The Schizosaccharomyces pombe hst4(+) gene is a SIR2 homologue with silencing and centromeric functions.

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4(+) gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Delta mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4(+) appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Delta mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4(+) is most similar to HST4. Furthermore, hst4(+) is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4(+) rescues the temperature-sensitivity and telomeric silencing defects of an hst3Delta hst4Delta double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.

The conserved core of a human SIR2 homologue functions in yeast silencing.

Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2's silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein's core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast-human chimeras' ability to function at only a subset of Sir2p's target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.

Resolution-dependent estimates of multiple sclerosis lesion loads.

BACKGROUND: Changes in brain lesion loads assessed with magnetic resonance imaging obtained at 1.5 Telsa (T) are used as a measure of disease evolution in natural history studies and treatment trials of multiple sclerosis. METHODS: A comparison was made between the total lesion volume and individual lesions observed on 1.5 T images and on high-resolution 4 T images. Lesions were quantified using a computer-assisted segmentation tool. RESULTS: There was a 46% increase in the total number of lesions detected with 4 T versus 1.5 T imaging (p < 0.005). The 4 T also showed a 60% increase in total lesion volume when compared with the 1.5 T (p < 0.005). In several instances, the 1.5 T scans showed individual lesions that coalesced into larger areas of abnormality in the 4 T scans. The relationship between individual lesion volumes was linear (slope 1.231) showing that the lesion volume observed at 4 T increased with the size of the lesion detected at 1.5 T. The 4 T voxels were less than one quarter the size of those used at 1.5 T and there were no consistent differences between their signal-to-noise ratios. CONCLUSIONS: The increase in signal strength that accompanied the increase in field strength compensated for the loss in signal amplitude produced by the use of smaller voxels. This enabled the acquisition of images with improved resolution, resulting in increased lesion detection at 4 T and larger lesion volumes.

A critical evaluation of cysteamine as a tool to deplete somatostatin in the rat central nervous system.

The wide central nervous system (CNS) distribution of somatostatin (SRIF) as well as the well documented reduction in SRIF concentration in the cerebral cortex in patients with Alzheimer's disease have served as an impetus for studies of this peptide's neurobiological role in the brain. These studies were designed to evaluate the efficacy of centrally administered cysteamine (CYS) as a tool to deplete SRIF in the hypothalamus (HYP) and extrahypothalamic brain areas. Somatostatin was measured by RIA in the frontal cortex (COR), hippocampus (HIP), and HYP in rats after seven daily infusions of CYS into unilateral cannulae stereotaxically positioned into either the lateral ventricle (LV; 300 micrograms/2 microliters) or the dorsal HIP (100 micrograms/2 microliters), and after single (300 mg/kg) or daily (100 mg/kg) sc injections; rats were killed 4 or 24 h after the last injection. After LV infusions, the SRIF concentration was significantly reduced only in the HYP (35% at 4 h and 27% at 24 h). After HIP infusions, the SRIF concentration was significantly reduced only in the HYP at 4 h (23%); no reductions were observed at 24 h. Both a single and repeated sc administrations of CYS reduced SRIF in the HYP only 24 h after treatment (54% and 50%, respectively). Acute sc CYS reduced SRIF in the COR (23%) and the HYP (29%) 4 h after treatment; repeated sc CYS reduced SRIF in the COR (25%) and the HYP (63%). Although the reduction of SRIF in the HYP was increased by repeated sc dosing, the reduction of extrahypothalamic SRIF by sc CYS was relatively small in magnitude and was not enhanced by repeated dosing. These results suggest that CYS is not an ideal tool for depletion of extrahypothalamic SRIF after sc or CNS administration and, moreover, raise serious questions about studies in which behavioral or endocrine alterations after CYS treatment were attributed to specific actions on SRIF-containing neurons.

Possible brainstem involvement in the modification of thermoregulatory processes by chlordecone in rats.

The involvement of the central nervous system in the hypothermia induced by chlordecone was studied by evaluating the effects of infusions of chlordecone injected into the lateral and third ventricles and the cisterna magna on colonic temperature (Tcol). Compared to rats given vehicle, infusions of 40, 320 or 800 micrograms of chlordecone into the lateral ventricle or 320 or 800 micrograms of chlordecone into the third ventricle, through chronic indwelling cannulae, did not change significantly Tcol. However, intracisternal infusions of 80, 160, 320 or 800 micrograms of chlordecone produced significant hypothermia (maximally 2.2 degrees C) which persisted for as long as 6 hr. Intracisternal infusions of chlordecone also produced a rapid increase in the temperature of the tail skin (Tsk) which persisted throughout the period of hypothermia. This suggests that the hypothermia produced by central administration of chlordecone is related to peripheral vasodilation. Since chlordecone has been reported to induce release of NE in the brainstem, and NE is known to modulate tonic vasomotor control in the medulla, the effects of NE infused intracisternally were studied. Intracisternal infusions of NE (16 micrograms) significantly decreased Tcol and increased Tsk, supporting the hypothesis that hypothermia induced by chlordecone is associated with vasodilatory effects, mediated by an adrenergic mechanism in the brainstem.

Pharmacological evaluation of central adrenergic involvement in chlordecone-induced hypothermia.

Adrenergic involvement in the hypothermia produced by systemically administered chlordecone (CLD) was evaluated in the rat using intracisternal pretreatment with 6-hydroxydopamine (6-OHDA), alpha-adrenergic receptor antagonists (phenoxybenzamine and phentolamine) and beta-adrenergic antagonists (propranolol and atenolol). The effect of intraperitoneal administration of 75 mg/kg of chlordecone on colonic temperature (Tcol) in male Fischer-344 rats was measured 7 days after administration of 6-OHDA and 30 min following pretreatment with the receptor antagonists. Prior depletion of catecholamines in brain with 250 micrograms of 6-OHDA administered intracerebrally attenuated hypothermia induced by chlordecone, without affecting basal Tcol. Phenoxybenzamine (10 or 20 micrograms) and phentolamine (5 or 10 micrograms) also reduced the hypothermic response to chlordecone. The beta-adrenergic receptor antagonists propranolol (50 or 100 micrograms) and atenolol (10 or 20 micrograms) did not attenuate chlordecone-induced hypothermia. These data suggest that the hypothermia induced by chlordecone is a result of alterations in central alpha-adrenergic functions, possibly involved with the sympathetic control of vasomotor tone.

Agonistic behavior in groups of limbic epileptic male rats: pattern of brain damage and moderating effects from normal rats.

Different ratios of normal male rats and male rats in which limbic seizures had been induced by a single systemic injection of lithium and pilocarpine were housed in groups of six. The group ratios ranged along the continuum from all normal rats to all experimental rats. The average numbers of episodes of boxing, biting and mounting--thrusting per rat per hour per group were recorded by direct observation (red light) for 1 h during the midscotophase. Groups that contained less than two normal rats exhibited significantly elevated amounts of agonistic (boxing, biting) behavior but not mounting behavior. Multiple regression analyses showed that combinations of neuronal loss within only two to three areas accommodated at least 50% of the variance in the numbers of these behaviors.

Suppression of experimental allergic encephalomyelitis is specific to the frequency and intensity of nocturnally applied, intermittent magnetic fields in rats.

Female Lewis rats (n=72) were inoculated with an emulsion of spinal cord and complete Freund's adjuvant. They were then exposed for approximately 6 min every hour between midnight and 08:00 h for 2 weeks to either 7 or 40 Hz amplitude-modulated magnetic fields whose temporal pattern was designed to simulate a (geomagnetic) storm sudden commencement. The peak strengths of the fields averaged between either 30-50 nT (low intensity) or 500 nT (high intensity). Rats exposed to the 7 Hz, low intensity magnetic fields displayed significantly less severe overt signs of experimental allergic encephalomyelitis than rats exposed to either of the two intensities of the 40 Hz fields, the high intensity 7 Hz field, or the reference (<10 nT) condition. The latter groups did not differ significantly from each other. Predicted severity based upon the numbers of foci of infiltrations of lymphocytes within the brains of the rats also demonstrated the ameliorating effects of the low intensity, 7 Hz exposures. These results suggest very specific characteristics of complex, weak magnetic fields within the sleeping environment could affect the symptoms of autoimmunity.

Fulminant CNS perivascular lymphocytic proliferation: association with sargramostim, a hematopoietic growth factor.

Sargramostim (GM-CSF) therapy was instituted in a 49-year-old woman with hepatitis C on chronic interferon alpha-2b therapy. Within two weeks, she developed progressive confusion, lethargy, and gait disturbance. At autopsy 4 months later, diffuse perivascular nonmonoclonal lymphoid infiltrates were demonstrated throughout the central nervous system (CNS). As the use of hematopoietic growth factors in clinical practice increases, potential adverse effects, such as the fulminant CNS lymphocytic proliferation in this patient, are more likely to be encountered.

Infiltration of lymphocytes in the limbic brain following stimulation of subclinical cellular immunity and low dosages of lithium and a cholinergic agent.

This experiment was designed to investigate the hypothesis that single small dosages of lithium (1.5 mEq/kg), the muscarinic agent pilocarpine (15 mg/kg) and spinal cord emulsion encourage perivascular infiltration of lymphocytes into the brain even when overt symptoms of experimental allergic encephalomyelitis are not apparent. The brains of rats that had received this small dosage of lithium and pilocarpine exhibited discernable infiltrations of lymphocytes within limbic tracts but no discernable neuronal loss. Although the brains of the rats that displayed overt seizures following larger dosages of lithium (3 mEq/kg) and pilocarpine (30 mg/kg) exhibited the usual pattern of neuronal loss within multiple thalamic and limbic structures and conspicuous foci of lymphocytic infiltration (particularly within the hippocampal formation) the correlation between the numbers of foci and the proportions of neuronal damage in these structures was not significant statistically. These results indicate that infiltrations of lymphocytes into brain parenchyma are not simple artifacts of the amount of neuronal damage and may be sensitive toxicological markers for subclinical interactions between drugs and immune responses.

Differential effects of low frequency, low intensity (<6 mG) nocturnal magnetic fields upon infiltration of mononuclear cells and numbers of mast cells in Lewis rat brains.

Immediately after inoculation to induce experimental allergic encephalomyelitis, 64 female Lewis rats were exposed to either a reference condition (<10 nT) or to one of two frequencies (7 Hz, 40 Hz) of magnetic fields whose two intensities (either 50 nT or 500 nT) were amplitude-modulated for 6 min once per hour between midnight and 8 h for 15 nights. Rats that had been exposed to the 7 Hz, low intensity fields displayed fewer numbers of foci of infiltrations of mononuclear cells compared to all other groups that did not differ significantly from each other. Rats exposed to the 5 mG (500 nT), 40 Hz magnetic fields displayed more foci in the right thalamus while those exposed to the 5 mG, 7 Hz fields displayed more foci in the left thalamus. Numbers of mast cells within the thalamus were also affected by the treatments. These results suggest that weak magnetic fields can affect the infiltration of immunologically responsive cells and the presence of mast cells in brain parenchyma. Implications for the potential etiology of 'electromagnetic sensitivity' symptoms are discussed.

Serotonergic modulation of the acoustic startle response in rats during preweaning development.

The involvement of serotonin (5-HT) in modulating the acoustic startle response (ASR) is well established in adult rats, but 5-HT involvement during the preweaning period, when 5-HT neurons undergo extensive development, has not previously been described. Three 5-HT receptor subtypes are reported to modulate the ASR in adult rats: 5-HT1A and 5-HT2 receptor agonists facilitate the ASR, whereas 5-HT1B agonists decrease the response. In the present study, the effects of 5-HT agonists and generalized 5-HT depletion on the ASR were studied in preweanling animals, using independent groups of Long-Evans rats tested on postnatal day (PND) 13, 17 and 21. 8-Hydroxy-2-(di-n-propylamino) tetralin (8OHDPAT, 62-1000 micrograms/kg), a 5-HT1A receptor agonist, and 5-methoxy-N,N-dimethyl tryptamine (MeODMT, 2-4 mg/kg), a nonselective 5-HT agonist, had no effect on PND 13 and then increased the ASR on PND 17 and 21. The 5-HT2 receptor antagonists cyproheptadine (5 mg/kg) and ketanserin (5 mg/kg) blocked the effect of MeODMT at both ages, providing some evidence that MeODMT increased the ASR through 5-HT2 receptors. 1-(m-Chlorophenyl) piperazine (mCPP, 1-5 mg/kg), a 5-HT1B agonist, had no effect on ASR amplitude on PND 13 or 17 and then produced a dose-related decrease in the response on PND 21. Generalized depletion of 5-HT by 80-90% in whole-brain and spinal cord, using p-chlorophenylalanine (PCPA, 300 mg/kg 24 hr prior to testing), did not alter ASR amplitude at any age.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute behavioral toxicity of carbaryl and propoxur in adult rats.

Motor activity and neuromotor function were examined in adult CD rats exposed to either carbaryl or propoxur, and behavioral effects were compared with the time course of cholinesterase inhibition. Rats received an IP injection of either 0, 2, 4, 6 or 8 mg/kg propoxur or 0, 4, 8, 16 or 28 mg/kg carbaryl in corn oil 20 min before testing. All doses of propoxur reduced 2 hr activity in a figure-eight maze, and crossovers and rears in an open field. For carbaryl, dosages of 8, 16 and 28 mg/kg decreased maze activity whereas 16 and 28 mg/kg reduced open field activity. In order to determine the time course of effects, rats received a single IP injection of either corn oil, 2 mg/kg propoxur or 16 mg/kg carbaryl, and were tested for 5 min in a figure-eight maze either 15, 30, 60, 120 or 240 min post-injection. Immediately after testing, animals were sacrificed and total cholinesterase was measured. Maximum effects of propoxur and carbaryl on blood and brain cholinesterase and motor activity were seen within 15 min. Maze activity had returned to control levels within 30 and 60 min whereas cholinesterase levels remained depressed for 120 and 240 min for propoxur and carbaryl, respectively. These results indicate that both carbamates decrease motor activity, but behavioral recovery occurs prior to that of cholinesterase following acute exposure.

Accuracy of frozen section diagnosis of the salivary gland.

Three hundred and one salivary gland lesions (162 benign, 72 malignant, and 67 benign non-neoplastic) of 677 cases were evaluated by use of intraoperative frozen sections by 66 pathologists. In seven patients, the diagnosis was deferred for permanent sections. In four cases (1.3%), the diagnosis at permanent section changed from one category of benign tumor to another, and in five cases (1.7%), from one category of malignant tumor to another. In four tumors, a frozen section diagnosis of benign was changed to malignant on permanent sectioning; all four involved acinic cell carcinomas. Only two tumors were incorrectly diagnosed as malignant. We conclude that diagnoses of most salivary gland lesions based on frozen section examination are reliable and accurate. However, the literature does indicate that caution should be exercised when malignant tumors are dealt with.

Comparison of four automated hematology analyzers.

OBJECTIVE: To compare four automated hematology analyzers for efficiency and sensitivity. DESIGN: Four automated hematology analyzers were compared in a side by side study: Bayer ADVIA 120 (Bayer Diagnostic Division, Tarrytown, NY), Beckman Coulter GEN S (Beckman Coulter, Brea, CA), Abbott CELL DYN 3500 and CELL DYN 4000 (Abbott Diagnostics, Santa Clara, CA). 164 specimens were analyzed for cell counts, indices, and the automated WBC differential (DLC). Tallies were kept of all interventions, defined as any parameter necessitating examination of a stained blood smear by a clinical laboratory scientist. A 400-cell manual differential was performed on each specimen and used as the reference to prepare truth tables for each type of WBC. PATIENTS: Specimens comprised regular runs from this tertiary care teaching hospital. These included inpatients, outpatients, and oncology patients, including bone marrow transplant patients. MAIN OUTCOME MEASURES: Results from the truth tables were used for calculating sensitivity and efficiency for each analyzer. Each DLC parameter was analyzed for variance using the one-way ANOVA test. RESULTS: No intervention was required for 103 of 164 specimens for the CELL DYN 3500; the ADVIA gave 70 reportable DLCs without intervention, the GEN S provided 91 and the CELL DYN 4000 resulted in 117 of 164 DLCs without intervention. Agreement or efficiency was 65% for the CELL DYN 3500, 41% for the ADVLA, 58% for the GEN S, and 79% for the CELL DYN 4000. Sensitivity was 67% for the CELL DYN 3500, 86% for the ADVIA, 76% for the GEN S, and 71% for the CELL DYN 4000. Probability of significant variation was as follows for each parameter: % neutrophil 0.8747, % lymphocyte 0.8830, % monocyte 0.0296, % eosinophil 0.7903, and % basophil <.0001. CONCLUSION: The analyzers tested were acceptable for routine laboratory work. Selection would depend on individual need with respect to sensitivity and efficiency. The clinical significance of disagreement between the DLC and the manual differential remains to be determined.

Long-term consequences of subtle stimuli during the first twenty-four hours of seizure-induced brain injury.

Chronically epileptic (induced by a single systemic injection of lithium and pilocarpine about 30 days before the experiment began) male rats were trained within a radial maze while they were administered either GABA-pentin (Neurontin), or prednisolone or given no treatment. There was no significant improvement in learning or memory between the groups. Numbers of trials per day were positively correlated with the time required to display the overt stereotyped forelimb clonus after the single pilocarpine injection. The numbers of correct trials completed during the first few days of acquisition were significantly greater for the rats that had receive weak (1 microT) complex, pulsed magnetic fields over the right hemisphere during the first 24 hr. after seizure induction than for those who received the same field over the left hemisphere or that had been exposed to reference conditions. Implications of the enhanced sensitivity of limbic neurons to subtle electromagnetic interaction during electrical lability are discussed.

Demands during maze. learning in limbic epileptic rats: selective damage in the thalamus?

A qualitatively evident enhancement of chromolytic neurons within the lateral posterior thalamus of rats in which limbic seizures had been induced by lithium and pilocarpine and who were later trained for spatial memory was assessed quantitatively. The significant increase in the numbers of chromolytic neurons and the decrease in the numbers of normal neurons for these rats compared to the reference brains suggested these morphological changes were recent. The hypothesis that excessive stimulation of the lateral posterior nucleus by daily training in a radial maze may have facilitated the necrosis was supported by the inverse relationship between a linear combination of the numbers of normal neurons and oligodendroglia and the rate of learning during the earlier but not the later sessions. An implication for iatrogenic effects from rehabilitation of humans following brain injury was suggested.

"Subclinical" dosages of lithium and pilocarpine that do not evoke overt seizures affect long-term spatial memory but not learning in rats.

After training is an automated radial maze, 11 male rats were injected with either "subclinical" dosages of lithium and pilocarpine or saline and then tested 5 days or 4 months later. When employed as their own controls or when compared with a saline-injected reference group, the rats that had received the lithium and pilocarpine displayed memory deficits but not learning learning deficits after the longest of the two delays (effect size was 41%). These results suggest that subtle disruption in memory but not learning to criterion could be associated with "subclinical electrical seizures" or the micromorphological changes associated with this activity.

Emergent properties following brain injury: the claustrum as a major component of a pathway that influences nociceptive thresholds to foot shock in rats.

Flinch (pain) thresholds for electric current delivered to the feet were correlated with the amount of necrosis within the diencephalon and telencephalon for rats in which seizures had been induced by lithium and pilocarpine about two months before the testing. The shared variance of the quantitative damage within the claustrum, the anterior part of the paraventricular nucleus of thalamus, (central) mediodorsal thalamus, and lateral amygdala (ventromedial part) explained 81% of the variance in the nociceptive (flinch) thresholds. A primary role of the claustrum within the neuropathways that mediate the response to the interoceptive and "painful" characteristics of stimuli is indicated. The concept of primary pathways versus "emergent" pathways subsequent to excitotoxic damage within the neuromatrix is discussed.