Multi-center reproducibility of neurochemical profiles in the human brain at 7 T.

The purpose of this work was to harmonize data acquisition and post-processing of single voxel proton MRS ((1) H-MRS) at 7 T, and to determine metabolite concentrations and the accuracy and reproducibility of metabolite levels in the adult human brain. This study was performed in compliance with local institutional human ethics committees. The same seven subjects were each examined twice using four different 7 T MR systems from two different vendors using an identical semi-localization by adiabatic selective refocusing spectroscopy sequence. Neurochemical profiles were obtained from the posterior cingulate cortex (gray matter, GM) and the corona radiata (white matter, WM). Spectra were analyzed with LCModel, and sources of variation in concentrations ('subject', 'institute' and 'random') were identified with a variance component analysis. Concentrations of 10-11 metabolites, which were corrected for T1 , T2 , magnetization transfer effects and partial volume effects, were obtained with mean Cramér-Rao lower bounds below 20%. Data variances and mean concentrations in GM and WM were comparable for all institutions. The primary source of variance for glutamate, myo-inositol, scyllo-inositol, total creatine and total choline was between subjects. Variance sources for all other metabolites were associated with within-subject and system noise, except for total N-acetylaspartate, glutamine and glutathione, which were related to differences in signal-to-noise ratio and in shimming performance between vendors. After multi-center harmonization of acquisition and post-processing protocols, metabolite concentrations and the sizes and sources of their variations were established for neurochemical profiles in the healthy brain at 7 T, which can be used as guidance in future studies quantifying metabolite and neurotransmitter concentrations with (1) H-MRS at ultra-high magnetic field.

Ultra high spatial and temporal resolution breast imaging at 7T.

There is a need to obtain higher specificity in the detection of breast lesions using MRI. To address this need, Dynamic Contrast-Enhanced (DCE) MRI has been combined with other structural and functional MRI techniques. Unfortunately, owing to time constraints structural images at ultra-high spatial resolution can generally not be obtained during contrast uptake, whereas the relatively low spatial resolution of functional imaging (e.g. diffusion and perfusion) limits the detection of small lesions. To be able to increase spatial as well as temporal resolution simultaneously, the sensitivity of MR detection needs to increase as well as the ability to effectively accelerate the acquisition. The required gain in signal-to-noise ratio (SNR) can be obtained at 7T, whereas acceleration can be obtained with high-density receiver coil arrays. In this case, morphological imaging can be merged with DCE-MRI, and other functional techniques can be obtained at higher spatial resolution, and with less distortion [e.g. Diffusion Weighted Imaging (DWI)]. To test the feasibility of this concept, we developed a unilateral breast coil for 7T. It comprises a volume optimized dual-channel transmit coil combined with a 30-channel receive array coil. The high density of small coil elements enabled efficient acceleration in any direction to acquire ultra high spatial resolution MRI of close to 0.6 mm isotropic detail within a temporal resolution of 69 s, high spatial resolution MRI of 1.5 mm isotropic within an ultra high temporal resolution of 6.7 s and low distortion DWI at 7T, all validated in phantoms, healthy volunteers and a patient with a lesion in the right breast classified as Breast Imaging Reporting and Data System (BI-RADS) IV.

Pushing the limits of high-resolution functional MRI using a simple high-density multi-element coil design.

Recent studies have shown that functional MRI (fMRI) can be sensitive to the laminar and columnar organization of the cortex based on differences in the spatial and temporal characteristics of the blood oxygenation level-dependent (BOLD) signal originating from the macrovasculature and the neuronal-specific microvasculature. Human fMRI studies at this scale of the cortical architecture, however, are very rare because the high spatial/temporal resolution required to explore these properties of the BOLD signal are limited by the signal-to-noise ratio. Here, we show that it is possible to detect BOLD signal changes at an isotropic spatial resolution as high as 0.55 mm at 7 T using a high-density multi-element surface coil with minimal electronics, which allows close proximity to the head. The coil comprises of very small, 1 × 2-cm(2) , elements arranged in four flexible modules of four elements each (16-channel) that can be positioned within 1 mm from the head. As a result of this proximity, tissue losses were five-fold greater than coil losses and sufficient to exclude preamplifier decoupling. When compared with a standard 16-channel head coil, the BOLD sensitivity was approximately 2.2-fold higher for a high spatial/temporal resolution (1 mm isotropic/0.4 s), multi-slice, echo planar acquisition, and approximately three- and six-fold higher for three-dimensional echo planar images acquired with isotropic resolutions of 0.7 and 0.55 mm, respectively. Improvements in parallel imaging performance (geometry factor) were up to around 1.5-fold with increasing acceleration factor, and improvements in fMRI detectability (temporal signal-to-noise ratio) were up to around four-fold depending on the distance to the coil. Although deeper lying structures may not benefit from the design, most fMRI questions pertain to the neocortex which lies within approximately 4 cm from the surface. These results suggest that the resolution of fMRI (at 7 T) can approximate levels that are closer to the spatial/temporal scale of the fundamental functional organization of the human cortex using a simple high-density coil design for high sensitivity.

Analysis of handling noises on wound strings.

This study analyzes the handling noises that occur when a finger is slid along a wound string. The resulting noise has a harmonic structure due to the periodic texture of the wound string. The frequency of the harmonics and the root-mean-square amplitude of the noise were found to be linearly proportional to the sliding speed. In addition, the sliding excites the longitudinal modes of the string, thus resulting in a set of static harmonics in the noise spectrum. The sliding excites different longitudinal modes depending on the sliding location.

Chlorambucil pharmacokinetics and DNA binding in chronic lymphocytic leukemia lymphocytes.

Chlorambucil (CLB) uptake by chronic lymphocytic leukemia lymphocytes was studied using a radiometric and a newly developed high-performance liquid chromatography assay. CLB labeled with 14C in either the chloroethyl group or phenyl ring was used with identical results. Drug accumulation by the cells was found to peak at 30 s, was independent of temperature, and was proportional to medium CLB concentration over a wide range. Efflux from cells loaded with CLB and resuspended in drug-free medium was nearly complete at 30 s. The metabolic inhibitors 2-deoxyglucose and NaN3, the nitrogen mustard transport inhibitor hemicholinium-3, and another alkylating agent, melphalan, had no effect on drug uptake. We conclude that CLB enters and exits chronic lymphocytic leukemia lymphocytes by simple diffusion. Cells from 17 patients with all stages of chronic lymphocytic leukemia were studied including three with CLB-resistant disease, and no heterogeneity was found in the peak cell-associated CLB content or in metabolite pattern on high-performance liquid chromatography. These findings make it unlikely that transport or cellular drug metabolism are factors in drug resistance. Drug-DNA binding was found to be temperature-sensitive and increased with time of incubation. Gel filtration of DNA before and after enzymatic digestion indicated the presence of drug-DNA adducts. High-performance liquid chromatography analysis of digested DNA and DNA treated by neutral thermal hydrolysis suggested the presence of multiple adducts. Most of the radioactivity was found as purine adducts. Studies with CLB labeled at two different sites revealed the presence of the phenyl group and ethyl chains in the adducts. A survey of patients showed increased drug-DNA binding in cells from patients with clinical CLB resistance.

Lack of drug-induced DNA cross-links in chlorambucil-resistant Chinese hamster ovary cells.

Chlorambucil (CLB) is an alkylating agent commonly used in the treatment of several neoplastic disorders. The mechanisms underlying resistance to this drug are not fully defined. We used the DNA alkaline elution technique to study cross-link formation in the wild type (K1) and a CLB-resistant (ChlR) Chinese hamster ovary cell line. [14C]CLB was used to measure drug uptake. The CLB-resistant cells were found to have negligible DNA cross-link formation compared to K1 cells at all time points tested. There was a correlation between the resistance to CLB and the decreased ability of resistant cells to form DNA cross-links. Results of drug uptake experiments excluded altered CLB accumulation as the basis for these findings. Assays of O6-alkylguanine transferase and topoisomerase. II provide evidence against a role of these enzymes in CLB resistance. These studies suggest that the mechanism of CLB cytotoxicity involves the formation of DNA cross-links. Reduced cross-link formation may confer resistance to CLB.

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II.

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.

Learning-induced activation of protein kinase C. A molecular memory trace.

PKC activation has been shown to mimic the biophysical consequences of classical conditioning in both rabbit hippocampus and Hermissenda type B cells. Furthermore, conditioning in rabbits results in the 24 h translocation of PKC from cytosol to membrane, which is probably responsible for mediating the biophysical consequences of conditioning. A model has been presented that suggests that long-term translocation of PKC occurs via the synergistic activation of a DG dependent pathway that activates PKC and a calcium dependent pathway that activates CaM kinase. Translocation of PKC to the plasma membrane, by altering ion channel properties, could subserve memory lasting for days, whereas translocation to the nuclear membrane could induce cellular change, by genomic regulation, lasting beyond days. We are, therefore, suggesting that protein kinase C may play a critical role in the formation of short, intermediate, and long-term associative memory.

Studies of chlorambucil-DNA adducts.

Chlorambucil (CLB) is a bifunctional nitrogen mustard whose therapeutic and major side-effects are thought to be caused by binding to DNA. HPLC analysis of hydrolyzed DNA from L1210 cells incubated with [14C]CLB generated two peaks of radioactivity, indicating the formation of two or more major adducts. Since DNA incubated with [14C]CLB in a cell-free system gave rise to the same profile, experiments were conducted with DNA from cells exposed to radiolabeled DNA precursors, which was then reacted with CLB. DNA containing [8-14C]guanine gave rise to one peak of radioactivity, while DNA containing [2,8-3H]adenine gave rise to two peaks. These peaks corresponded to the peaks seen in the experiment with intact L1210 cells treated with [14C]CLB. Experiments with DNA containing [5-3H]cytosine indicated that no cytosyl adducts were formed. No adducts were seen in hydrolysates prepared from labeled DNA incubated with drug solvent alone. These data indicate that the majority of adducts induced by CLB are guanyl adducts, but a substantial quantity of adenyl adducts has also been identified.

Decreased phosphorylation of synaptic glycoproteins following hippocampal kindling.

Rats with chronic electrode implants to region CA3 of the hippocampus were rapidly kindled by stimulation with a 10 s, 10 Hz train of biphasic square waves presented every 5 min, until generalized seizures developed (60-70 stimulations). The hippocampi were isolated from the brains of control animals (implanted but not stimulated), and experimental animals which had developed generalized seizures. Synaptic membranes (SM) were prepared. SM were incubated with [gamma-32P]ATP and the incorporation of 32P into proteins and glycoproteins isolated by affinity chromatography on concanavalin-A-agarose was investigated. There was no difference in the phosphorylation pattern of total SM proteins between groups. In contrast, the phosphorylation of a glycoprotein with an apparent molecular weight of 180,000 was decreased 20-40% in kindled animals. This result was replicated in three independent experiments. The results suggest that the phosphorylation of glycoprotein 180 may be related to neuroplastic events.

Studies on drug resistance in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a neoplastic disease in which drug resistance invariably occurs. We have studied the uptake and interaction with molecular targets of two drugs, chlorambucil and adriamycin, in CLL lymphocytes and CHO cell lines. Resistance does not appear related to uptake for either drug. Exposure to CLB causes DNA cross-links in the sensitive but not in the resistant cell line. The GSH content of B-CLL lymphocytes is depleted after a 20-hr incubation. An inability to maintain its GSH content may contribute to this cell's vulnerability to CLB. The resistance of CLL lymphocytes to ADR may be related to the undetectable levels of its target enzyme DNA topoisomerase II. Future approaches may involve study of novel anthracyclines, DNA topoisomerase I inhibitors and the development of in vitro predictive tests.

Social determinants of adolescent drinking; what they think, what they do and what I think and do.

Four social determinants (parental and peer modeling, parental and peer norms) were studied for their effects on adolescents' norms, preferences and reported drinking behavior.

[Noroviruses and sapoviruses in man and farm animals]. / Noroviren und Sapoviren beim Menschen und bei Nutztieren.

Noro- and Sapoviruses belong to the virus family Caliciviridae and are important causative agents of acute epidemic gastroenteritis in man. In many cases transmission of Noro- and Sapoviruses occurs via contaminated food or water and the respective diseases are therefore designated as food borne. Recently, the presence of Noro- and Sapoviruses in farm animals has attracted increasing attention. Clinical symptoms were observed after experimental infection of cattle and swine with members of the two virus groups. Thus far it is not known, whether virus transmission from animals to man does occur.

Transient and persistent depolarization-induced changes of protein phosphorylation in a molluscan nervous system.

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea-water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ [( Ca2+]o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10-fold by high K+, irrespective of the level of [Ca2+]o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF- and EDTA-sensitive protein phosphatase capable of dephosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on [Ca2+]o even in normal low K+ (10 mM) medium. Removal of [Ca2+]o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long-lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms of associative learning in mammal and mollusc.

Recent work in this laboratory has begun to cast light on the biochemical mechanisms by which a cell stores associatively acquired information. This appears to occur principally via two general pathways. The first seems to be a long-term activation of protein kinase C (resulting in long-term alterations in protein phosphorylation) while the second involves changes in RNA synthesis. One striking aspect of these mechanisms in that they seem to be conserved across the species we have studied (rabbit and Hermissenda). In the present paper we review some of the studies that support the role of protein kinase C activation and RNA synthesis in memory formation.

Epileptogenic effects of electrolytic lesions in the hippocampus: role of iron deposition.

Small, anodal electrolytic lesions in the dorsal hippocampus were produced in rats, and continuous electrographic records were made from contralateral hippocampal electrodes for as long as 48 h following the lesion. Intense spontaneous epileptiform activity was observed, which developed from isolated spiking and spindling, to phasic spike trains, recurrent paroxysmal discharges and, in some cases, continuous ictal activity lasting to 7 h. Spontaneous clonic convulsions were observed in two cases. Epileptiform activity usually subsided after 12 h, but electrographic abnormalities such as spontaneous spiking and high-amplitude spindling persisted as long as 12 days. Such effects were reliably produced with iron-depositing lesions using stainless-steel electrodes, but not with platinum electrodes, suggesting that ionic deposition is critical for the lesion-induced seizure development. These results pose interpretive difficulties for the use of ion-depositing lesions in the study of limbic brain function.

Classical conditioning induces long-term translocation of protein kinase C in rabbit hippocampal CA1 cells.

The role of the Ca2+/phospholipid-dependent, diacylglycerol-activated enzyme protein kinase C (PKC) in rabbit eyelid conditioning was examined. PKC was partially purified from the CA1 region of hippocampal slices from naive, pseudoconditioned, and conditioned rabbits 24 hr after the rabbits were well conditioned. Crude membrane and cytosol fractions were prepared. In conditioned rabbits, significantly more PKC activity (63.3%) was associated with the membrane fraction (and significantly less with the cytosol fraction) compared to naive (42.0%) and pseudoconditioned (44.7%) animals. These differences in distribution of enzyme activity were paralleled by differences in stimulation of enzyme activity by Ca2+, phospholipid, and diacylglycerol. There were no between-group differences in basal protein kinase activity. These results suggest that there is a long-term translocation of PKC from cytosol to membrane as a result of conditioning. Autoradiographic binding of radioactive phorbol 12,13-dibutyrate to PKC demonstrated that almost all specific binding was in the stratum radiatum, a region containing the proximal apical dendrites of CA1 pyramidal neurons. Therefore, this may be the site of the conditioning-specific PKC translocation, a locus well-suited to underlie the biophysical effects of conditioning.

Inhibition of protein synthesis prolongs Ca2+-mediated reduction of K+ currents in molluscan neurons.

Elevated intracellular Ca2+ concentration within the Hermissenda type B cell has previously been shown to cause transient reduction of both the early K+ current IA and the delayed, Ca2+-dependent K+ current ICa2+-K+, a reduction that is more permanent with classical conditioning. Other earlier experiments suggested that Ca2+-mediated reduction of K+ currents initially involves the dual activation of Ca2+/calmodulin-dependent and Ca2+/lipid-dependent protein kinases. In the present study, voltage-clamp conditions that cause substantial increases in intracellular Ca2+ concentration (i.e., a Ca2+ "load") were used to produce IA and ICa2+-K+ reduction with and without the protein synthesis inhibitor anisomycin or cycloheximide or the control substance deacetylanisomycin in the bathing medium. Anisomycin (100 microM) and cycloheximide (100 microM) caused no significant change of resting membrane potential, holding current, or the non-voltage-dependent "leak" current. However, inhibition of protein synthesis prevented recovery from Ca2+-mediated K+-current reduction. This effect resembled the effect of injecting purified Ca2+-dependent kinases and was blocked by the presence of trifluoperazine in the bathing medium. Activation of protein kinase C with a water-soluble phorbol ester caused marked reduction of protein synthesis in Hermissenda neurons as monitored by two-dimensional gel electrophoresis. Synthesis of new proteins therefore may be important for reversal of initial steps during memory storage, and Ca2+-activated phosphorylation pathways may initiate long-term changes by turning off (as well as by turning on) the synthesis of particular proteins.

Regulation of Hermissenda K+ channels by cytoplasmic and membrane-associated C-kinase.

Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low-molecular-weight protein was changed only within the eyes of conditioned Hermissenda. To examine the role of PKC in causing K+ current changes as well as changes of phosphorylation during conditioning (and possibly other physiologic contexts), we studied here the effects of endogenous PKC activation and exogenous PKC injection on phosphorylation and K+ channel function. Several phosphoproteins (20, 25, 56, and 165 kilodaltons) showed differences in phosphorylation in response to PKC activators applied to intact nervous systems or to isolated eyes. Specific differences were observed for membrane and cytosolic fractions in response to both the phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA) or exogenous PKC in the presence of Ca2+ and phosphatidylserine/diacylglycerol. Type B cells pretreated with DPBA responded to PKC injection with a persistent reduction of K+ currents. In the absence of DPBA, PKC injection also caused K+ current reduction only following Ca2+ loading conditions. However, the direct effect of PKC injection in the absence of DPBA was only to increase ICa2+-K+. According to a proposed model, the amplitude of the K+ currents would depend on the steady-state balance of effects mediated by PKC within the cytoplasm and membrane-associated PKC. The model further specifies that the effects on K+ currents of cytoplasmic PKC require an intervening proteolytic step. Such a model predicts that increasing the concentration of cytoplasmic protease, e.g., with trypsin, will increase K+ currents, whereas blocking endogenous protease, e.g., with leupeptin, will decrease K+ currents. These effects should be opposed by preexposure of the cells to DPBA. Furthermore, prior injection of leupeptin should block or reverse the effects of subsequent injection of PKC into the type B cell. All of these predictions were confirmed by results reported here. Taken together, the results of this and previous studies suggest that PKC regulation of membrane excitability critically depends on its cellular locus. The implications of such function for long-term physiologic transformations are discussed.