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PubMed2001

Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520.

A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduced in the cytosolic and secreted forms. The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts. The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis. The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time. The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied. Thermostability measurements showed that Chi40 has a T(m) of 60.7 degrees C at neutral pH. (13)C-(15)N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21trxB(DE3) cells grown in minimal medium in the presence of the paramagnetic elements [(13)C]glucose and (15)NH(4)Cl. The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy.

Carbon Isotopes↗

PubMed2001

Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage.

The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.

Adenoviridae↗

PubMed1976

Interchromosomal asynchrony of DNA replication in polytene chromosomes of Drosophila pseudoobscura.

Analysis of 3H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of 3H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.

Animals↗

PubMed1994

Muscarinic receptors binding in retinal pigment epithelium during rat development.

[3H]Quinuclidinyl benzylate (3H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of 3H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of 3H-QNB binding showed two high affinity sites to RPE, with KB = 2.6nM and 45 nM. Specific 3H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of 3H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.

Animals↗

PubMed2002

[Angiogenesis--anti-angiogenesis. Significance for tumor growth and metastasis].

Angiogenesis plays an important role in the local growth and metastasis of a variety of malignant tumors. During the past three decades so-called angiogenesis factors have been discovered and characterized in more detail. These are the major contributors to angiogenesis. The term angiogenesis factors includes other functionally heterogeneous molecules. The best characterized angiogenesis factors are endothelial growth factors, such as "vascular endothelial growth factor" (VEGF), "fibroblast growth factor" (FGF), "platelet-derived growth factor" (PDGF), Angiogenin and interleukin-8 (IL-8). However, members of the family of matrix metalloproteinases are also included. The list of factors involved in angiogenessis and their receptors are increasing steadily as does the list of molecules with antiangiogenic capacity. The latter comprises the endogenous factors, Angiostatin, Endostatin, Thrombospondin-1 and 2, and chemical compounds, such as the Fumagillin derivative, AGM-1470, or inhibitors of matrix metalloproteinases. These have attracted more interest during the past years, since investigations on these molecules foster hope for new therapeutic strategies. Some of these antiangiogenic factors have already been used in various therapeutic approaches to influence tumor growth. While impressive results have been obtained in well controlled experimental animal models, the results of clinical studies in humans fall short of these successes. This is at least in part due to the fact that angiogenesis of tumors is a complex process based on the interaction of a variety of factors. The present article summarizes the current knowledge about tumor angiogenesis with a particular emphasis on antiangiogenic molecules. The presented data open new interesting therapeutic perspectives for future dermatology.

Angiogenesis Inducing Agents↗

PubMed2000

[Detection of numerical chromosome aberrations in leukoplakia and squamous epithelial carcinomas of the head-neck area using fluorescence in situ hybridization].

The tumorigenesis of head and neck squamous cell carcinoma (HNSCC) has been proposed to represent a multistep process characterized by an accumulation of genetic alterations. To study numerical chromosomal aberrations and chromosomal imbalances, biopsies of 11 malignant tumours and biopsies of 16 oral premalignant lesions (leukoplakias) were analyzed by fluorescence in situ hybridization (FISH) using centromeric probes for chromosomes, 1, 7, 9, 10 and 17. The comparison of the alterations observed in simple leukoplakias (group 1, n = 8), dysplastic leukoplakias (group 2, n = 8) and malignant tumours (group 3, n = 11) by the Cochran-Armitage Trend Test revealed an increasing number of numerical chromosomal abberations. This difference was statistically highly significant (p < 0.001). The data open up the possibility that FISH analysis might help to better characterize the progression of premalignant oral leukoplakias.

Biopsy↗

PubMed2006

Differences in the cellular mechanism underlying the effects of amphetamine on prepulse inhibition in apomorphine-susceptible and apomorphine-unsusceptible rats.

BACKGROUND: Amphetamine is often used to mimic certain aspects of schizophrenia in laboratory animals, such as a decreased prepulse inhibition. MATERIALS AND METHODS: Apomorphine-susceptible and apomorphine-unsusceptible rats represent a well-characterized animal model for individual differences in the sensitivity to dopaminergic drugs. Moreover, apomorphine-susceptible rats show a wide variety of schizophrenia-like abnormalities. The differential response to administration of amphetamine (1-4 mg/kg, i.p.) was investigated in these two rat lines using the prepulse inhibition paradigm. Because amphetamine promotes dopamine release, the cellular mechanism underlying the line-specific effects of amphetamine was investigated by administration of alpha-methyl-para-tyrosine (aMpT) and reserpine, substances that are known to deplete the cytosolic dopamine pool and the vesicular dopamine pool, respectively, the former being primarily implicated in mediating the effects of amphetamine. RESULTS: All doses of amphetamine decreased prepulse inhibition in apomorphine-susceptible rats, whereas only the highest doses (2 and 4 mg/kg, i.p.) of amphetamine decreased prepulse inhibition in apomorphine-unsusceptible rats. Alpha-methyl-para-tyrosine, but not reserpine, blocked the amphetamine-induced disruption in prepulse inhibition in apomorphine-unsusceptible rats, whereas both substances alone had no effect in apomorphine-susceptible rats. However, the combination of alpha-methyl-para-tyrosine and reserpine did block the amphetamine-induced effects in the latter rat line. DISCUSSION: The present study suggests that apomorphine-susceptible rats are more sensitive to systemic administration of amphetamine than apomorphine-unsusceptible rats. In addition, the data show that the cellular mechanism underlying the effects of amphetamine differs between apomorphine-susceptible and apomorphine-unsusceptible rats. Whereas the effects of amphetamine on prepulse inhibition in apomorphine-unsusceptible rats just require the alpha-methyl-para-tyrosine sensitive dopamine pool, the effects in apomorphine-susceptible rats require both the alpha-methyl-para-tyrosine sensitive and the reserpine sensitive dopamine pool. Because apomorphine-susceptible rats share many features with schizophrenic patients, these data open the perspective that in these patients amphetamine may induce dopamine release from both types of dopamine pool. This might provide an explanation for the increased dopamine release after this psychostimulant drug in patients vs controls.

Amphetamine↗

PubMed2002

Movement mapping as dynamic preoperative surgical planning in total hip replacement. A precondition to navigation?

The purpose of the present study was to develop and present a computer program for preoperative simulation of implant components placement in total hip alloarthroplasty, and its influence on the possible range of motion (ROM). We evaluated a computer simulation for preoperative estimation of range of motion (movement mapping) in total hip replacement. The computer program was based on Borland C++. The system had an open data port, so the data could be transferred to an Excel spreadsheet for statistical evaluation. With the developed virtual computer simulation, a practical model was established. The model showed range of motion patterns which correspond to clinical experience. ROM was best at a shaft anteversion between 20 degrees and 30 degrees. ROM had its maximum with a CCD angle of 120 degrees -130 degrees. Acetabular cup anteversion was optimal between 10 degrees and 20 degrees, and cup inclinations were optimal below 40 degrees. The presented movement mapping system seems to be a reliable option for dynamic preoperative planning, which may be a prerequisite for the use of intraoperative navigation systems.

Acetabulum↗

PubMed2006

Vagus nerve stimulation (VNS) for depression: what do we know now and what should be done next?

Vagus nerve stimulation (VNS) therapy is the first US Food and Drug Administration-approved somatic clinical intervention for treatment-resistant depression (TRD). Long-term open data suggest a sustainable antidepressant response over time. Here we review the clinical data that exist so far and their limitations. We also discuss guidelines that may inform the clinical utilization of this procedure. Further clinical studies, in addition to prospective cost utilization and health economic investigations, are needed to better understand VNS therapy and the impact it holds on TRD care.

Bipolar Disorder↗

PubMed1983

Relationship of masseter electromyographic activity to mandible position in the freely-moving rat.

Simultaneous records of masseter-EMG activity and position of the mandible were made on 4 Wistar rats eating four food types: standard food pellets (large pellets), 45 mg precision food pellets (small pellets), bread and pudding. Masseter activity was recorded with chronic bipolar silver EMG electrodes placed within the muscle and led subcutaneously to a pedestal on the head. The transducer indicating jaw position consisted of two tiny inductance coils, placed on the head and under the mandibular symphysis. The lower coil acted as a receiving aerial for the carrier signal supplied via the upper coil, so that distance between the coils, and thus relative jaw position, was traced on the oscilloscope screen. A silent period was found in the masseter-EMG bursts similar to that found in man, where the silent period represents tooth contact and the occlusal phase of chewing (time during which teeth are in the intercuspal position) is defined as the interval between the silent period and the termination of the EMG activity in the muscle. Applying this to the rat data, opening represented 70-75 per cent of the total cycle while the combined closing and occlusion phases comprized 25-30 per cent when either type of pellet was eaten. With bread, closing and occlusal phases represented 56 per cent of the total cycle. For all three food types, muscular force, as indicated by the jaw-closing movement registered by the transducer, continued beyond the masseter EMG. Irregular jaw movements seen when the animals lapped pudding were not correlated directly with masseter activity.

Animals↗

PubMed1986

Origin of differences of inhibitory potency of cardiac glycosides in Na+/K+-transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle.

The inhibitory potency of altogether 95 steroidal compounds (including cardenolides, bufadienolides and their glycosides) on the Na/K-ATPases (Na+/K+-transporting ATPases, EC 3.6.1.37) from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle was compared to probe the complementary chemotopology of the inhibitor binding site areas on the three enzyme variants. The changes of potency, resulting from systematic variations of the geometry of steroid skeleton and the character as well as the structure of side chains at C3 or/and C17 of steroid backbone, allowed the following major conclusions. With the human cardiac and cerebral enzyme forms, the paired K0.5 (K'D) values for 77 steroid derivatives, covering seven orders of ten, were highly correlated. On an average, the total of compounds showed a 1.5-fold higher affinity to the cardiac enzyme. This tiny differentiation did not appear to be connected with an important difference in the chemotopology of the complementary subsites for steroid nucleus binding on the two enzyme forms. With the human and guinea-pig cardiac enzyme variants, the K0.5 values for 69 steroid derivatives, covering six orders of ten, were determined. For 41 5 beta, 14 beta-androstane derivatives only, the paired K0.5 values showed a close correlation. Here, the human enzyme variant exhibited 27-fold higher affinity. However, the paired K0.5 values determined on both enzymes for 28 steroid derivatives of differing structural features were but poorly correlated. Essentially, the geometries of the steroid nucleus determined the differential contributions of the side chains at C3 and C17 to the integral inhibitory potency on the two enzyme variants. Thus, the species differences in the potency of cardiac glycosides were traced to species differences in the complementarity of the steroid binding subsites. Hence, estimates of the potency of new steroidal compounds obtained on the guinea-pig cardiac enzyme can be neither quantitatively nor qualitatively easily extrapolated to the human cardiac enzyme. The extrathermodynamic analysis of the data opened major new insights in the structure-activity relationships concerning the role of C14 beta-OH, the character of the lead structure in cardioactive steroid lactones, and the significance of the configuration of A/B ring junction.

Animals↗

PubMedDate not supplied

Nonstationary properties of postural sway.

Postural sway during quite stance is usually assumed to be a stationary stochastic process. We tested this assumption by investigating the time invariance of the average value and variance of the postural sway of three subjects. The sway was measured with a force plate under three conditions: subject standing on two feet with eyes open; subject standing on two feet with eyes closed; and subject standing on one foot with eyes open. Data were collected in 1 min runs. More than 50 min of data were collected for each subject under each test condition. The data were averaged across all runs for each subject and condition. Trends were found to be present in the data. In addition, there were initial transient increases in the second-order moments about the trends. The transient changes in first- and second-order moments usually disappeared during the first 20 s. In light of these findings, we can reject the hypothesis that postural sway is a stationary process. The results imply that the usual methods to parameterize postural sway have to be either changed or reinterpreted.

Adult↗

PubMed1995

Ontogenesis of goal-directed behavior: anatomo-functional considerations.

Recent neuroanatomical and neurophysiological studies in man have revealed ontogenetic events which coincide with broadly defined phases of behavioral and cognitive development. During the early fetal period, early produced neurons make initial synapses which form the basis for the earliest electrical activity of the human brain. The overall immaturity of neuronal connections, in particularly in cortical areas, correlates with the absence of any behavioral pattern or goal-directed movements. In the late fetus and preterm infant, transient accumulation of major afferent pathways, the presence of transient layers (subplate zone) and transient pattern of transmitter-related organization form the neurological basis of cortical electric responses as well as transient behavioral states and sleep patterns. Parallel to the profound structural and chemical reorganization of the human cerebrum during the first 6 postnatal months there is a disappearance of transient behavioral and motor patterns. The previously close spatio-temporal correlation between these events becomes progressively looser. The overproduction of circuitry elements during the subsequent period peaks in associative cortex between 1 and 2 years of age, corresponding to the emergence of skilled actions and cognitive functions. After the elimination of some circuitry elements after the second year of life, the prolonged maturation of goal-directed behavior and the protracted emergence of different cognitive functions correlates with the development plateau of synapse production which can be seen up to 16 years of age. Parallel to the prolonged maturation of postsynaptic elements, there are well defined maturational changes in the chemical properties of associative pyramidal neurons of cortical layer III. These findings correspond to the prolonged maturation of movement-related brain macropotentials as well as other cognition-related potentials, where the last prominent changes were seen after 10 years of age. Although the coincidence of the developmental events does not necessarily mean a causal relationship, the combination of structural and physiological data opens new vistas for the further investigation of the neurobiological basis of goal-directed movement and cognitive behavior.

Adolescent↗

PubMed1996

Dose-dependent delivery of colchicine to the rat hippocampus by microdialysis.

The time courses of the colchicine delivery and diffusion rate in the brain were studied by microdialysis in the rat. Microdialysis allowed the exposure of the brain tissue to colchicine to be regulated, unlike a bolus injection. Colchicine was infused directly into the dorsal hippocampus at 40 ng/ml and 40 micrograms/ml, for 8 h. The amount of colchicine delivered to the brain and the diffusion rate from the probe were dose-dependent: colchicine diffusion into the brain was linear at 40 ng/ml but tended to plateau after 4 h at 40 micrograms/ml. The drug actually delivered with the higher dosage was only about 50% of that predicted from a constant diffusion. The total amount delivered at 40 ng/ml was 3.73 +/- 0.14 ng and at 40 micrograms/ml, it was 2.06 +/- 0.20 micrograms. Thus tissues surrounding the infusion site were saturated at high concentration and no more colchicine was diffused. Postmortem measurements of colchicine concentration in the forebrain confirmed these findings. Hence, the way in which colchicine is delivered to the brain is a critical factor for induction of its neurotoxic effects. These data open the way to a research on the correlation between local brain concentrations of colchicine and neurodegenerescence.

Animals↗

PubMed1991

Taurine receptors in membranes from retinal pigment epithelium cells in culture.

[3H]Taurine-specific binding to membranes from retinal pigment epithelium was demonstrated. A single saturable system was found, with KB = 237 nM and Bmax = 2.8 pmol/mg protein. Binding to freshly prepared membranes showed partial Na(+)-dependence while in frozen/thawed membranes, binding remained unchanged in the absence or presence of this ion. A 30-40% increase in binding was observed at physiological temperature (37 degrees C) compared to 4 degrees C in fresh but not in frozen membranes. Accumulation of taurine was followed during differentiation in vitro; results showed that changes in uptake and receptor binding to frozen membranes are not parallel, discarding the possibility of an interaction with uptake sites. Pharmacology of these binding sites suggests that they could be common to other amino acids, since displacement experiments showed that glycine, beta-alanine and strychnine were as potent as taurine itself in displacing [3H]taurine. Our data open the possibility of taurine being involved in the communication between the retina and the retinal pigment epithelium through an interaction with specific receptors.

Amino Acids↗

PubMed1995

Effects of Ca2+ channel antagonist subtypes on mitochondrial Ca2+ transport.

This study was carried out to define the effects of various Ca2+ channel modulatory drugs on mitochondrial Ca2+ movements. Bovine adrenal medulla mitochondria took up Ca2+ at an initial rate of 6.8 nmol mg protein-1 5 s-1, with a Km of 15 microM and a Bmax of 30 nmol mg protein-1. At 30 microM, neither verapamil, diltiazem, nitrendipine nor Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] affected the initial rate of Ca2+ uptake. Ca(2+)-loaded mitochondria retained their Ca2+ contents in the presence of ruthenium red for at least 30 min. Cinnarizine and flunarizine, but not verapamil, diltiazem, isradipine, Bay K 8644 or nitrendipine, caused a fast and dramatic Na(+)-independent Ca2+ loss. Other Ca2+ channel antagonists assayed such as penfluridol, R56865 [N-[1-(4-(4-fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2- benzothiazolamine], lidoflazine, R87926 [(+)-(S)-4-(2-benzothiazolyl-methylamino)-alpha-[(3,4-difluorophenoxy ) methyl] 1 piperidine] and sabeluzole, also had a mitochondrial Ca2+ depleting effect which seemed to be directly related to their octanol/water partition coefficient. The Na(+)-dependent Ca2+ efflux from mitochondria was completely inhibited by diltiazem and greatly blocked by nitrendipine. Isradipine caused a moderate blockade and Bay K 8644 and verapamil had no effect. All these data open the possibility of developing novel Ca2+ channel antagonists having selective actions on plasmalemmal Ca2+ channels, and others with additional and different effects on mitochondrial Ca2+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2004

Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation.

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.

Aldrin↗

PubMed2005

Synthesis and anti-inflammatory activity of a series of N-substituted naproxen glycolamides: nitric oxide-donor naproxen prodrugs.

A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been synthesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds 4a, 4b, 5a, 5b, 7b, and 7c exhibited anti-inflammatory activity equivalent to that of the parent NSAID, naproxen-Na, in the rat carrageenan paw edema model. At equimolar doses relative to naproxen-Na, the NO-donor glycolamide derivatives 4a, 4b, 5a, 5b, 7b, and 7c were gastro-sparing in the rat. Naproxen formation from these NO-donor glycolamides varied among the structures examined, with the N-substituent on the amide group having a particular influence, and demonstrated their prodrug nature. Compound 7b was selected for exemplary demonstration that the glycolamide nitrates can be bioactivated to release NO. These data open the possibility that naproxen glycolamide nitrates may represent a safer alternative to naproxen as anti-inflammatory medicines.

Amides↗