EXPERIMENTAL STUDIES ON THE INTERRELATIONS OF CONDYLAR GROWTH AND ALVEOLAR BONE FORMATION.
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Alveolar Process↗
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Vitellogenin of the cockroach, Leucophaea maderae: nucleotide sequence, structure and analysis of processing in the fat body and oocytes.
A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced. The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide 'subunits' following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights = 112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A)+ RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.
Adipose Tissue↗
Continuous processing of fusion protein expressed as an Escherichia coli inclusion body.
In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.
Blotting, Western↗
Colonization of ephemeral detrital patches by vagile macroinvertebrates in a brackish lake: a body size-related process?
The mechanisms regulating the build-up of invertebrate assemblages on ephemeral detritus patches are still poorly understood. Here, the daily colonization of decaying reed leaves by vagile macroinvertebrates was monitored in an brackish lake in Italy. The highly variable abundance patterns of dominant taxa were analysed by spectral and geostatistical techniques to test for nonrandomness and to further determine whether they were related to body size. Comparisons between two contrasting sites allowed an assessment of the generality of our observations. At both sites, the macroinvertebrate assemblage was dominated by three detritivorous taxa, i.e. the isopod Lekanesphaera monodi, the amphipod Microdeutopus gryllotalpa and the polychaete Neanthes caudata. Overall, their abundance patterns were characterised by short-term fluctuations of a nonrandom, autocorrelated nature. In addition, a significant covariation was observed between the average body mass of each taxon and the complexity of the respective abundance pattern, expressed by the fractal dimension D. The covariation was observed at both study sites, notwithstanding the diverging outcomes of bivariate pattern comparisons for similar-sized taxa. Our findings indicate that the size of macroinvertebrates is strongly related to the short-term dynamics of their abundance patterns on reed detritus, suggesting that the interaction between vagile consumers and ephemeral resource patches might be influenced by individual energetics. The implications of size-related constraints for the coexistence of species on decaying detrital patches are discussed.
Analysis of Variance↗
Distribution and properties of beta-adrenergic receptors in human iris-ciliary body.
beta-Adrenergic receptors from the iris-ciliary body of human eyes removed shortly after death were studied using membranes prepared by isopycnic centrifugation of tissue homogenates. This procedure separates uveal melanin pigment from plasma membranes and reduces nonspecific binding of 125I-iodopindolol. The observed binding of 125I-iodopindolol was of high affinity (Kd = 78 +/- 6.6 pM), saturable, and fully reversible (t1/2 = 4.6 min). Scatchard plots were linear and revealed a Bmax of 134 +/- 20 fmol/mg of protein from the whole iris-ciliary body. The affinities of the receptors for a series of agonists and antagonists were determined. The order of potency for the inhibition of the binding of the radioligand by antagonists was ICI 118,551 greater than MK950 greater than propranolol greater than ICI 89,406 greater than metoprolol. This order of potency is characteristic of beta-adrenergic receptors of the beta 2 subtype. Preparations of iris-ciliary body were also subjected to microdissection prior to density gradient centrifugation to permit the study of beta-adrenergic receptors in the ciliary processes, ciliary body, and iris. Each of these regions was found to contain approximately one third of the total number of beta-adrenergic receptors in the human iris-ciliary body. The highest density of receptors was located in the ciliary processes (180 +/- 40 fmol/mg of protein), while the density of receptors in the iris (98 +/- 7.5 fmol/mg of protein) and ciliary body (less the processes) (42 +/- 17 fmol/mg of protein) was notably lower. Only beta 2-adrenergic receptors are detectable by competition experiments in the iris-ciliary body as a whole, or in the individual preparations of iris, ciliary processes, or ciliary body; however, microdissection and analysis of beta-adrenergic receptor subtypes in isolated ciliary muscle permitted detection of a small number of beta 1-adrenergic receptors. beta 1-Adrenergic receptors comprised about 10% of the total number of beta-adrenergic receptors in the whole iris-ciliary body. The finding that most of the beta-adrenergic receptors in the human iris-ciliary body are of the beta 2 subtype may be of significant therapeutic importance in the medical management of glaucoma.
Adolescent↗
Hunger, caloric preloading and the selective processing of food and body shape words.
Fifty-three female subjects performed a modified Stroop colour-naming task 30 minutes after consuming a high-calorie preload, a low-calorie preload or water. Half of the subjects were 18 hours food deprived prior to testing. Food restriction and the caloric density of the preload had no effect on the colour-naming of food-related words. However, impairments in the colour-naming of food-related words did vary according to the subjects' self-reported hunger level. This relationship between hunger and colour-naming impairment was not linear, with the most hungry subjects displaying the smallest impairment in the colour naming of food words. The results are interpreted in terms of recent thought on the relationship between attentional processing and fear arousal.
Adolescent↗
Immunolocalization of tripeptidyl peptidase II, a cholecystokinin-inactivating enzyme, in rat brain.
Tripeptidyl peptidase II (EC 3.4.14.10) is a serine peptidase apparently involved in the inactivation of cholecystokinin octapeptide [Rose C. et al. (1996) Nature 380, 403-409]. We have compared its distribution with that of cholecystokinin in rat brain, using a polyclonal antibody raised against a highly purified preparation for immunohistochemistry at the photon and electron microscope levels. Tripeptidyl peptidase II-like immunoreactivity was mostly detected in neurons, and also in ependymal cells and choroid plexuses, localizations consistent with a possible participation of the peptidase in the inactivation of cholecystokinin circulating in the cerebrospinal fluid. Immunoreactivity was mostly detected in cell bodies, large processes and, to a lesser extent, axons of various neuronal populations. Their localization, relative to that of cholecystokinin terminals, appears to define three distinct situations. The first corresponds to neurons with high immunoreactivity in areas containing cholecystokinin terminals, as in the cerebral cortex or hippocampal formation, where pyramidal cell bodies and processes surrounded by cholecystokinin axons were immunoreactive. A similar situation was encountered in many other areas, namely along the pathways through which cholecystokinin controls satiety, i.e. in sensory vagal neurons, the nucleus tractus solitarius and hypothalamic nuclei. The second situation corresponds to cholecystokinin neuronal populations containing tripeptidyl peptidase II-like immunoreactivity, as in neurons of the supraoptic or paraventricular nuclei, axons in the median eminence or nigral neurons. In both situations, localization of tripeptidyl peptidase II-like immunoreactivity is consistent with a role in cholecystokinin inactivation. The third situation corresponds to areas with mismatches, such as the cerebellum, a region devoid of cholecystokinin, but in which Purkinje cells displayed high tripeptidyl peptidase II-like immunoreactivity, possibly related to a role in the inactivation of neuropeptides other than cholecystokinin. Also, some areas with cholecystokinin terminals, e.g., the molecular layer of the cerebral cortex, were devoid of tripeptidyl peptidase II-like immunoreactivity, suggesting that processes other than cleavage by tripeptidyl peptidase II may be involved in cholecystokinin inactivation. Tripeptidyl peptidase II-like immunoreactivity was also detected at the ultrastructural level in the cerebral cortex and hypothalamus using either immunoperoxidase or silver-enhanced immunogold detection. It was mainly associated with the cytoplasm of neuronal somata and dendrites, often in the vicinity of reticulum cisternae, Golgi apparatus or vesicles, and with the inner side of the dendritic plasma membrane. Hence, whereas a fraction of tripeptidyl peptidase II-like immunoreactivity localization at the cellular level is consistent with its alleged function in cholecystokinin octapeptide inactivation, its association with the outside plasma membrane of neurons remains to be confirmed.
Aminopeptidases↗
Macroglia distribution in the developing and adult inferior colliculus.
Macroglia distribution in the developing and adult gerbil inferior colliculus (IC) was investigated using two oligodendrocytic [myelin-associated-glycoprotein (MAG) and oligodendrocyte-specific molecule (Rip)] and two astrocytic [glial fibrillary acidic protein (GFAP) and S100] markers and immunohistochemistry. There was a spatio-temporal pattern of myelin marker expression starting in the ventral area of the IC and continuing to the dorsal part of the nucleus. Myelination, as revealed by MAG and Rip markers, starts in the IC during the second postnatal week. The intensity of myelination increased between stages P15 and P21 and extended to the whole IC. The appearance of myelin proteins in the IC may suggest a possible axonal outgrowth inhibition by oligodendrocytes in this structure. A differential pattern of staining was obtained with S100 and GFAP antibodies. Astrocytes identified as S100 immunoreactive cells were observed in the IC by birth and the staining was localized to their cell body and processes. S100 positive cells were homogeneously distributed within the IC nucleus. S100 pattern of staining remained the same in stages P7, P15 and P21. In adult IC, S100 positive cell processes were in contact with neuronal cell bodies, other S100 positive cells and blood vessels. Quantitative analysis showed an increase in the density of positive cells during the first postnatal week and a decrease then after through to adulthood. Unlike S100, GFAP immunoreactivity showed a different pattern of staining. At birth GFAP positive astrocytes were observed along the collicular brain midline and around the IC nucleus delimiting its boundaries. The GFAP pattern of labelling remained the same during development and in the adult. This data suggests the presence of two astrocytes subtypes with different locations in the IC nucleus. The GFAP positive astrocytes were located along the edge of the nucleus, while the S100 positive ones displayed a homogeneous distribution across the nucleus.
Animals↗
Separation of a process from the neuronal cell body using a thin aluminum foil separator.
A new technique using a simple-structured separator is described to separate the end of the process from the cell body (soma) of cultured cells. By this method, stimulation limited to neuronal processes is experimentally possible. The separator is of an oblong shape and consists of two parts: a square bracket-shaped frame made of Teflon and a thin aluminum foil which is stuck on both free ends of frame (bracket) coated with epoxy-resin. The separator is horizontally placed on the dish under microscope to divide the process on the way, so that the free end of the process is outside the oblong separator. This separator is simple and low cost, and does not need a long process, unlike other methods requiring a special culture dish with a micro-groove for growth of the neuronal process (neurite). The new technique could be useful in basic pain research and neurophysiology.
Aluminum↗
Phenotypic plasticity in adult sympathetic neurons: changes in neuropeptide expression in organ culture.
Vasoactive intestinal peptide (VIP)-like immunoreactivity is present at low levels in the superior cervical ganglion of the adult rat, where immunostained neural processes, but only an occasional immunostained cell body, are found. However, when ganglia are maintained for 24 or 48 hr in organ culture, their content of VIP-like immunoreactivity increases 6- or 31-fold, respectively. When examined at 24 hr, the increase in VIP-like immunoreactivity is totally blocked by an inhibitor of RNA or protein synthesis. Many neuronal cell bodies and processes with immunoreactivity for VIP and the related peptide histidine isoleucine amide (PHI) are seen in cultured ganglia. In addition, VIP/PHI mRNA is abundant in cultured ganglia but only barely detectable in ganglia prior to culture. Under the same culture conditions, neuropeptide Y-like immunoreactivity increases to a small extent, and tyrosine hydroxylase activity and total ganglion protein remain unchanged. These results support the idea that adult sympathetic neurons exhibit plasticity in neuropeptide expression and that this plasticity, in the case of VIP, depends on changes in gene expression.
Age Factors↗
Tuning p-wave interactions in an ultracold Fermi gas of atoms.
We have measured a p-wave Feshbach resonance in a single-component, ultracold Fermi gas of 40K atoms. We have used this resonance to enhance the normally suppressed p-wave collision cross section to values larger than the background s-wave cross section between 40K atoms in different spin states. In addition to the modification of two-body elastic processes, the resonance dramatically enhances three-body inelastic collisional loss.
Journal Article↗
[The glioneuronal interrelations in the nervous system of the crayfish].
The glio-neuronal interrelations in the nerves, connective and ganglia were studied in the crayfish Astacus leptodactylus as a representative of invertebrates. The outer membranes of the ganglia, connectives and nerves are formed by specialized cells with morphological features of the loose connective tissue cells. In the ganglia between the outer membrane and the neuropile there is another type of the connective tissue cells--Leydig's 1st type cells. The sheaths of axon bundles in the connectives and neuropile are formed by the bodies and processes of glial cells. Their processes also cover solitary axons. The neuron bodies are surrounded by glial cells forming trophospongium. Axons of greater diameter are surrounded by a greater amount of glial layers. Specific features of the structure of myelin-like neurite sheaths were found. The ratio of the length of the membrane of gliocytes covering the neurite to the length of the neurite circle on sections of motor fibers of the nerve is 1:15.7; of sensory fibers of the nerve--1:2.1; of connective neurites--1:1.9; of axons inside the ganglion--1:0.4; in the neuropile--1:0.01.
Animals↗
Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co-localizes with tubulin.
Differentiation of oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we have shown the presence of MAP1B in oligodendrocytes grown in primary glial cultures by double-label immunofluorescence using antibodies to galactocerebroside (GC) and MAP1B. The staining of the cultures showed that GC-positive oligodendrocytes were also stained with MAP1B antibodies. However, MAP1B stain was limited to cell bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta-tubulin in secondary glial cultures that were enriched for oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double-labeling of the cells showed that MAP1B was co-localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)-positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MAP1B (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.
Animals↗
Monoclonal antibodies to human neuron-specific enolase reveal heterogeneity of the enzyme in neurons of the central nervous system.
Three monoclonal antibodies to human neuron-specific enolase (NSE) were used to survey the human brain and spinal cord for immunoreactivity. Two of the antibodies (EB and CF) recognized the same population of cells and cell processes. Reactivity was restricted to myelinated axons, basket cell bodies and processes, and a small population of pyramidal cell bodies in the visual cortex. The third antibody (AD) reacted with some, but not all, of the neuronal cell bodies in cerebral cortex, hippocampus, midbrain, and spinal cord. Many neurons did not react with any of the antibodies. The epitope recognized by AD was trypsin-sensitive, while those recognized by EB and CF were not. These studies suggest that NSE may have multiple conformational or structural forms which are segregated between the cell body and axon.
Animals↗
Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons.
Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.
Animals↗
Distribution of neuropeptide-like immunoreactivity in intact and chronically decentralized middle cervical and stellate ganglia of dogs.
Neuropeptide-like immunoreactivity to antisera raised against Leu- and Met-enkephalin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and substance P (SP) have been studied immunohistochemically in middle cervical and stellate ganglia of dogs. To investigate the relationship of the peptides to one another as well as to preganglionic and postganglionic neurons, intact and chronically decentralized middle cervical and stellate ganglia were studied. Ganglia were processed for immunohistochemistry in unoperated dogs and in dogs two weeks after unilateral ganglionic decentralization. The immunoreactivity for each peptide had a characteristic distribution in the ganglia. These distributions differed from one another and from the distribution of cardiac postganglionic sympathetic neurons. Camera lucida drawings of peptide distributions were made to compare different peptides and counts were made to determine the percentages of cells immunoreactive for a given peptide. The results demonstrated that enkephalin-like immunoreactivity in axons was present in both the stellate and middle cervical ganglia, but was heaviest in the caudal 2/3 of the stellate ganglia. Enkephalin-like immunoreactive fibers formed pericellular baskets around stellate ganglion neurons. VIP-like immunoreactive cell bodies and processes were distributed sparsely, but widely, in the stellate ganglia and to a lesser extent in the middle cervical ganglia. One of two commercial antisera to SP resulted in immunoreactive staining of cell bodies and processes in the stellate ganglia. SP-like immunoreactivity in neurons represented about 10% or less of the cells in the stellate ganglia. At least 80-85% of the neurons in the stellate and middle cervical ganglia were immunoreactive for NPY antisera. Decentralization eliminated enkephalin-like immunoreactive staining in the middle cervical and stellate ganglia, but not the VIP-, NPY- and SP-like immunoreactive staining of neurons in these ganglia. In summary, the enkephalin-like immunoreactive axons in the thoracic autonomic ganglia appear to be derived from extrinsic neurons, most likely from preganglionic spinal neurons. VIP-, SP- and NPY-like immunoreactivity were not significantly affected by decentralization. The results provide anatomical evidence for substrates related to neuropeptidergic synaptic mechanisms in thoracic autonomic ganglia.
Animals↗
AMPA glutamate receptor subunits are differentially distributed in rat brain.
To demonstrate the regional, cellular and subcellular distributions of non-N-methyl-D-aspartate glutamate receptors in rat brain, we generated antipeptide antibodies that recognize the C-terminal domains of individual subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring glutamate receptors (i.e. GluR1, GluR4, and a region highly conserved in GluR2, GluR3 and GluR4c). On immunoblots, antibodies detect distinct proteins with mol. wts ranging from 102,000 to 108,000 in homogenates of rat brain. Immunocytochemistry shows that glutamate receptor subunits are distributed abundantly and differentially within neuronal cell bodies and processes in cerebral cortex, basal ganglia, limbic system, thalamus, cerebellum and brainstem. The precise patterns and cellular localizations of glutamate receptor subunit immunoreactivities are unique for each antibody. In neocortex and hippocampus, pyramidal neurons express GluR1 and GluR2/3/4c immunoreactivities; many non-pyramidal, calcium-binding, protein-enriched neurons in cerebral cortex are selectively immunoreactive for GluR1. In striatum, the cellular localizations of GluR1, GluR2/3/4c and GluR4 immunoreactivities are different; in this region, GluR1 co-localizes with many cholinergic neurons but is only present in a minor proportion of nicotinamide adenine dinucleotide phosphate diaphorase-positive striatal neurons. GluR1 co-localizes with most dopaminergic neurons within the substantia nigra. In several brain regions, astrocytes show GluR4 immunoreactivity. Within the cerebellar cortex, cell bodies and processes of Bergmann glia express intense GluR4 and GluR1 immunoreactivities; perikarya and dendrites of Purkinje cells show GluR2/3/4c immunoreactivity but no evidence of GluR1 or GluR4. Ultrastructurally, GluR subunit immunoreactivities are localized within cell bodies, dendrites and dendritic spines of specific subsets of neurons and, in the case of GluR1 and GluR4, in some populations of astrocytes. This investigation demonstrates that individual AMPA-preferring glutamate receptor subunits are distributed differentially in the brain and suggests that specific neurons and glial cells selectively express glutamate receptors composed of different subunit combinations. Thus, the co-expression of all AMPA receptor subunits within individual cells may not be obligatory for the functions of this glutamate receptor in vivo.
Amino Acid Sequence↗
End-to-end signal processing from the embedded body sensor to the medical end user through QoS-less public communication channels: the U-R-SAFE experience.
The URSafe project (IST-2001-33352) aims at creating a telemedicine care environment for the elderly and convalescent. The idea is to provide a portable device which monitors autonomously different biomedical signals and is able to send an alarm to a medical center if an abnormality is detected. In the initial version of URSafe, three sensors where included in the platform: electrocardiogram (ECG), oxygen rate (SpO2) and Fall Detector. This paper presents the signal processing algorithms involved in the project. These are at two levels: ECG analysis and signal transmission.
Accidental Falls↗