p97 and close encounters of every kind: a brief review.

The AAA (ATPase associated with various cellular activities) ATPase, p97, is a hexameric protein of chaperone-like function, which has been reported to interact with a number of proteins of seemingly unrelated functions. For the first time, we report a classification of these proteins and aim to elucidate any common structural or functional features they may share. The interactors are grouped into those containing ubiquitin regulatory X domains, which presumably bind to p97 in the same way as the p47 adaptor, and into non-ubiquitin regulatory X domain proteins of different functional subgroups that may employ a different mode of interaction (assuming they also bind directly to p97 and are not experimental artifacts). Future studies will show whether interacting proteins direct p97 to different cellular pathways or a common one and structural elucidation of these interactions will be crucial in understanding these underlying functions.

Learning and memory in streptozotocin-induced diabetic rats in a novel spatial/object discrimination task.

Diabetes mellitus is associated with disturbances of cognitive functioning. The aim of this study was to examine cognitive functioning in diabetic rats using the 'Can test', a novel spatial/object learning and memory task, without the use of aversive stimuli. Rats were trained to select a single rewarded can from seven cans. Mild water deprivation provided the motivation to obtain the reward (0.3 ml of water). After 5 days of baseline training, in which the rewarded can was marked by its surface and position in an open field, the animals were divided into two groups. Diabetes was induced in one group, by an intravenous injection of streptozotocin. Retention of baseline training was tested at 2-weekly intervals for 10 weeks. Next, two adapted versions of the task were used, with 4 days of training in each version. The rewarded can was a soft-drink can with coloured print. In a 'simple visual task' the soft-drink can was placed among six white cans, whereas in a 'complex visual task' it was placed among six soft-drink cans from different brands with distinct prints. In diabetic rats the number of correct responses was lower and number of reference and working memory errors higher than in controls in the various versions of the test. Switches between tasks and increases in task complexity accentuated the performance deficits, which may reflect an inability of diabetic rats to adapt behavioural strategies to the demands of the tasks.

Non-linear effects in the retention of an avoidance task induced by anabolic steroids.

The results of the study reported in Brain Research in 1995 by Isaacson et al. [Isaacson, R.L., Varner, J.A., Baars, J.-M., de Wied, D., 1995. The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance task. Brain Res. 689, 79-84] have been re-examined with special emphasis placed on the distributions of latencies found in the passive avoidance task using rats. This study used two retention tests, one 24 h after training the other at 48 h after training. In the first experiment in that study a range of doses of two anabolic steroids, pregnenolone sulfate and ethylestrenol, were given s.c. just after the footshock training trial. In experiment 2 a similar range of doses of both steroids was given to the rats 1 h before the first retention test. Placing emphasis on the distributions rather than measures of central tendencies revealed that, in contrast to the vehicle treated animals, the anabolic steroid treated animals exhibited bimodal distributions of response latencies. These differences between control and hormone treated animals were observed in both experiments. The new information was interpreted in terms of non-linear dynamics including some aspects of Chaos theory.

Elucidating the mechanism of familial amyloidosis- Finnish type: NMR studies of human gelsolin domain 2.

Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type.

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloidosis-Finnish type (FAF). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild-type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curvature with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations D187N and D187Y destabilize gelsolin domain 2 by 1.22 and 2.16 kcal. mol(-1) (1 kcal = 4.18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilization conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation.

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony-forming-unit fibroblast (CFU-F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10-fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre-B-cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs.

Immunohistological markers of cerebrovascular integrity.

The brain depends on other organ systems of the body for oxygen, nutrients, and the elimination of metabolic byproducts. The primary route for such transfer of these essentials is the cerebrovasculature. The cerebrovasculature also participates in metabolizing or excluding xenobiotics, segregating components of the immune response, regulating pH and osmolarity of the cerebrospinal fluid, selectively distributing hormones, and impeding pathogenic invasion. Various aspects of these diverse functions are attributed to the complex structural and molecular properties of cerebral endothelial cells collectively referred to as the blood-brain barrier (1-7). The hallmark structural specialization of the blood-brain barrier is the tight junction between the endothelial cells, which prevents diffusion of plasma proteins and molecules of a similar size or larger (8-12). Other structural specializations include close apposition of astrocytic endfeet, sparsely distributed pericytes, and extensive association with microglia. Molecular specializations include endothelial expression of transporters and enzymes, such as those involved in xenobiotic metabolism (12-27).

Chronic administration of aluminum-fluoride or sodium-fluoride to rats in drinking water: alterations in neuronal and cerebrovascular integrity.

This study describes alterations in the nervous system resulting from chronic administration of the fluoroaluminum complex (AlF3) or equivalent levels of fluoride (F) in the form of sodium-fluoride (NaF). Twenty seven adult male Long-Evans rats were administered one of three treatments for 52 weeks: the control group was administered double distilled deionized drinking water (ddw). The aluminum-treated group received ddw with 0.5 ppm AlF3 and the NaF group received ddw with 2.1 ppm NaF containing the equivalent amount of F as in the AlF3 ddw. Tissue aluminum (Al) levels of brain, liver and kidney were assessed with the Direct Current Plasma (DCP) technique and its distribution assessed with Morin histochemistry. Histological sections of brain were stained with hematoxylin & eosin (H&E), Cresyl violet, Bielschowsky silver stain, or immunohistochemically for beta-amyloid, amyloid A, and IgM. No differences were found between the body weights of rats in the different treatment groups although more rats died in the AlF3 group than in the control group. The Al levels in samples of brain and kidney were higher in both the AlF3 and NaF groups relative to controls. The effects of the two treatments on cerebrovascular and neuronal integrity were qualitatively and quantitatively different. These alterations were greater in animals in the AlF3 group than in the NaF group and greater in the NaF group than in controls.

The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance task.

Two experiments using male rats evaluated the effects of a range of doses of the neurosteroid, pregnenolone sulfate (PS), or of the synthetic neurosteroid, ethylestrenol (E), on the retention of a passive avoidance task. The steroids either were given immediately after the training trial or 1 h before the first retention test. Retention tests were given both 24 h and 48 h after acquisition. In both experiments, separate groups of animals were trained under low or moderate footshock conditions. At all doses tested both PS and E improved retention under the low footshock conditions. In groups trained with the higher footshock, the steroid-treated groups performed no better than the vehicle controls. Indeed, there were suggestions that some doses impaired retention. These results seem best understood as an induction of bimodality or 'turbulence' in behavior as used in Chaos theory rather than a shift in an inverted U-shaped retention function. In the second experiment in which the steroids were given before retention testing, they were generally without effect.

Behavioral and neuroanatomical consequences of a unilateral intraventricular infusion of AF64A and limitations on the neuroprotective effects of nimodipine.

The monoethylcholine aziridinium ion, AF64A, (3 nmol in 1 microliter) or artificial CSF (1 microliter) was infused unilaterally into the right dorsal lateral ventricle of male adult rats. Treatment with the L-type calcium channel antagonist, nimodipine (70 micrograms/kg b.wt.) or its vehicle was administered beginning before and for seven days following surgery. The infusion of AF64A reduced spontaneous alternation rates in the T-maze when compared to CSF and sham infused animals. AF64A-treated animals also took longer to reach the goal area in a complex maze task on specific trials relative to CSF and sham-infused animals. Locomotion and habituation to the open field did not differ between surgery groups. Unilateral AF64A significantly depleted acetylcholinesterase (AChE) positive terminals in the ipsilateral hippocampus and cell bodies in the ipsilateral medial septal area (MSA). Receptors for nerve growth factor (NGF-R), often colocalized with cholinergic cell bodies and terminals, also were depleted in the ipsilateral MSA of AF64A infused animals. Treatment with nimodipine did not have a neuroprotective effect on AF64A animals in either behavioral or histological results. However, some degree of protection was found in the vehicle-treated rats. This effect was likely a consequence of the stress of the injection procedure rather than the content of the vehicle, largely polyethylene glycol 400. Nimodipine-treated animals, regardless of surgery group, exhibited fewer emotional responses and had lower spontaneous alternation rates than untreated animals. The behavioral alterations found in the nimodipine groups are most easily explained in terms of altered emotionality. Overall our findings indicate that AF64A is a potent cholinotoxin that can selectively eliminate the ipsilateral septohippocampal cholinergic system when unilaterally infused into the lateral ventricle. It is possible that the mechanism of action of AF64A, like other nitrogen mustard analogues, involves disruption of basic processes involved in protein synthesis and DNA activities. Because of this, the toxic effects of the aziridinium mustard are independent of extracellular calcium and thus may not be susceptible to protection by calcium channel antagonists.

Chronic aluminum fluoride administration. I. Behavioral observations.

This study examined the behavioral effects of chronic ingestion of various monofluoroaluminum complexes (AlF3) in drinking water. Forty young adult male Long-Evans rats were divided into four groups of 10 rats each. The groups received different concentrations of AlF3 in the drinking water from three sample solutions having a total Al concentration of 0.5, 5.0, and 50 ppm, respectively, or double-distilled deionized water on an ad lib. basis for 45 weeks. General decline of bodily appearance was observed in the lowest concentration AlF3 group, and animals in this group succumbed in greater numbers during the course of the study than those in any other group. Examinations of performance in an open field, an analysis of walking patterns, and a balance beam test did not find any difficulties indicative of motor disorder. Indeed, on the initial trial on the balance beam, the AlF3-treated animals exhibited superior performance. No group differences were found in behavior assessed by spontaneous alternation or by a modified Morris water maze test. When retested in the Morris maze after a low dose of scopolamine (0.4 mg/kg), the control animals took longer to reach the platform while the AlF3-treated rats were not affected. In an olfactory preference test, the AlF3-treated animals failed to show preferences exhibited by the controls, indicating a possible olfactory impairment. The level of Al in the brains of the AlF3-exposed rats, as determined by direct current plasma analysis, was almost double that of the control animals. There was a similar trend for the Al content found in the kidneys.

The effects of pregnenolone on acquisition and retention of a food search task.

Two experiments were undertaken in which the effects of semichronic administration of the precursor steroid, pregnenolone, were examined in a food search task. In both experiments male rats were required to find a food reward in a designated hole in an arena with 16 equally spaced holes. Hormone administration began 8 days before the onset of training. Training was given on an every-other-day schedule for five sessions. Animals were deprived of food for 18 h before training or testing. Retention testing occurred 10 days after acquisition and this was followed by 2 days of training using a different hole for the reward. The two experiments differed only in the method of hormone administration. In one experiment the rats received an implanted (sc) slow release pellet containing pregnenolone before training. In the second experiment the animals received ip injections of pregnenolone sulfate before and during initial training and then had the slow release pellet implanted between acquisition and retention. Significant enhancement of retention was found during the middle trials of the retention test when the treated and control groups from the two experiments were combined. No differences were found during acquisition training in either experiment. On the first day of training the animals to find the reward in a new location, the group injected with pregnenolone sulfate and later implanted with pregnenolone slow-release pellets exhibited performance superior to that of their matched control group.

Nimodipine's functional benefits depend on lesion completeness in medial septal area.

The effects of a 4-day nimodipine treatment (70 micrograms/kg IP beginning on the day of surgery) given to rats with lesions directed at the medial septal area were monitored for 120 days. Body weight, water intake, open-field activity, rearing, hole-poking, and repetitive motor acts were periodically measured through 120 postsurgical days. Although no differences were found in water intake between any of the groups, the body weights of rats with any medial septal damage, whether treated with nimodipine or not, were lower than rats with control operations by postsurgery day 120. Rats with any medial septal damage, whether treated with nimodipine or not, had lower rearing frequencies, rearing durations, and hole-poking frequencies than controls on all test days. However, rats with complete medial septal lesions treated with nimodipine exhibited movement in the open field and frequencies of stereotyped, species-typical acts similar to those of control rats by postsurgery day 60. This nimodipine effect was not observed in rats with partial lesions of the medial septal region. This study emphasizes that a brief administration of nimodipine shortly after brain damage can influence behavioral changes 40-60 days after surgery, but that this effect was not apparent in rats with only partial medial lesions.

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity.

The possible role of extracellular calcium ([Ca+2]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca+2]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca+2]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca+2]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca+2]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca+2]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca+2]e was approximately 2 microM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca+2]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca+2]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca+2]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca+2]e can contribute to cytotoxicity, the presence of [Ca+2]e might not influence cryopreservation-induced cytotoxicity.

Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine.

The immortalized septal cell line, SN56 B5 G4, generated by the fusion of mouse septal area cells and neuroblastoma cells, was used to determine if nimodipine, an antagonist of voltage sensitive calcium 'L' channels, might act in a neuroprotective fashion when intracellular calcium levels were raised by incubation in ouabain and monensin. Fluorescent indicator dyes and the automated spectrofluorometer, the CytoFluor 2300, were used to analyze specific cellular targets and functions affected by ouabain and monensin and possible protection by prior incubation with nimodipine. Ouabain and monensin were used together to create a time- and dose-dependent toxic episode. Increases in the emission intensity of Fluo3-AM demonstrated that the concentration of intracellular calcium was monotonically increased by increasing levels of ouabain-monensin. The calcein-AM fluorescent probe indicated that there were no changes in plasma membrane permeability during the toxic episode. Lysosomal integrity decreased as indicated by decreases in neutral red retention. The concentration of free radicals increased as shown by the increase in emission intensity of 2',7'-dichlorfluorescein. Nimodipine pretreatment of the cells incubated with ouabain and monensin resulted in apparent protection of lysosomes and a reduction in the level of free radicals. While nimodipine, by itself, produced a small decrease in intracellular calcium, it actually augmented the ouabain-monensin induced increase in intracellular calcium. The data suggest that in immortalized septal cells, (a) nimodipine offers protection to certain of the responses induced by ouabain-monensin, (b) the protection offered by nimodipine may be independent of antagonism of voltage sensitive calcium channels, and (c) that the protective changes can occur at the same time that intracellular calcium is increasing. These latter observations question the hypothesis that the protection against cell death and dysfunction offered by nimodipine is due solely to maintaining calcium homeostasis.

Cardiac inotropic effects of ethanol and calcium-channel modulators.

Our objective was to analyze the influence of ethanol ingestion on the in vitro inotropic effects of dihydropyridines alone, or in combination with ethanol, on atrial muscle from rats offered a liquid diet with ethanol ("ethanol rats," ER) or without ethanol ("normal rats," NR). Left atria from NR or ER were superfused with Tyrode's solution (36 degrees C) and driven at 1.5 Hz while recording tension. Bay K 8644 (BAYK) increased, while nimodipine or ethanol decreased, the tension developed and the velocity of development of tension. The preparations recovered rapidly from the effects of ethanol, but not from those of the dihydropyridines. The effects of ethanol and dihydropyridines in combination were the result of the additive or counteractive actions of the drugs. The effects of ethanol and nimodipine on ER preparations were not different from those observed in NR. The action of BAYK was significantly smaller in ER than in NR. In other words, chronic ingestion of ethanol reduced the positive inotropic effect of BAYK, but it did not modify the negative inotropic action of nimodipine or ethanol.

Behavioral and anatomical consequences of unilateral fornix lesions and the administration of nimodipine.

Male Wistar rats subjected to unilateral fimbria-fornix transection by mechanical knife cut or to sham operations were tested in a water maze and in an open field. Half the animals in each group were treated with either 0.06 mg/kg nimodipine or vehicle, administered i.p. for 7 days, beginning the day of surgery. Animals were sacrificed and brains were processed for acetylcholine esterase (AChE) histochemistry. In the water maze, lesioned rats showed a significant impairment relative to the sham-operated animals. Nimodipine treatment did not improve performance. There were no differences among the groups in the observed frequencies of the open field behaviors of locomotion, hole-poke, rearing and grooming. A significant reduction of AChE-positive cell bodies was found in the medial septal region on the side of the lesion. There were no differences in water maze performance among groups of rats treated with 0.0, 0.5, 1.0, or 5.0 mg/kg nimodipine for 7 days, beginning the day of fimbria-fornix transection, in an attempt to determine any dose-dependent effect of the drug.