elicited by fur touch (0/5 positive responses), drape removal (0/7 positive responses) and cage movement (0/7 positive responses). Muscimol injections into the amygdala reduced the mean ear flow coefficient of variation for a 15 min observation period from 47+/-5 before injection to 15+/-33% after injection (P<0.01, n=7 rabbits). Muscimol injections into the amygdala did not alter the vigorous ear pinna vasoconstriction elicited by ear pinch (7/7 positive responses). Our results indicate that neuronal function in the amygdala, probably the central nucleus of the amygdala, is necessary for the occurrence of ear pinna vasoconstriction episodes elicited by unconditioned salient stimuli but not for the occurrence of corresponding vasoconstriction elicited by nociceptive stimuli.

Amygdala↗

PubMed1995

Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516.

The phosphorylation of threonine residues in the catalytic core of several protein kinases is important for the functional integrity of these enzymes. The corresponding residues of cGMP-dependent protein kinase I alpha (cGMP kinase) are Thr-514 and/or Thr-516. The in vivo phosphorylation and functional role of these residues was studied. cGMP kinase was overexpressed and purified as a catalytically active and inactive enzyme in Sf9 insect cells and in Escherichia coli, respectively. The enzymological and physicochemical properties of the Sf9 cGMP kinase were indistinguishable from that of the purified bovine lung enzyme. The cysteines of cGMP kinase including Cys-518 were labeled with vinylpyridine. Amino acid sequencing and mass spectroscopy of the labeled peptides showed that Thr-516 was phosphorylated in the enzyme purified from Sf9 cells but not in that from E. coli. The functional importance of phosphothreonine-516 was investigated by substitution of Thr-516 by alanine (T516A) or by glutamate (T516E). Expression in insect cells of the T516A mutant resulted in a protein lacking detectable kinase activity, whereas the T516E mutant retained basal phosphotransferase activity. In E. coli, the exchange of Thr-516 by glutamate did not lead to the synthesis of a catalytically active enzyme. These results demonstrate that phosphothreonine-516 of cGMP kinase is crucial for the formation of an enzymatically active protein kinase.

Amino Acid Sequence↗

PubMed1976

Selective hypoaldosteronism: a study of steroid biosynthetic pathways under adrenocorticotrophin and angiotensin II infusion.

1. The functional integrity of the adrenal cortex has been tested in a case of selective hypoaldosteronism by adrenocorticotrophin (ACTH) and angiotensin II (AII) infusion. 2. During ACTH infusion a normal functioning zona fasciculata was indicated by the impressive increase of the ACTH-dependent plasma steroids; the aldosterone response was moderate. 3. During AII infusion the plasma aldosterone response was blunted with an unexpected dose-dependent increase in pregnenolone, resulting in abnormal decreasing progesterone/pregnenolone ratios during the infusion, suggesting a slow-down in the conversion of pregnenolone into progesterone. 4. This defect, a probable consequence of chronic renin deficiency on the zona glomerulosa, could be a contributing factor to the hypoaldosteronism.

Adrenal Cortex↗

PubMed2000

Neuropsychological consequences of chronic bilateral stimulation of the subthalamic nucleus in Parkinson's disease.

The aim of this study was to examine possible neuropsychological changes in patients with advanced idiopathic Parkinson's disease treated with bilateral deep brain stimulation (DBS) of the subthalamic nucleus (STN). Eleven patients (age = 67 +/- 8 years, years with Parkinson's disease = 15 +/- 3, verbal IQ = 114 +/- 12) were evaluated (in their best 'on state') with tests assessing processes reliant on the functional integrity of frontal striatal circuitry, prior to the procedure (n = 11), at 3-6 months (n = 11) and at 9-12 months (n =10) post-operatively. Six of these patients were older than 69 years. Despite clinical motor benefits at 3-6 months post-operative, significant declines were noted in working memory, speed of mental processing, bimanual motor speed and co-ordination, set switching, phonemic fluency, long-term consolidation of verbal material and the encoding of visuospatial material. Declines were more consistently observed in patients who were older than 69 years, leading to a mental state comparable with progressive supranuclear palsy. 'Frontal' behavioural dyscontrol without the benefit of insight was also reported by half (three of six) of the caregivers of the elderly subgroup. At 9-12 months postoperative, only learning based on multiple trials had recovered. Tasks reliant on the integrity of frontal striatal circuitry either did not recover or gradually worsened over time. Bilateral STN DBS can have a negative impact on various aspects of frontal executive functioning, especially in patients older than 69 years. Future studies will evaluate a larger group of patients and examine the possible reversibility of these effects by turning the DBS off.

Affect↗

PubMed2004

A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek's disease virus.

Genetic resistance to Marek's disease (MD) has been proposed as a method to augment current vaccinal control of MD. Although it is possible to identify QTL and candidate genes that are associated with MD resistance, it is necessary to integrate functional screens with linkage analysis to confirm the identity of true MD resistance genes. To help achieve this objective, a comprehensive 2-hybrid screen was conducted using genes unique to virulent Marek's disease virus (MDV) strains. Potential MDV-host protein interactions were tested by an in vitro binding assay to confirm the initial two-hybrid results. As a result, 7 new MDV-chicken protein interactions were identified and included the chicken proteins MHC class II beta (BLB) and invariant (Ii) chain (CD74), growth-related translationally controlled tumor protein (TPT1), complement component Clq-binding protein (C1QBP), retinoblastoma-binding protein 4 (RBBP4), and alpha-enolase (ENO1). Mapping of the encoding chicken genes suggests that BLB, the gene for MHC class II beta chain, is a positional candidate gene. In addition, the known functions of the chicken proteins suggest mechanisms that MDV might use to evade the chicken immune system and alter host gene regulation. Taken together, our results indicate that integrated genomic methods provide a powerful strategy to gain insights on complex biological processes and yield a manageable number of genes and pathways for further characterization.

Animals↗

PubMed2001

Spinal cord and nerve root monitoring during surgical treatment of lumbar stenosis.

The author describes application of intraoperative neurophysiologic monitoring to surgical treatment of lumbar stenosis. Benefits of somatosensory and motor evoked potential studies during surgical correction of spinal deformity are well known and documented. Free-running and evoked electromyographic studies during pedicle screw implantation is an accepted practice at many institutions. However, the functional integrity of spinal cord, cauda equina, and nerve roots should be monitored throughout every stage of surgery including exposure and decompression. Somatosensory evoked potentials monitor overall spinal cord function. Intraoperative electromyography provides continuous assessment of motor root function in response to direct and indirect surgical manipulation. Electromyographic activities observed during exposure and decompression of the lumbosacral spine included complex patterns of bursting and neurotonic discharge. In addition, electromyographic activities at distal musculature were elicited by impacting a surgical instrument or graft plug against bony elements of the spine. All electromyographic events provided direct feedback to the surgical team and were regarded as a cause for concern. Simultaneously monitored evoked potential and electromyographic studies protect spinal cord and nerve roots during seemingly low-risk phases of a surgical procedure when neurologic injury may occur and the patient is placed at risk for postoperative myelopathy or radiculopathy.

Decompression, Surgical↗

PubMed1994

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.

Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity.

Antineoplastic Agents↗

PubMed1999

Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida.

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.

Culture Media↗

PubMed1999

Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation.

Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these "functional" in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of -60 mV evoked in control neurons potassium currents with a peak current of 10.0 +/- 2.5 nA and a large steady state current of 8.5 +/- 2.6 nA, which was still activated from a holding potential of -40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 +/- 2.4 nA; steady state: 3.6 +/- 2.0 nA). This current was inactivated at a holding potential of -40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42 degrees C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices.

Animals↗

PubMedDate not supplied

Positional candidate gene approach and functional genomics strategy in atopy gene discovery.

As part of our effort in searching for genetic factors contributing to the susceptibility to atopy and asthma, we have focused on a 'positional candidate' approach in identifying CC chemokine gene polymorphisms and their functional correlates. To date, a single-nucleotide polymorphism was found in the RANTES proximal promoter region, and a high degree of sequence variation was identified in the 3'-untranslated region -of the eotaxin gene. Also, we are pursuing a series of functional genomics' studies designed to identify differentially expressed genes in a panel of allergen-specific human Th2 cells and in antigen-induced hyperreactive murine airways. This is performed using a combination of protocols including suppression-subtractive hybridization and cDNA array hybridizations with 18,363 nonredundant sequences. A data base is being generated from a list of subtracted cDNA sequences and array-positive clones to categorize differentially expressed genes. Sequences are being placed in biologically relevant categories on the basis of function (i.e., receptor, signal transduction pathways, transcription, and translation). With the increasing amount of sequence information compiled by the Human Genome Project, it will be particularly challenging to integrate functional gene-mapping efforts to define and compare aberrant genotypes/phenotypes in atopic diseases.

Chemokine CCL5↗

PubMed1995

Time series forecasts of poison center call volume.

We tested the hypothesis that time series analysis can provide accurate predictions of future poison center telephone call volume by a prospective stochastic time series modeling of calls to a university-based regional poison center. All callers evaluated and managed during two sequential years had the time and date of the call recorded in a computer database. Time series variables were formed for poison center calls per hour. Prediction models were developed from the 1992 data and included four types: raw observations, moving average, means with moving average smoothing, and autoregressive integrated moving average. Forecasts from each model were tested against observations from the first 26 weeks of 1993. Each model's adequacy was tested on residuals by autocorrelation functions, integrated periodograms, linear regression, and differences among the variances. A total of 44,584 calls were received in 1992 and 24,781 in the first half of 1993. Large periodic variations in call volume with time of day were found (p < 0.001). The model based on arithmetic means of each hour of the week with three-point moving average smoothing yielded the most accurate forecasts and explained 58.5% of the variation observed in the 1993 test series (p < 0.001). Time series analysis can provide powerful, accurate short range forecasts of future poison center telephone call volume. Simpler, less expensive models performed best in this study.

Computers↗

PubMed1992

Improved integration potential for calcium-phosphate-coated implants after glow-discharge and water-storage.

Laboratory and clinical data support the conclusion that Radio-Frequency-Glow-Discharge-Treatment (RFGDT) of calcium-phosphate-coated implants can accelerate their functional integration with bone at host sites. In addition to the benefits of surface cleaning and activation associated with RFGDT, a period of water-storage prior to implantation also seems to be beneficial in eluting easily solubilized alkaline calcium components.

Dental Implantation, Endosseous↗

PubMed1981

The concept of health and the role of health education.

The field of health education needs to establish a conceptual basis that provides a legitimate focus and direction for health education. Health education should recognize that notions of health are human constructs which are restricted by man's interpretation of the functional integrity of the body and mind and relative to the activities and interests of individual societies. Health education should define health as that state of body and mind functioning which affords man the ability to strive towards his functional objectives and his culturally desired goals. The function of health education should be to impart knowledge for the purpose of developing the powers of reason and judgement in order to help people make their own choices about patterns of living which enhance the well functioning of the body and mind.

Attitude to Health↗

PubMed2002

Evaluation of mitochondrial function and membrane integrity by dual fluorescent staining for assessment of sperm status in rats.

Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.

Administration, Oral↗

PubMed2003

Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization.

The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C(10)) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C(10)). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C(10)-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (alpha or beta), 18C(10) did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies.

Bacteria↗

PubMed1982

[Effect of normoxic and anoxic liver perfusion as well as ischemic liver storage on parameters of mitochondrial respiration and swelling].

Using the model of Hb-free perfused rat liver, the influence of normoxic or anoxic perfusion, as well as ischemic storage of the liver on parameters of respiration and on swelling has been studied comparatively. The system used for Hb-free perfusion ensures at normoxic perfusion of the liver the functional integrity of isolated mitochondria for a period of investigation of 180 min. Normothermal ischemia of the liver leads, already after 20 min, to a significant decline of the ADP/O quotient and of the respiration control coefficient of the isolated mitochondria, as well as to mitochondrial swelling. Hypothermal ischemia of the liver (25 degrees C) clearly diminishes the mitochondrial functional loss. As far as the liver disposes of an endogenous glycogen store, the integrity of the mitochondria isolated after anoxic liver perfusion (25 degrees C) is retained and longer than after hypothermal ischemia of the liver. Mitochondria isolated after anoxic perfusion from glycogen-deprived liver swell and uncouple as fast as mitochondria prepared after ischemic storage of the glycogen-containing liver. Normoxic reperfusion of the ischemically pre-damaged liver leads to partial recovery of the mitochondria, if reoxygenation of the liver is started no later than after 80 min of ischemic storagel.

Animals↗