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PubMed1993

Modulation of murine immunological responses by salivary gland extract of Simulium vittatum (Diptera: Simuliidae).

The influence of Simulium vittatum Zetterstedt salivary gland extract on several immunological mechanisms was investigated in murine model hosts (laboratory mice). These mechanisms included the expression of major histocompatibility complex class II cell surface molecules, the in vitro mitogen responsiveness of lymphoid cells, and the antibody responses to heterologous foreign antigens (sheep erythrocytes). Experiments were designed to determine the influence of salivary gland extract following in vivo inoculation or in vitro inclusion in cell culture. In vivo inoculation of salivary gland extract reduced the percentage of Ia+ cells in spleen cell populations, although this difference was ameliorated by a 2 d in vitro culture period, regardless of whether salivary gland extract was included in culture. Salivary gland extract had no effect on Ia expression by cells derived from regional lymph nodes or the skin (epidermis). In vivo inoculation with salivary gland extract did not affect the responsiveness of splenic lymphocytes to mitogens, whereas in vitro exposure to salivary gland extract reduced both T and B cell mitogenesis. Finally, antibody responses to sheep erythrocytes were enhanced if salivary gland extract was included as a coinoculant, although this was expressed only at the systemic level regardless of the route of antigen delivery. In light of these results, immunomodulatory functions of black fly saliva are postulated; they are operative at different levels on different subcompartments of the immune system, possibly via cytokine modulation.

Animals↗

PubMed1992

Biotin in human milk: methods, location, and chemical form.

For infants, no Recommended Dietary Allowance for biotin has been published; the estimated safe and adequate intake seems to be based on measurements of human milk. However, published estimates of the biotin content disagree substantially. We sought to address several of the potential sources of disagreement by defining the conditions for the collection, storage, and subcompartment distribution of biotin in human milk using the [125I]avidin assay. Composition of the collection vessel (glass vs. common plastics) had no effect on biotin content of human milk. The biotin content of milk did not change during storage at room temperature for at least 1 wk, at 5 degrees C for at least 1 mo, or at -20 degrees C or -70 degrees C for at least 1.5 y. Biotin in the cell pellet and fat fraction accounted for less than 5% of that in the skim fraction. Of the biotin in the skim fraction, none (less than 3%) was reversibly bound to macromolecules, and less than 5% was covalently bound to macromolecules. We conclude that assay of free biotin will detect almost all of the biotin present in a sample of mature human milk.

Adult↗

PubMed1995

The ribonuclease activity of nucleolar protein B23.

Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.

Animals↗

PubMed2001

Development of a physiologically based pharmacokinetic model of isopropanol and its metabolite acetone.

A physiologically based pharmacokinetic (PBPK) model for isopropanol (IPA) and its major metabolite, acetone, is described. The structure of the parent chemical model, which can be used for either IPA or acetone by choosing the appropriate chemical-specific parameters, is similar to previously published models of volatile organic chemicals such as styrene. However, in order to properly simulate data on the exhalation of IPA and acetone during inhalation exposures, it was necessary to expand the description of the lung compartment to include a subcompartment for the upper respiratory tract mucus layer. This elaboration is consistent with published PBPK models of other water-soluble vapors in which the mucus layer serves to absorb the chemical during inhalation and then release it during exhalation. In the case of IPA exposure, a similar PBPK structure is used to describe the kinetics of the acetone produced from the metabolism of IPA. The resulting model is able to provide a coherent description of IPA and acetone kinetics in the rat and human for exposures to IPA by several routes: intravenous, intraperitoneal, oral, inhalation, and dermal. It is also able to consistently reproduce kinetic data for exposures of rats or humans to acetone. Thus, the model provides a validated framework for performing chemical-specific route-to-route extrapolation and cross-species dosimetry, which can be used in place of generic default calculations in support of risk assessments for IPA and acetone.

2-Propanol↗

PubMed2003

PBPK predictions of perchlorate distribution and its effect on thyroid uptake of radioiodide in the male rat.

Due to perchlorate's (ClO4-) ability to competitively inhibit thyroid iodide (I-) uptake through the sodium-iodide symporter (NIS), potential human health risks exist from chronic exposure via drinking water. Such risks may include hypothyroidism, goiter, and mental retardation (if exposure occurs during critical periods in neurodevelopment). To aid in predicting perchlorate's effect on normal I- kinetics, we developed a physiologically-based pharmacokinetic (PBPK) model for the adult male rat. The model structure describes simultaneous kinetics for both anions together with their interaction at the NIS, in particular, the inhibition of I- uptake by ClO4-. Subcompartments and Michaelis-Menten (M-M) kinetics were used to describe active uptake of both anions in the thyroid, stomach, and skin. Separate compartments for kidney, liver, plasma, and fat were described by passive diffusion. The model successfully predicts both 36ClO4- and 125I- kinetics after iv doses of 3.3 mg/kg and 33 mg/kg, respectively, as well as inhibition of thyroid 125I- uptake by ClO4- after iv doses of ClO4- (0.01 to 3.0 mg/kg). The model also predicts serum and thyroid ClO4- concentrations from 14-day drinking water exposures (0.01 to 30.0 mg ClO4-/kg/day) and compensation of perchlorate-induced inhibition of radioiodide uptake due to upregulation of the thyroid. The model can be used to extrapolate dose metrics and correlate observed effects in perchlorate toxicity studies to other species and life stages, such as rat gestation (Clewell et al., 2003). Because the model successfully predicts perchlorate's interaction with iodide, it provides a sound basis for future incorporation of the complex hypothalamic-pituitary-thyroid feedback system.

Animals↗

PubMed2003

Predicting neonatal perchlorate dose and inhibition of iodide uptake in the rat during lactation using physiologically-based pharmacokinetic modeling.

Perchlorate (ClO4-), a contaminant in drinking water, competitively inhibits active uptake of iodide (I-) into various tissues, including mammary tissue. During postnatal development, inhibition of I- uptake in the mammary gland and neonatal thyroid and the active concentration ClO4- in milk indicate a potentially increased susceptibility of neonates to endocrine disruption. A physiologically based pharmacokinetic (PBPK) model was developed to reproduce measured ClO4- distribution in the lactating and neonatal rat and predict resulting effects on I- kinetics from competitive inhibition at the sodium iodide symporter (NIS). Kinetic I- and ClO4- behavior in tissues with NIS (thyroid, stomach, mammary gland, and skin) was simulated with multiple subcompartments, Michaelis-Menten (M-M) kinetics and competitive inhibition. Physiological and kinetic parameters were obtained from literature and experiment. Systemic clearance and M-M parameters were estimated by fitting simulations to tissue and serum data. The model successfully describes maternal and neonatal thyroid, stomach, skin, and plasma, as well as maternal mammary gland and milk data after ClO4- exposure (from 0.01 to 10 mg/kg-day ClO4-) and acute radioiodide (2.1 to 33,000 ng/kg I-) dosing. The model also predicts I- uptake inhibition in the maternal thyroid, mammary gland, and milk. Model simulations predict a significant transfer of ClO4- through milk after maternal exposure; approximately 50% to 6% of the daily maternal dose at doses ranging from 0.01 to 10.0 mg ClO4-/kg-day, respectively. Comparison of predicted dosimetrics across life-stages in the rat indicates that neonatal thyroid I- uptake inhibition is similar to the adult and approximately tenfold less than the fetus.

Animals↗

PubMed1994

A role for synaptic vesicles in non-neuronal cells: clues from pancreatic beta cells and from chromaffin cells.

Synaptic vesicles, vesicular carriers highly specialized for the secretion of fast nonpeptide neurotransmitters, until recently were considered neuron-specific organelles. The identification and characterization of several of the most abundant synaptic vesicle proteins have led to the identification in peptide-secreting endocrine cells of a class of microvesicles, referred to as synaptic-like microvesicles (SLMVs), which are similar to synaptic vesicles in membrane composition, biogenesis, and life cycle. Studies of pancreatic beta cells and of a chromaffin cell-derived cell line (PC12 cells) have suggested that SLMVs, like synaptic vesicles, store and secrete neurotransmitter-like substances that act as paracrine/endocrine signaling molecules. SLMVs of pancreatic beta cells store GABA; SLMVs of PC12 cells contain acetylcholine. Both synaptic vesicles and SLMVs are recycling organelles that represent a specialized subcompartment of the receptor-mediated recycling pathway of all cells. Recently, homologues of membrane proteins of synaptic vesicles and SLMVs have been identified in the endocytic/recycling compartment of non-neuronal, nonendocrine cells. Tracing the evolution of synaptic vesicles from vesicular carriers present in all cells will help to advance our understanding of neurotransmitter release as well as of molecular mechanisms of vesicular traffic.

Animals↗

PubMed2001

Activity-dependent accumulation of Ca2+ in axon and dendrites of the leech Leydig neuron.

We have investigated Ca2+ changes evoked by single action potentials (APs) in axon and dendrites of leech Leydig neurons. Dendritic Ca2+ transients induced by an AP were twice as large as in the axon, and Ca2+ recovery was significantly faster in the dendrites as compared to the axon. The AP-induced Ca2+ transients were blocked by Co2+ and suppressed in Ca2+-free saline, indicating Ca2+ influx through voltage-activated channels. During a train of APs, Ca2+ accumulated significantly more in the axon than in the dendrites. Suppression of the Ca2+ influx changed the shape of the action potential and increased the firing frequency. The results suggest a functional role of Ca2+ influx and Ca2+ accumulation during electrical activity in different neuronal subcompartments.

Action Potentials↗

PubMed2004

Organisation of the human dorsomedial hypothalamic nucleus.

This study used acetylcholinesterase (AChE) histochemistry to reveal the organization of the dorsomedial hypothalamic nucleus (DM) in the human. Topographically, the human DM is similar to DM in the monkey and rat. It is wedged between the paraventricular nucleus, dorsally, and the ventromedial nucleus, ventrally. Laterally, DM borders the lateral hypothalamic area while medially it approaches the 3rd ventricle. The AChE staining distinguished two subcompartments of the human DM: the larger diffuse and the smaller compact DM. The subcompartmental organization of the human DM appears homologous to that found in the monkey and less complex than that reported in rats. Understanding of the organization of DM creates meaningful anatomical reference for physiological and pharmacological studies in the human hypothalamus.

Acetylcholinesterase↗

PubMed2002

Pharmacokinetic modeling of Gd-DTPA extravasation in brain tumors.

RATIONALE AND OBJECTIVES: The endothelial permeability of brain tumors affecting the blood-brain barrier can be quantified using dynamic contrast enhanced MR imaging. MATERIALS AND METHODS: The dynamics of contrast medium (CM) exchange was investigated in 31 brain tumors (intra- and extra-axial) by fast repeated T1-weighted imaging in order to determine the exchange rates and saturation concentrations. RESULTS: The analysis of CM exchange reveals two different transport processes from the blood into two separate interstitial subcompartments for intra- and extra-axial tumors, a rapid one with a transfer constant of 1/ =7.0 seconds and a slow one with 1/ =133.7 seconds. Highly significant differences exhibit gliomas and meningiomas with respect to the saturation concentrations of fast and slowly enhancing compartments. CONCLUSIONS: The fast component is probably caused by extravasation into viable tissue (enhanced in meningiomas) while the slow one probably reflects increased diffusion distances into poorly perfused tissue such as necrotic areas in glioblastomas.

Adolescent↗

PubMed2000

Neuromagnetic recordings in temporal lobe epilepsy.

The introduction of whole-head magnetoencephalography (MEG) systems facilitating simultaneous recording from the entire brain surface has established MEG as a clinically feasible method for the evaluation of patients with temporal lobe epilepsy (TLE). In mesial TLE, two types of MEG spike dipoles could be identified: an anterior vertical and an anterior horizontal dipole. Dipole orientations can be used to attribute spike activity to temporal lobe subcompartments. Whereas the anterior vertical dipole is compatible with epileptic activity in the mediobasal temporal lobe, the anterior horizontal dipole can be explained by epileptic activity of the temporal tip cortex. In nonlesional TLE, medial and lateral vertical dipoles were found which could distinguish between medial and lateral temporal seizure onset zones as evidenced from invasive recordings. In lesional TLE, MEG could clarify the spatial relationship of the structural lesion to the irritative zone. Evaluation of patients with persistent seizures after epilepsy surgery may represent another clinical important application of MEG because magnetic fields are less influenced than electric fields by the prior operation. Simultaneous MEG and invasive EEG recordings indicate that epileptic activity restricted to mesial temporal structures cannot reliably be detected on MEG and that an extended cortical area of at least 6 to 8 cm2 involving also the basal temporal lobe is necessary to produce a reproducible MEG signal. In lateral neocortical TLE MEG seems to be more sensitive than scalp-EEG which further underlines the potential role of MEG for the study of nonlesional TLE. Whole-head MEG therefore can be regarded as a valuable and clinically relevant noninvasive method for the evaluation of patients with TLE.

Atrophy↗

PubMedDate not supplied

MEG and EEG in epilepsy.

Both EEG and magnetoencephalogram (MEG), with a time resolution of 1 ms or less, provide unique neurophysiologic data not obtainable by other neuroimaging techniques. MEG has now emerged as a mature clinical technology. While both EEG and MEG can be performed with more than 100 channels, MEG recordings with 100 to 300 channels are more easily done because of the time needed to apply a large number of EEG electrodes. EEG has the advantage of the long-term video EEG recordings, which facilitates extensive temporal sampling across all periods of the sleep/wake cycle. MEG and EEG seem to complement each other for the detection of interictal epileptiform discharges, because some spikes can be recorded only on MEG but not on EEG and vice versa. Most studies indicate that MEG seems to be more sensitive for neocortical spike sources. Both EEG and MEG source localizations show excellent agreement with invasive electrical recordings, clarify the spatial relationship between the irritative zone and structural lesions, and finally, attribute epileptic activity to lobar subcompartments in temporal lobe and to a lesser extent in extratemporal epilepsies. In temporal lobe epilepsy, EEG and MEG can differentiate between patients with mesial, lateral, and diffuse seizure onsets. MEG selectively detects tangential sources. EEG measures both radial and tangential activity, although the radial components dominate the EEG signals at the scalp. Thus, while EEG provides more comprehensive information, it is more complicated to model due to considerable influences of the shape and conductivity of the volume conductor. Dipole localization techniques favor MEG due to the higher accuracy of MEG source localization compared to EEG when using the standard spherical head shape model. However, if special care is taken to address the above issues and enhance the EEG, the localization accuracy of EEG and MEG actually are comparable, although these surface EEG analytic techniques are not typically approved for clinical use in the United States. MEG dipole analysis is approved for clinical use and thus gives information that otherwise usually requires invasive intracranial EEG monitoring. There are only a few dozen whole head MEG units in operation in the world. While EEG is available in every hospital, specialized EEG laboratories capable of source localization techniques are nearly as scarce as MEG facilities. The combined use of whole-head MEG systems and multichannel EEG in conjunction with advanced source modeling techniques is an area of active development and will allow a better noninvasive characterization of the irritative zone in presurgical epilepsy evaluation. Finally, additional information on epilepsy may be gathered by either MEG or EEG analysis of data beyond the usual bandwidths used in clinical practice, namely by analysis of activity at high frequencies and near-DC activity.

Brain↗

PubMed2006

Revisiting the role of magnetoencephalography in epilepsy.

PURPOSE OF REVIEW: This review considers the current role of magnetoencephalography in clinical epileptology. RECENT FINDINGS: While magnetoencephalography and electroencephalography complement each other for interictal spike detection, magnetoencephalography is more sensitive in neocortical epilepsy. In temporal lobe epilepsy, magnetoencephalography can attribute epileptic activity to subcompartments of the temporal lobe and differentiate between patients with mesial, lateral and diffuse seizure onsets. In extratemporal epilepsy, magnetoencephalography provides unique information in nonlesional cases and helps to define the relationship of epileptic activity with respect to lesions and eloquent cortex. Magnetoencephalography also contributes to the clinical decision process in patients with cortical dysplasias, Landau-Kleffner syndrome and recurrent seizures after prior epilepsy surgery. Magnetoencephalography-guided re-evaluation of magnetic resonance imaging helps to reveal previously unrecognized lesions. In a presurgical setting interictal magnetoencephalography was superior to scalp electroencephalography. Complete resection of the magnetoencephalography-defined irritative zone has prognostic implications on postoperative seizure control. Magnetoencephalography can reliably localize sensorimotor and language cortex. Disadvantages of this technique include the difficulties in obtaining ictal recordings and the considerable costs involved. SUMMARY: Magnetoencephalography has been developed to a valuable noninvasive tool in clinical epileptology. The development of approaches which take into account both magnetoencephalography and electroencephalography simultaneously should provide more useful information in the future.

Brain Mapping↗

PubMed1992

Dynamics of Nitrogenous Assimilate Partitioning between Cytoplasmic and Vacuolar Fractions in Carrot Cell Suspension Cultures.

Bulk vacuole isolation, gas chromatography-mass spectrometry,, and high-performance liquid chromatography have been used to investigate the accumulation and partitioning of assimilated nitrogen supplied as (15)NH(4)Cl between vacuolar and extravacuolar (cytoplasmic) fractions of protoplasts from suspension cultures of carrot (Daucus carota L. cv Chantenay). Glutamine was the most abundant amino acid in the vacuole of protoplasts from late-exponential phase cells, whereas alanine, glutamate, and gamma-aminobutyric acid were located primarily in the cytoplasmic fraction. In (15)N-feeding studies, newly synthesized glutamine partitioned strongly to the vacuole, whereas glutamate partitioned strongly to the cytoplasm, gamma-aminobutyric acid was totally excluded from the vacuole, and alanine was distributed in both compartments. Comparison of the (15)N-enrichment patterns suggests that initial assimilation to glutamine occurs within a subcompartment of the cytoplasmic fraction. The protoplast-feeding technique may be extended to investigate cytoplasmic compartmentation further.

Journal Article↗

PubMed2003

Peroxisomal ascorbate peroxidase resides within a subdomain of rough endoplasmic reticulum in wild-type Arabidopsis cells.

Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167-2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20-27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2+-induced shift from a distribution throughout gradients (approximately 18%-40% [w/w] Suc) to > or =42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX.

Arabidopsis↗

PubMed1997

Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals.

Pulse-chase labeling of carnation (Dianthus caryophyllus L. cv Improved White Sim) petals with [14C]acetate has provided evidence for a hydrophobic subcompartment of lipid-protein particles within the cytosol that resemble oil bodies, are formed by blebbing from membranes, and are enriched in lipid metabolites (including fragrance volatiles) derived from membrane fatty acids. Fractionation of the petals during pulse-chase labeling revealed that radiolabeled fatty acids appear first in microsomal membranes and subsequently in cytosolic lipid-protein particles, indicating that the particles originate from membranes. This interpretation is supported by the finding that the cytosolic lipid-protein particles contain phospholipid as well as the same fatty acids found in microsomal membranes. Radiolabeled polar lipid metabolites (methanol/water-soluble) were detectable in both in situ lipid-protein particles isolated from the cytosol and those generated in vitro from isolated radiolabeled microsomal membranes. The lipid-protein particles were also enriched in hexanal, trans-2-hexenal, 1-hexanol, 3-hexen-1-ol, and 2-hexanol, volatiles of carnation flower fragrance that are derived from membrane fatty acids through the lipoxygenase pathway. Therefore, secondary lipid metabolites, including components of fragrance, appear to be formed within membranes of petal tissue and are subsequently released from the membrane bilayers into the cytosol by blebbing of lipid-protein particles.

Journal Article↗

PubMed2003

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.

Base Sequence↗

PubMed1978

A new analytical method for determining duration of phases, rate of DNA synthesis and degree of synchronization from flow-cytometric data on synchronized cell populations.

L-cells synchronized by mitotic selection were investigated by flow-cytometry and the fractions of cells in the various cell cycle compartments were determined as a function of time. A new analytical evaluation procedure was developed, by which the mean transit-times of cells through various cell cycle phases can be calculated from these data. Three examples for application of the method are presented: (1) determination of the duration of G1, S, G2 + M and of the whole cell cycle; (2) calculation of the rate of DNA synthesis in several subcompartments of the S-phase; and (3) evaluation of the degree of synchronization at different stages of the cell cycle.

Cell Cycle↗